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G LOBAL GAJ ANTIVIRAL JOURNAL ™ 2008 HIV December 9-12, 2008 Wyndham Grand Rio Mar Hotel Puerto Rico Final Program and Abstract Book This program is sponsored by Emory University School of Medicine GAJ Volume 4, Supplement 1 GAJ GLOBAL ANTIVIRAL JOURNAL Aims and Scope Global Antiviral Journal publishes peer-reviewed original works related to international efforts to advance antiviral discovery and development, including full-length articles and short papers, as well as solicited review articles, conference reports, letters and book reviews. Occasional supplements contain conference abstracts, presentations and/or posters from international meetings in the fields of virology and antiviral research. The scope of the journal encompasses chemistry and biological advances in the fundamental and clinical study of antiviral diseases and their treatment. Areas covered include HIV, hepatitis B, hepatitis C and emerging viruses, co-infections, vaccines, animal models, pharmacology, microbicides, alternative therapies, viral dynamics and resistance issues. The journal is published online by IHL Press at www.ihlpress.com/gaj.html. All printed supplements are also made available online. Publication Policy Global Antiviral Journal publishes only original, documented research of high scientific quality, following accepted ethical standards of research. Submission of a manuscript signifies that it has been neither copyrighted, published, nor submitted or accepted for publication elsewhere. Editor-in-Chief Raymond F. Schinazi, Emory University School of Medicine and Veterans Affairs Medical Center, Department of Pediatrics, Medical Research 151H, 1670 Clairmont Road, Decatur, Georgia, 30033, USA Editorial Office IHL Press, 26 Bailey Road, Arlington, MA, 02476, USA Telephone: +1 781 648 1933 Fax: +1 781 646 2699 [email protected] Subscription Details Subscription prices are available upon request from the Publisher. All inquiries should be directed to the Editorial Office. Advertising and Supplements All advertising enquiries and supplement proposals, including advertising within supplements, should be directed to the Editorial Office. Copyright © 2008 IHL Press. All rights reserved. No part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, diagnoses, drug dosages, instructions or ideas contained in the material herein. ISSN (print): 1556-9047 ISSN (online): 1556-9055 ™ HIV 2008 December 9-12, 2008 • Wyndham Grand Rio Mar Hotel • Puerto Rico Final Program and Abstract Book HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies i Table of Contents Page Corporate Supporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv Continuing Medical Education. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Scholarships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Conference Committees . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi Social Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Scientific Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Abstracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv Pathogenesis and Targeted Design. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Toward HIV Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Prevention and Drug Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 HIV Therapy in Developing Countries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 HIV/Hepatitis and Other Co-infections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Novel Therapeutic Approaches: Antiviral Mechanism and Predictive Toxicology. . . . . . . . . . 47 Pharmacology and Drug Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Author Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Late Breaker Abtracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 CME Program Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies iii HIV DART 2008 is supported by independent educational grants from the following: Gold Support Silver Support Bronze Support RFS Pharma, LLC • Samchully Pharm. Co., Ltd Additional Support ACLIRES International Ltd. • American Academy of HIV Medicine Department of Veterans Affairs • Presidio Pharmaceuticals, Inc. HIV DART 2008 is also supported in part by Grant Number 5R13AI065203 from the National Institute of Allergy and Infectious Diseases. The views expressed in written conference materials or publications and by speakers and moderators do not necessarily reflect the official policies of the Department of Health and Human Services; nor does mention of trade names, commercial practices, or organizations imply endorsement by the U.S. Government. The Organizers would like to thank The NAMES Project Foundation for partnering with HIV DART 2008 to display sections of the AIDS Quilt memorializing Puerto Ricans who lost their lives to AIDS. “A memorial, a tool for education and a work of art, the Quilt is a unique creation, an uncommon and uplifting response to the tragic loss of human life.” –The NAMES Project, www.aidsquilt.org iv Global Antiviral Journal Volume 4, Supplement 1 Continuing Medical Education HIV DART 2008 is sponsored by Emory University School of Medicine. Conference Objectives HIV DART was designed to interest physicians, clinicians, scientists and clinical researchers in the HIV/co-infection arena. The educational objectives of this program are as follows: • Understand the role of viral targets in the drug discovery and development process • Identify novel therapeutic agents and viral targets • Develop improved strategies to reduce or eliminate drug-resistance and toxicity • Provide insights on novel immunological approaches compatible with clinical drug development • Develop approaches to eliminate or eradicate HIV from viral reservoirs • Understand the consequences of co-infection with hepatitis B & C on the management of subjects with HIV infection Continuing Medical Education Accreditation Statement: Emory University School of Medicine is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. Credit Designation Statement: Emory University School of Medicine designates this educational activity for a maximum of 17 AMA PRA Category 1 Credit(s)TM. Physicians should only claim credit commensurate with the extent of their participation in the activity. Scholarships Conference scholarships were provided for post-doctoral fellows, nurses, assistant professors, underrepresented minorities, and residents of developing countries. 2008 Scholarship Recipients Jules Levin, NATAP, USA Terence McPhaul, National AIDS Education & Services for Minorities, USA Andy Mtambo, BC Centre for Excellence in AIDS/HIV, Canada Mrunalee Patil, Emory University School of Medicine, USA Maria Sandu, Clinical Hospital for Infectious Diseases, Romania Tracy Swan, Treatment Action Group, USA HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies v Organizing Committee David Cooper, Co-chair José Rodriguez-Orengo, Local Chair Joep Lange, Co-chair Raymond F. Schinazi, Chair Robert Murphy, Chair Jean-Pierre Sommadossi, Emeritus Chair University of New South Wales, Australia University of Amsterdam, the Netherlands Northwestern University, USA Instituto de Ciencias Forenses, Puerto Rico, USA Emory University/VA Medical Center, USA Idenix Pharmaceuticals, USA Scientific Committee Françoise Brun-Vézinet Alain Lafeuillade Sal Butera Hiroaki Mitsuya Pedro Cahn Julio Montaner Bonaventura Clotet Alan Perelson Steven Deeks Praphan Phanuphak Courtney Fletcher Richard Pollard Joel Gallant Bruce Polsky José Gatell François Raffi Brian Gazzard Douglas Richman Matthias Götte Ian Sanne Eric Hunter Charles van der Horst John Idoko Stefano Vella Christine Katlama Mark Wainberg Hôpital Bichat Claude Bernard, France Centers for Disease Control and Prevention, USA Fundación Huesped, Argentina Fundacio IRSI Caixa, Spain University of California, San Francisco, USA University of Nebraska Medical Center, USA Johns Hopkins University School of Medicine, USA Hospital Clinic, Spain Chelsea & Westminster Hospital, UK McGill University, Canada Emory Vaccine Center, USA Jos University Teaching Hospital, Nigeria Groupe Hospitalier Pitié-Salpêtrière, France Chalucet Hospital, France Kumamoto University School of Medicine, Japan BC Centre for Excellence in HIV/AIDS, Canada Los Alamos National Laboratory, USA Thai Red Cross AIDS Research Centre, Thailand University of California, Davis Medical Center, USA St. Luke’s-Roosevelt Hospital Center, USA Centre Hospitalier Universitaire de Nantes, France University of California, San Diego/VA Medical Center, USA Wits Health Consortium, South Africa University of North Carolina at Chapel Hill, USA Istituto Superiore di Sanità, Italy McGill AIDS Centre, Canada Paolo La Colla Università degli Studi di Cagliari, Italy vi Global Antiviral Journal Volume 4, Supplement 1 Social Functions Welcome Reception Tuesday, December 9, 2008 18:00 Pool Patio East, Wyndham Grand Rio Mar Poster Session Reception Wednesday, December 10, 2008 16:00 - 18:30 Caribbean Ballroom Gala Party Thursday, December 11, 2008 20:00 Vista Verde Garden, Wyndham Grand Rio Mar Conference Secretariat 1631 Phoenix Boulevard, Suite 4 College Park, Georgia 30349 USA Telephone: +1 770 997 2484 Facsimile: +1 770 997 2488 E-mail: [email protected] Website: www.informedhorizons.com HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies vii HIV DART 2008 Frontiers in Drug Development for Antiretroviral Therapies Scientific Program Tuesday, December 9, 2008 Abstract 18:00 Welcome Reception Wednesday, December 10, 2008 8:00 Opening Remarks and Presentation of the Gertrude Elion Distinguished Lecturer Award Raymond F. Schinazi Emory University/Veterans Affairs Medical Center, USA Robert Murphy Northwestern University, USA 8:15 Gertrude Elion Distinguished Lecturer Award Approaches towards HIV-1 Eradication: Harnessing Cellular Restrictions Mario Stevenson University of Massachusetts Medical School, USA Pathogenesis and Targeted Design Chairs: Daria Hazuda Leonid Margolis 01 Merck Research Labs, USA National Institute of Child Health and Human Development, USA 8:55 Understanding HIV Maturation using Electron Cryotomography Elizabeth Wright California Institute of Technology, USA 02 9:15 Vpu and its Cellular Targets Paul Spearman Emory University School of Medicine, USA 03 9:35 Cell Penetrating Peptides: From Molecular Mechanisms to Therapeutics Gilles Divita CNRS, France 04 9:55 Panel Discussion 10:15 Break Toward HIV Eradication I Chairs: Bruce Polsky Matthias Götte St. Luke's Roosevelt Hospital Center, USA McGill University, Canada 10:35 Complementary Mechanisms of Nucleoside Analog Resistance from Structural Studies of HIV-1 Reverse Transcriptase Eddy Arnold Rutgers University, USA 06 10:55 Dose Response Curve Slope Sets Class Specific Limits on Inhibitory Potential of Anti-HIV Drugs Robert Siliciano Johns Hopkins University School of Medicine and Howard Hughes Medical Institute, USA 07 11:15 Pharmacology, Metabolism and Transport of HIV Drugs into Viral Reservoirs Saye Khoo University of Liverpool, UK 08 11:35 Panel Discussion HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies ix Abstract Oral Abstract Session I Chairs: Charles Boucher Adrian Ray Erasmus Medical Center Rotterdam and University Medical Centre Utrecht, the Netherlands Gilead Sciences, Inc., USA 11:55 Immunotoxins to Selectively Deplete Reservoirs of HIV-infected Cells that Persist in the Face of Highly Suppressive Antiretroviral Therapy Edward Berger National Institutes of Health, USA 09 12:05 Development of Pyrimidinedione NNRTIs with a High Genetic Barrier to Resistance Robert Buckheit ImQuest BioSciences, USA 10 12:15 Development of VS411, a Virostatic Drug Fixed Dose Combination (FDC) Designed to Inhibit both HIV and Immune Hyperactivation Franco Lori ViroStatics Srl, Italy 11 12:25 Inhibiting Vpu Function with the Novel Compound BIT225, Results in Inhibition of HIV-1 Release from Human Macrophage Reservoirs John Wilkinson Biotron Limited, Australia 12 12:35 Lunch 14:00 – 16:00 Free Afternoon 16:00 – 18:30 Poster Session Toward HIV Eradication II Chairs: Raymond F. Schinazi Emory University/Veterans Affairs Medical Center, USA Ian Sanne Wits Health Consortium, South Africa 18:30 State of the Art Lecture Integrase Inhibitors: New Insights into Mechanism of Action and Pharmacology with Implications for Treatment and Prevention Strategies Daria Hazuda Merck Research Labs, USA 13 19:00 A Non-human Primate Model for Development of AIDS Eradication Strategies Thomas North University of California, Davis, USA 14 19:20 Diminution of HIV Latency through Blockage of Arginine Methylation of Viral Proteins Mark Wainberg McGill University AIDS Centre, Canada 15 19:40 Panel Discussion x Global Antiviral Journal Volume 4, Supplement 1 THURSDAY, DECEMBER 11, 2008 Prevention and Drug Resistance Chairs: Mark Wainberg Douglas Mayers Abstract McGill University AIDS Centre, Canada Idenix Pharmaceuticals, Inc., USA 8:00 Presentation of David Barry DART Achievement Award Robert Murphy Northwestern University, USA The Award will be given to Dr. Larder by Mrs. Gracia Barry 8:10 David Barry DART Achievement Award Milestones in HIV Drug Resistance: From in Vitro to in Silico 19 Brendan Larder HIV Resistance Response Database Initiative (RDI), UK 8:50 STOP HIV/AIDS: Seek and Treat for Optimal Prevention of HIV/AIDS Julio Montaner British Columbia Centre for Excellence in HIV/AIDS, Canada 9:10 Transmission of Drug Resistant HIV: Patterns of Resistance to New Antiretroviral Agents and their Clinical Relevance Charles Boucher Erasmus Medical Center Rotterdam and University Medical Centre Utrecht, the Netherlands 9:30 Panel Discussion 9:50 Break HIV Therapy in Developing Countries Chairs: Christine Katlama José Gatell 20 21 Hôpital Pitié-Salpêtrière, France University of Barcelona, Spain 10:10 Experiences in HIV Treatment in Developing Countries Ian Sanne Wits Health Consortium, South Africa 10:30 To Wean or not to Wean: Prevention of Mother-to-child Transmission through Breast Milk Charles van der Horst University of North Carolina at Chapel Hill, USA 30 10:50 Topical Microbicides: Moving towards Topical Pre-exposure Prophylaxis (PrEP) Sharon Hillier University of Pittsburgh School of Medicine, USA 31 11:10 Panel Discussion Oral Abstract Session II Chairs: Charles van der Horst Paul Spearman 11:30 University of North Carolina at Chapel Hill, USA Emory University, USA Glycan Deletions in the HIV-1 gp120 V1/V2 Domain Compromise Viral Infectivity, Sensitize the Mutant Virus Strains to Carbohydrate Binding Agents and Represent a Specific Target for Therapeutic Intervention Joeri Auwerx K.U. Leuven, Rega Institute for Medical Research, Belgium 11:40 Gag NC/p1 Protease Resistance Mutations can cause Selection of Additional NC/p1 Changes to Optimize Cleavage Efficiency and Replicative Capacity Monique Nijhuis University Medical Centre Utrecht, the Netherlands HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 32 33 xi Abstract 11:50 Safety, Tolerability and Efficacy of Darunavir/ritonavir in Treatment-experienced Women with HIV Infection: Interim Analysis of GRACE (Gender, Race, And Clinical Experience) Carmen Zorrilla University of Puerto Rico School of Medicine, USA 34 12:00 96-Week (wk) Efficacy and Safety of Raltegravir (RAL) in Treatment-experienced Patients José Gatell University of Barcelona, Spain 35 12:10 Lunch on your own 13:30 – 17:00 Free Afternoon HIV/Hepatitis and Other Co-infections Chairs: Tracy Swan Treatment Action Group, USA Joep Lange University of Amsterdam, the Netherlands 17:00 State of the Art Lecture The Aging Liver in the HIV Population Douglas Dieterich Mount Sinai School of Medicine, USA 17:30 Co-infection with HCV and HBV: When and How to Treat? Bruce Polsky St. Luke's Roosevelt Hospital Center, USA Poster Discussion 17:50 Poster Review, Part I Joep Lange University of Amsterdam, the Netherlands 18:20 Poster Review, Part II Adrian Ray Gilead Sciences, Inc., USA 37 38 20:00 Gala Party FRIDAY, DECEMBER 12, 2008 Novel Therapeutic Approaches: Antiviral Mechanism and Predictive Toxicology Chairs: Carmen Zorrilla University of Puerto Rico School of Medicine, USA Richard Pollard University of California, Davis Medical Center, USA 8:00 State of the Art Lecture Optimizing HAART Therapy in the Era of Integrase and Entry Inhibitors Christine Katlama Hôpital Pitié-Salpêtrière, France 8:30 IDX-899 -- A Novel Once-a-day Second Generation NNRTI for the Treatment of HIV/AIDS Douglas Mayers Idenix Pharmaceuticals, Inc., USA 43 8:50 Vicriviroc: A Next Generation CCR5 Antagonist for Treatment of HIV Lisa Dunkle Schering-Plough Research Institute, USA 44 9:10 Amdoxovir Combined with Low Dose AZT for HIV-1 Therapy Robert Murphy Northwestern University, USA 45 9:30 Break 9:50 A New Era in HIV-antiretroviral Therapy: Darunavir, Etravirine and TMC278 Eric Lefebvre Tibotec, the Netherlands xii 42 46 Global Antiviral Journal Volume 4, Supplement 1 Abstract 10:10 Low Potential for Class Related Toxicity of Next Generation Nucleotide Analog GS-9148 Adrian Ray Gilead Sciences, Inc., USA 47 10:30 Mechanisms Associated with Delayed HIV RT Chain-termination Matthias Götte McGill University, Canada 48 10:50 Panel Discussion 11:10 Break 11:30 Special Lecture Acyclovir as an HIV-1 RT Inhibitor in Herpesvirus-infected Human Tissues Leonid Margolis National Institute of Child Health and Human Development, USA Oral Abstract Session III Chairs: Lisa Dunkle José Rodriguez-Orengo 49 Schering-Plough Research Institute, USA Instituto de Ciencias Forenses, Puerto Rico, USA 11:50 Comparison of Two Human Pancreatic Cell Lines for Predicting Mitochondrial Toxicity by Nucleoside Analogs Leda Bassit Emory University School of Medicine and VA Medical Center, USA 50 12:00 ARTEMIS: Efficacy and Safety of Darunavir/ritonavir (DRV/r) 800/100 mg Once-daily vs. Lopinavir/ritonavir (LPV/r) in Treatment-naïve, HIV-1-infected Patients at 96 Weeks Dushyantha Jayaweera University of Miami, USA 51 12:10 Etravirine (ETR; TMC125) Demonstrates Favorable Efficacy and Safety in the Phase III DUET Trials Regardless of Geographic Location Christine Katlama Hôpital Pitié-Salpêtrière, France 52 12:20 Pharmacokinetics and Short-term Safety and Efficacy of Once-daily Etravirine Without and With Once-daily Darunavir/ritonavir in Antiretroviral-naïve HIV-1 Infected Adults James Witek Tibotec Therapeutics, USA 53 12:30 Late Breaker Presentations 12:50 Closing Remarks Raymond F. Schinazi Emory University/Veterans Affairs Medical Center, USA 13:10 Lunch HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies xiii Abstracts page session title and author abstract 1 Pathogenesis and Targeted Design 3 Approaches towards HIV-1 Eradication: Harnessing Cellular Restrictions 01 M Stevenson 3 Understanding HIV Maturation using Electron Cryotomography 02 ER Wright 4 Vpu and Its Cellular Targets P Spearman 03 5 Cell Penetrating Peptides: From Molecular Mechanisms to Therapeutics G Divita 04 6 Foot Fractures in HIV-infected Patients Previously Treated with Tenofovir (TDF)- versus Non-TDF-containing Highly Active Antiretroviral Therapy (HAART) G Pakes 05 9 Toward HIV Eradication 11 Complementary Mechanisms of Nucleoside Analog Resistance from 06 Structural Studies of HIV-1 Reverse Transcriptase E Arnold 11 Dose Response Curve Slope Sets Class Specific Limits on Inhibitory Potential of Anti-HIV Drugs RF Siliciano 07 12 Pharmacology, Metabolism and Transport of HIV Drugs into Viral Reservoirs S Khoo 08 13 Immunotoxins to Selectively Deplete Reservoirs of HIV-infected Cells that Persist in the Face of Highly Suppressive Antiretroviral Therapy EA Berger 09 14 Development of Pyrimidinedione NNRTIs with a High Genetic Barrier to Resistance RW Buckheit Jr. 10 14 Development of VS411, a Virostatic Drug Fixed Dose Combination (FDC) Designed to Inhibit both HIV and Immune Hyperactivation F Lori 11 15 Inhibiting Vpu Function with the Novel Compound BIT225, Results in Inhibition of HIV-1 Release from Human Macrophage Reservoirs J Wilkinson 12 HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies xv page session title and author abstract 16 Integrase Inhibitors: New Insights into Mechanism of Action and Pharmacology with Implications for Treatment and Prevention Strategies D Hazuda 13 17 A Non-human Primate Model for Development of AIDS Eradication Strategies TW North 14 17 Diminution of HIV Latency through Blockage of Arginine Methylation of Viral Proteins MA Wainberg 15 18 Combination Therapy for Treating HIV Latency TP Castor 16 19 Inhibition of Late Stage Events in HIV-1 Replication: Discovery and Development of Therapeutic Compounds with Effects on Viral RNA Synthesis Which Potentially Target Rev Function TL Hartman 17 20 HIV Infected Refugees: ART Therapy and Its Affect on CD4 Counts at 3 and 6 Months M Patil 18 21 Prevention and Drug Resistance 23 Milestones in HIV Drug Resistance: From In Vitro to In Silico B Larder 19 23 STOP HIV/AIDS: Seek and Treat for Optimal Prevention of HIV/AIDS J Montaner 20 24 Transmission of Drug Resistant HIV: Patterns of Resistance to New Antiretroviral Agents and their Clinical Relevance CAB Boucher 21 25 Ninety-nine Is Not Enough: Kinetic, Structural and Thermodynamic Characterization of HIV Protease with Insertion in the Flap Region Isolated from an HIV-positive Patient M Kožíšek 22 25 TOPO Cloning and Pyrosequencing Revealed a Novel S68 Deletion in HIV-1 Reverse Transcriptase Selected by Dexelvucitabine in Human Lymphocytes I Massud 23 26 Operation Sweet Tooth: Effective Use of Social Marketing Campaigns in Non-Traditional Social Settings T McPhaul 24 xvi Global Antiviral Journal Volume 4, Supplement 1 page session title and author abstract 27 Utilizing Mental Health Counseling in African American Men who have Sex with Men Minority Populations as a Primary and Secondary HIV Prevention Tool T McPhaul 25 28 Sex is Good: An Investigation into the Quality of Life and Sexual Practices Among Individuals on HAART in British Columbia A Mtambo 26 29 Vertical Transmission of HIV Infection in Western Romania M Sandu 27 29 Development of the Dual Acting Pyrimidinedione IQP-0528 as a Vaginal Topical Anti-HIV Microbicide KM Watson 28 30 Development of Resistance to Enfuvirtide in Cerebrospinal Fluid in a Patient with Suppressed Plasma HIV-1 RNA AMJ Wensing 29 33 HIV Therapy in Developing Countries 35 To Wean or Not to Wean: Prevention of Mother-to-child Transmission through Breast Milk C van der Horst 30 36 Topical Microbicides: Moving Towards Topical Pre-exposure Prophylaxis (PrEP) S Hillier 31 36 37 Glycan Deletions in the HIV-1 gp120 V1/V2 Domain Compromise Viral Infectivity, Sensitize the Mutant Virus Strains to Carbohydrate Binding Agents and Represent a Specific Target for Therapeutic Intervention J Auwerx 32 Gag NC/p1 Protease Resistance Mutations can cause Selection of Additional NC/p1 Changes to Optimize Cleavage Efficiency and Replicative Capacity M Nijhuis 33 38 Safety, Tolerability and Efficacy of Darunavir/ritonavir in Treatment- experienced Women with HIV Infection: Interim Analysis of GRACE (Gender, Race, And Clinical Experience) C Zorrilla 34 39 96-Week (wk) Efficacy and Safety of Raltegravir (RAL) in Treatment- experienced Patients J Gatell 35 40 Valacyclovir as Antiretroviral Therapy M Pop 36 HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies xvii page session title and author abstract 41 HIV/Hepatitis and Other Co-infections 43 The Aging Liver in the HIV Population D Dieterich 37 43 Co-infection with HCV and HBV: When and How to Treat? B Polsky 38 44 Antimicrobial Molecules for Treatment of Multi-drug Resistant (MDR) and Extensively Drug Resistant (XDR) Strains of Mycobacterium Tuberculosis D Miller 39 45 Progressive Multifocal Leukoencephalopathy – A Case Report M Pop 40 45 Accelerated Approval and Post-marketing Commitments: A Delicate Balance T Swan 41 47 Novel Therapeutic Approaches: Antiviral Mechanism and Predictive Toxicology 49 Optimizing HAART Therapy in the Era of Integrase and Entry Inhibitors C Katlama 42 50 IDX899 - A Novel Once-a-day Second Generation NNRTI for the Treatment of HIV/AIDS D Mayers 43 51 Vicriciroc: A Next-generation CCR5 Antagonist for Treatment of HIV LM Dunkle 44 51 Amdoxovir Combined with Low Dose AZT for HIV-1 Therapy RL Murphy 45 52 A New Era in HIV-antiretroviral Therapy: Darunavir, Etravirine and TMC278 E Lefebvre 46 53 Low Potential for Class Related Toxicity of Next Generation Nucleotide Analog GS-9148 AS Ray 47 54 Mechanisms Associated with Delayed HIV RT Chain-termination M Götte 48 55 Acyclovir as an HIV-RT Inhibitor in Herpesvirus-infected Human Tissues L Margolis 49 56 Comparison of Two Human Pancreatic Cell Lines for Predicting Mitochondrial Toxicity by Nucleoside Analogs L Bassit 50 xviii Global Antiviral Journal Volume 4, Supplement 1 page session title and author abstract 57 ARTEMIS: Efficacy and Safety of Darunavir/ritonavir (DRV/r) 800/100 mg Once-daily vs Lopinavir/ritonavir (LPV/r) in Treatment-naïve, HIV-1-infected Patients at 96 Weeks D Jayaweera 51 58 Etravirine (ETR; TMC125) Demonstrates Favorable Efficacy and Safety in the Phase III DUET Trials Regardless of Geographic Location C Katlama 52 59 Pharmacokinetics and Short-term Safety and Efficacy of Once-daily Etravirine Without and With Once-daily Darunavir/ritonavir in Antiretroviral-naïve HIV-1 Infected Adults J Witek 53 60 Activation of Different Cell Death Pathways in Patients on Antiretroviral Therapy with Long-term Viral Suppression but Unfavorable Immunologic Response J Blanco 54 61 Safety and Tolerability of the Lopinavir/ritonavir (LPV/r) Tablet in Comparison to other Protease Inhibitors (PIs): The SAPIKAT trial UF Bredeek 55 62 Analysis of Virologic Endpoints Versus Standard Intent to Treat, in ARTEMIS and TITAN Trials L Chen 56 63 Improvement of the Transglycosylation Reaction for the Synthesis of β-D-3’-Azido-2’,3’-Dideoxypurine Nucleoside Analogs by Conventional and Microwave Assisted Heating S Coats 57 64 The Phtalocyanine Prototype Derivative Alcian Blue: The First Synthetic Agent with Selective Anti-human Immunodeficiency Virus Activity Due to Its Lectin-like Properties KO François 58 64 Long-time Virologic Efficacy of Boosted Double Protease Inhibitor Therapy H Knechten 59 65 Etravirine Demonstrates a Favorable Safety and Tolerability Profile at Week 48 in the Pooled DUET Trials Irrespective of Gender, Age, Ethnicity and Weight E Lefebvre 60 67 Adherence to Darunavir/ritonavir (DRV/r) and Lopinavir/r (LPV/r) in Treatment-naïve HIV Patients in ARTEMIS E Lefebvre 61 HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies xix page session title and author abstract 67 Irreversible Pepsin Fraction (IPF) Displays Significant Antiretroviral Activity via Specific Novel Cytokine Stimulation In Vitro Investigation of Activity on Human Lymphocytes D Miller 62 68 Successful Use of Dual Therapy with Etravirine and Raltegravir in Patients with HIV Infection G Pierone 63 69 HIV-1 Co-receptor Use in Heavily Treatment-experienced Spanish Patients R Sanchez-de la Rosa 64 70 Cost-effectiveness of Maraviroc plus Optimized Background Therapy in Treatment-experienced Patients with R5 HIV-1 in Spain R Sánchez-de la Rosa 65 71 Once Daily Darunavir/ritonavir: A Single Centre Cohort Experience C Scott 66 73 Pharmacology and Drug Metabolism 75 Etravirine (ETV, TMC-125) Plasma Levels in Decompensated Liver Disease: 67 A Case Report M Aboud 75 Virologic Efficacy of Dual-boosted Once-daily (QD) Atazanavir, Fosamprenavir, and Ritonavir (ATV/FPV/RTV): 48 Week Results UF Bredeek 68 76 A Kinetic Simulation of In Vitro Pharmacodynamics for HIV Drugs HL De Bondt 69 77 Lower Levels of Nucleoside Analog Triphosphates in Primary Human Macrophages Compared to Human Lymphocytes Could Impair Potency of Antiretroviral Drugs in Human Viral Reservoirs C Gavegnano 70 78 Lack of Pharmacokinetic Interaction for Low and Normal Dose Zidovudine with Amdoxovir in HIV-1 Infected Individuals SJ Hurwitz 71 79 Metabolism of Highly Active and Selective 3’-Azido-2’,3’-Dideoxypurine Nucleosides in Primary Human Lymphocytes and MT-2 Cells A Obikhod 72 xx Global Antiviral Journal Volume 4, Supplement 1 Pathogenesis and Targeted Design HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 1 Abstract 01 Approaches towards HIV-1 Eradication: Harnessing Cellular Restrictions M Stevenson University of Massachusetts Medical School, Worcester, MA, USA The replication of primate lentiviruses is dependent upon their ability to commandeer cellular factors at various stages in the replication cycle. Research over the past several years however, has revealed the presence of “cellular restrictions” that potently antagonize viral replication. For example, the Apobec 3 proteins are cytidine deaminases that compromise the formation and integrity of viral cDNA while Bst2/ tetherin prevents detachment of budding viruses from the surface of the infected cell. In order to counteract these restrictions, primate lentiviruses have evolved accessory proteins: the Vif protein targets Apobec 3 for proteasomal destruction while Vpu mislocalizes Bst2/tetherin away from sites of virus budding. We have recently obtained evidence for a novel restriction that is expressed in cells of macrophage lineage. This restriction potently antagonizes the replication of primate lentiviruses including HIV-1/2 and SIV as well as retroviruses such as MLV. We have evidence that this restriction is counteracted by the viral accessory proteins Vpx/ Vpr. These proteins commandeer a damaged DNA response protein (DDB1) to target the restriction to the proteasome. We have further obtained evidence that this restriction dictates the cell cycle dependence of lentivirus and retrovirus infection. Therefore when the restriction is neutralized, macrophages, which are normally refractory to MLV infection, are rendered permissive to MLV transduction. With a view to targeting accessory proteins for drug discovery, we have identified small molecule inhibitors of HIV-1 Vif. These Vif antagonists exhibit antiviral effects only in cells that express Apobec 3 proteins and increase the extent of cytidine deamination in nascent viral cDNA. Given our increasing understanding of the HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies function of the viral accessory proteins, they remain highly attractive targets for therapeutic intervention in HIV/AIDS. Abstract 02 Understanding HIV Maturation using Electron Cryotomography ER Wright1, JB Schooler1, HJ Ding1, C Kieffer2, C Fillmore2, WI Sundquist2, and GJ Jensen1 1 Division of Biology, California Institute of Technology, Pasadena, CA, USA; 2 Department of Biochemistry, University of Utah, Salt Lake City, UT, USA BACKGROUND: HIV maturation is a dynamic process, which occurs once the virus buds and is released from the infected cell. The unprocessed Gag polyprotein is composed of polypeptide segments, termed MA, CA, SP1, NC, SP2, and p6 that are arranged radially, with the N-terminal MA region at the membrane and the C-terminal NC-SP2-p6 region nearest the center [1, 2]. Maturation involves the proteolytic processing of Gag and Gag-Pol polyproteins by protease (PR). During maturation, MA remains bound to the membrane, while the processed CA subunits condense to form a central conical capsid that encases NC, the RNA genome, and other viral enzymes. The structures of nearly every component of HIV have been determined, but it is still unclear how individual subunits come together to form the intact virion. We are studying the maturation process of HIV-1 by electron cryotomography (cryo-ET) in order to understand the structural changes that occur within the virus. METHODS: HIV-1 virions were made non-infectious and/or halted in the immature state by the inactivation of the proteins PR, RT, and RNase H [3, 4]. Solutions of the purified HIV-1 virions were flash-frozen onto EM grids in liquid ethane. The frozen-hydrated grids were imaged in a 300 kV “G2 Polara” FEI TEM under low dose conditions. Single and dual-axis tilt series were collected using the predictive UCSF tomography package [5]. All images were energy-filtered, recorded 3 with a defocus ranging from -4 to -8 μm, and with CCD pixels representing 0.46 or 0.56 nm on the specimen. Images were binned by a factor of 2, and three-dimensional reconstructions of the virions were calculated with IMOD [6]. Individual virions were selected and denoised by non-linear anisotropic diffusion in the BSOFT program [7, 8]. Additional processing and analysis steps were performed using either the Amira software package (Visage Imaging, Inc.) or UCSF Chimera [9]. RESULTS: The capsid of the mature virus can have numerous morphologies, the most common being a somewhat asymmetrical cone. Cryo-ET examinations of mature HIV-1 virions revealed that the conical cores were unique in structure and position (Fig. 1), but they also demonstrated certain similarities with respect to size and shape, the distance of the cone’s base from the envelope/MA layer, the range of the cone angle. It was also observed that the conical capsid shape was preferred in vivo, which argues in favor of the template-directed model of capsid formation [4]. Examinations of immature virions revealed the concentric shells of the Gag polyprotein (Fig. 1). Upon further analysis, only the CA and SP1 shells contained patches of hexagonal order. Averaging well-ordered unit cells led us to propose a model for the immature lattice in which each CA hexamer is stabilized by a bundle of six SP1 helices (Fig. 2). References: [1] S. D. Fuller et al., Curr. Biol. 7 (1997) 729. [2] T. Wilk et al., J. Virol. 75 (2001) 759. [3] D. J. Wyma et al., J. Virol. 74 (2000) 9381. [4] J. Benjamin et al., J. Mol. Biol. 346 (2005) 577. [5] Q. S. Zheng et al., J. Struct. Biol. 147 (2004) 91. [6] J. R. Kremer et al., J. Struct. Biol. 116 (1996) 71. [7] J. B. Heymann, J. Struct. Biol. 133 (2001) 156. [8] A. S. Frangakis and R. Hegerl, J. Struct. Biol. 138 (2002) 105. [9] E. F. Pettersen et al., J. Comput. Chem. 25 (2004) 1605. 4 Fig 1. Tomographic reconstruction of two isolated HIV-1 virions (top, immature; bottom, mature) preserved in vitreous ice. Scale bar 100 nm. Fig 2. Model for the roles of Gag subunits in the immature and mature lattices. Top and side views (top and bottom, respectively) of low-pass filtered atomic models of the immature (left) and mature (right) lattices. Scale bar 8 nm. Abstract 03 Vpu and Its Cellular Targets P Spearman, J Hammonds, S Ali, D Shaw, L Ding, and JJ Wang Emory University School of Medicine, Atlanta, GA, USA BACKGROUND: Vpu is a small accessory protein that functions in the downregulation of CD4 and enhances particle release. The mechanism by which Vpu enhances particle release has remained mysterious until recent years, when it was shown that there is a dominant host cell restriction in many human cells that is not present in simian cells. In the past year, tetherin (BST2, CD137) has been identified as the molecule responsible for particle tethering at Global Antiviral Journal Volume 4, Supplement 1 the plasma membrane of cells. Calcium modulating cyclophilin ligand (CAML) was also identified by our group as a host restriction factor overcome by Vpu. Additional studies are uncovering the mechanism of action of these restriction factors, and should provide insights into how host restriction may be turned to therapeutic advantage. METHODS: We identified CAML as a direct interactor of Vpu by yeast 2-hybrid screen, and confirmed the interaction by co-immunoprecipitation from mammalian cells. African green monkey CAML was then cloned from Cos-7 cells, sequenced, and expressed for comparisons with human CAML. Tetherin was expressed from its cDNA in tagged form. Both CAML and tetherin were analyzed for the ability to restrict particle release, using p24 antigen assay, Western blot, and electron microscopy as endpoints. The subcellular distribution of both molecules in the presence or absence of Vpu was compared using laser confocal fluorescence microscopy. RESULTS: Both CAML and tetherin restricted particle release, and were able to convert permissive cells to a restrictive phenotype. Tetherin was more potent in this effect on the basis of amount of cDNA required in transfection to confer restriction. Overexpression of either molecule resulted in a particle tethering that was apparent by electron microscopy. Tetherin more dramatically colocalized with Vpu in coexpression studies, and Vpu redistributed tetherin from the plasma membrane to intracellular sites. An African green monkey CAML molecule failed to confer restriction, and the difference in function of human and AGM CAML was mapped to a single residue change in the cytoplasmic domain. CONCLUSIONS: Vpu overcomes host cell restriction that is characterized by particle tethering at the membrane. Therapeutic strategies that target Vpu would result in a potent block to viral replication occurring after viral budding. HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies Abstract 04 Cell Penetrating Peptides: From Molecular Mechanisms to Therapeutics A Agopian1, E Gros1, P Clayette2, MC Morris1, G Aldrian-Herrada1, and G Divita1 1 CRBM, CNRS, UMR5237, Montpellier, France; 2 SPI-BIO CEA, Fontenais aux roses, France The rapid emergence of drug-resistant viruses against approved drugs together with their side effects limits the potency of existing anti-HIV-1 therapeutics. Therefore, there is an urgent demand for new and safer drugs and extensive efforts have been made in the design of molecules that specifically target protein/protein interfaces required during HIV-viral replication. Nowadays, delivery constitutes a major piece of the therapeutic puzzle, and the success of future therapies requiring large molecules will be conditioned by access to potent delivery systems. With the aim of designing specific antiviral molecules, we have validated a new concept that combines peptide-based nanoparticle delivery system with short peptides that target protein-protein interfaces required for reverse transcription. The whole process of reverse transcription is driven by different protein/protein interactions involving mainly reverse transcriptase (RT). RT is a heterodimer p51/p66, each consisting of distinct sub-domains: fingers, palm, connection, thumb and RNAse H, the latter only present in p66. The formation of the biologically active RT is a two-step mechanism, including a rapid protein/protein interaction “dimerization”, followed by conformational changes “maturation”. We have designed two families of short peptides targeting either dimerization or maturation steps. Pep-71 (9-mer) derived from the Trp-rich cluster of the connection subdomain blocks RTdimerization. Pep-71 interacts preferentially with Trp24 and Phe61, in a cleft between the connection and fingers domains of the small p51 within 5 heterodimeric-RT, and destabilizes the dimeric conformation, thereby triggering dissociation. Paw (15-mer) derived from the thumb domain prevents the dynamics of the fingers/thumb domains of p66, thereby inhibiting maturation of heterodimeric-RT and its association with Integrase. In order to evaluate these inhibitors in vivo, we have developed a peptidebased nanoparticle-system NANO-VEPEP, which forms stable complexes with peptides and improves their cellular uptake, as well as their in vivo stability/ biodistribution. NANO-VEPEP particles can be functionalized, which constitutes a major advantage for in vivo targeting. When delivered into cells using NANO-VEPEP, both Pep-71 and Paw dramatically abolish replication of HIV-1LAI with IC50 of 0.5 and 0.32 nM respectively, and a selectivity index of about 4000. Moreover, sub-nanomolar concentrations of both peptides block the replication of either multidrug resistant strains or primary HIV isolates from subtypes A to G. Preliminary evaluation of Pep71 in an HIV susceptible transgenic rat model has confirmed the potency of the NANO-VEPEP-Pep-71 particles in vivo. We have established a proof-of-concept that targeting conformational changes required for RT flexibility can lead to highly potent specific new antiviral drugs that can bypass resistance limitation. We believe that combining large molecules with modulable delivery systems will provide new perspectives for inhibitors of the “niche” of highly resistant strains and more generally raise new hope for more specific drugs to reach clinical evaluation. Abstract 05 Foot Fractures in HIV-infected Patients Previously Treated with Tenofovir (TDF)- versus NonTDF-containing Highly Active Antiretroviral Therapy (HAART) R Joseph1, A Horizon1, Q Liao2, S Ross2, and G Pakes2 1 Cedars-Sinai Health System, Los Angeles, CA, USA; 2 GlaxoSmithKline, Research Triangle Park, NC, USA BACKGROUND: Among HIV-infected patients, osteopenia has been reported in 22-50% and osteoporosis in 3-21%. Both conditions notably increase the risk of bone fractures, especially of hip and spine. However, little is known about the incidence of foot fractures in HIV-infected patients or about the association of particular antiretroviral drugs in the pathogenesis of these fractures. We characterized foot fractures diagnosed in HIV+ patients in the Los Angeles Cedars-Sinai Health System. METHODS: In this retrospective case series study, medical records of all male HIV-infected patients with MRI-confirmed foot fractures (n=30) were examined. Data regarding demographics, HIV history, co-morbidities, HAART/non-HIV drug prescription history, DEXA bone mineral density scores, and fracture type were collected on Excel sheets and analyzed by logistic regression with stepwise selection. RESULTS: Proportionally more foot fracture patients had received TDF-containing HAART (17 [57%]) than non-TDF-containing HAART (13 [43%]) prefracture. At fracture diagnosis, these 2 groups were similar regarding median age (49y/48y), HIV1RNA (both 1.7 log10c/mL), time between HIV diagnosis and foot fracture (both 17.0y); incidence of metatarsophalangial fracture (12%/15%) and vertebral fracture (12%/15%); family history of bone diseases (24% vs 23%); frequency of malabsorption syndrome, renal failure, calcium deficiency, and vitamin D deficiency; and concurrent use of bisphosphonates 6 Global Antiviral Journal Volume 4, Supplement 1 (65%/69%), calcitonin, and diuretics. However, the TDF-treated group had more osteoporosis (35%/8%), stress-type fractures (53%/31%), other concurrent fractures (12%/0%), wasting syndrome (29%/15%), centripetal obesity (18%/8%), chronic cigarette smoking of >1pack/day (35%/8%), DEXA T-scores <-2.4 (denoting osteoporosis) in femur (24%/9%) and spine (47%/36%), and a lower frequency of alcoholism (12%/31%) and anemia of chronic disease (0%/23%). More TDF-treated patients concurrently received protease inhibitors (71%/46%), NNRTIs (24%/0%), prednisone (24%/0%), calcium supplements (100%/85%), vitamin D (100%/85%), testosterone (47%/23%), and teriparatide (29%/8%). Median time from TDF initiation until fracture was 2.57y (range,1.17-5.69y). Logistic regression analysis showed relationships between femur DEXA T-scores <1.5 and body weight (p=0.036) and serum glucose (p=0.034). CONCLUSIONS: This small pilot study suggests a greater incidence of foot fractures in HIV-infected patients on TDF- than non-TDF-containing HAART. Co-morbidities and/or co-administered drugs may have influenced the occurrence of these fractures. HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 7 Toward HIV Eradication HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 9 Abstract 06 Complementary Mechanisms of Nucleoside Analog Resistance from Structural Studies of HIV-1 Reverse Transcriptase E Arnold1,2, K Das1,2, X Tu1,2, R Bandwar1,2, S Sarafianos1,2, S Tuske1,2, AD Clark, Jr.1,2, J Bauman1,2, PL Boyer3, Q Han2, B Gaffney2, RA Jones2, K White4, J Feng4, M Miller4, and SH Hughes3 1 Center for Advanced Biotechnology and Medicine; 2 Rutgers University Department of Chemistry and Chemical Biology, Piscataway, NJ, USA; 3 NCI Drug Resistance Program, Frederick, MD, USA; 4 Gilead Sciences, Foster City, CA, USA Nucleoside inhibitors (NRTIs) of HIV-1 reverse transcriptase (RT) are among the most important antiAIDS drugs and are prescribed in nearly all treatment regimens. We have used X-ray crystallography to study the structural basis for the mechanisms of resistance to AZT (zidovudine) and PMPA (tenofovir). A detailed understanding of the distinct mechanisms of resistance to the two drugs and their antagonistic relationship is critical for understanding the complexity of NRTI resistance and for designing new and broadly effective NRTIs. Resistance of HIV-1 RT to AZT has been shown to involve an ATP-mediated excision reaction in which AZTMP is removed from the terminated primer, forming a dinucleoside tetraphosphate product, AZTppppA. We have solved and analyzed a series of AZT-resistant HIV-1 RT structures, including ternary complexes with a template-primer and the excision product AZTppppA. The structures define the roles played by the major AZT-resistance mutations in the mechanism of AZT resistance. Primary mutations K70R and Y215Y help in binding the ATP molecule to the mutant RT and therefore can be classified as excision-enhancing mutations (EEMs). The mutation K65R causes resistance to tenofovir and has complex relationships with other NRTI-resistance HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies mutations. We have analyzed structures of K65R HIV1 RT in ternary complexes with tenofovir diphosphate and dATP. We propose that differential stacking of the guanidinium groups of K65R and R72 creates a “checkpoint” that reduces dNTP incorporation and allows the mutant RT to discriminate between tenofovir diphosphate and the normal substrate, dATP. The structural results also suggest potential mechanisms of complex relationships of K65R mutation with other NRTI-resistance mutations including the antagonistic relationship with EEMs. Abstract 07 Dose Response Curve Slope Sets Class Specific Limits on Inhibitory Potential of Anti-HIV Drugs L Shen1, S Peterson1, AR Sedaghat1, MA McMahon1, M Callender1, H Zhang1, Y Zhou1, KS Anderson2, EP Acosta3, and RF Siliciano1, 4 1 Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2 Yale University School of Medicine, New Haven, CT, USA; 3 University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA; 4 Howard Hughes Medical Institute, Baltimore, MD, USA Background: Preventing disease progression for HIV-1 infected patients requires regimens that maximally suppress virus replication. A comparative measure of antiviral activity under clinically relevant conditions would guide drug development and regimen selection but is currently lacking. We hypothesized that the slope parameter (or Hill coefficient), which is neglected in current measures of antiviral activity, might have a dramatic effect on antiviral activity of anti-HIV-1 drugs. To account for the effect of slope, we developed a new index, instantaneous inhibitory potential (IIP), as a new in vitro measure of antiviral activity. Method: A single round infectivity assay with wide dynamic range in primary CD4+ lymphoblasts was used to obtain dose response curves for anti-HIV-1 11 drugs. IC50 and slope parameters were determined by fitting the curves into the median effect model with least square regression analysis. IIP equals the number of logs by which single round infectivity is reduced at clinically relevant drug concentrations and was calculated based on the median effect equation. Result: We show that current measures of antiviral activity, IC50 and inhibitory quotient, neglect a critical dimension - the dose response curve slope, which has a dramatic effect on antiviral activity. Strikingly, slope values are class-specific for antiviral drugs and define intrinsic limitations on antiviral activity for some drug classes. Nucleoside reverse transcriptase inhibitors and integrase inhibitors have slopes of ~1, characteristic of non-cooperative reactions, while nonnucleoside reverse transcriptase inhibitors, protease inhibitors, and fusion inhibitors unexpectedly show slopes >1. IIP is strongly influenced by slope, varies by >8 logs for current anti-HIV drugs and provides a more accurate in vitro pharmacodynamic measure of antiviral activity than traditional IC50 or inhibitory quotient measures. Conclusion: Antiretroviral drugs acting through different mechanisms have different value for the slope parameter which has crucial influence on antiviral activity under clinically relevant conditions, as reflected in IIP. Only agents with slopes >1 achieve high level inhibition of single round infectivity, a finding with profound implications for drug and vaccine development. Abstract 08 Pharmacology, Metabolism and Transport of HIV Drugs into Viral Reservoirs S Khoo University of Liverpool, UK Failure of antiretroviral therapy (ART) to suppress viral replication within a reservoir of HIV infection 12 establishes a sanctuary. These sanctuaries arise in part because of physiochemical characteristics of drugs which limit their penetration into cells and tissue compartments, and also because of anatomical, biochemical and physiological barriers inherent to compartments. The brain and testes are recognised sanctuaries where discordance in viral load or viral quasispecies may arise in some individuals (although in most individuals, viral kinetics mimics that of peripheral blood). PIs (being lipophilic and highly protein bound) are most likely to be affected by processes which establish drug sanctuaries, and NRTIs least (although differences exist within this class). Significant differences have also been observed between nevirapine and efavirenz, with the latter accumulating to a lesser degree within brain and genital tract. Accumulating evidence also points to possible sanctuaries within the female genital tract, breast milk and placenta. The evidence for drug sanctuaries within cellular subsets is much less strong- due in large part to technical difficulties that these studies have encountered. A hierarchy of intracellular accumulation of HIV PIs has been observed but the clinical relevance is uncertain. Major questions remain around localisation (cell membrane-associated, or truly intracellular?) and intracellular free fraction of drug. Whilst the major determinants of this hierarchy are the physiochemical characteristics of PIs (lipophilicity, protein binding), the large inter-individual variability observed may be in part due to differences in drug transporters. Transporters may not only affect drug disposition in gut, liver and kidney, but also within anatomical compartments and cells. PIs are substrates for efflux (ABCB1, ABCC1/2, ABCG) transporters and recent data suggest they may also be substrates for influx (SLCO 1A2, 1B1) transporters. N(t)RTIs also undergo carrier-mediated transport (ABCC4/5; hCNT, hENT). The clinical relevance of these findings to drug sanctuaries depends on cell surface expression of these molecules within specific tissues, and also pharmacogenetic variability which may alter drug disposition between individuals. Although the liver and gut are major sites of drug metabolism by cytochrome P450 enzymes, some P450 isoforms may Global Antiviral Journal Volume 4, Supplement 1 also be present within the cytosol of PBMCs, adding another dimension of complexity to the study of intracellular pharmacology. New generations of existing classes, and new classes of anti-HIV compounds increase opportunities for maximising penetration of all components of the regimen into sanctuaries. Conventional clinical trial endpoints lack sensitivity to tease out differences in regimen potency outside of plasma, and although the effects of drug sanctuaries may not be immediately apparent, the fact that they exist at all gives cause for concern. Abstract 09 Immunotoxins to Selectively Deplete Reservoirs of HIVinfected Cells that Persist in the Face of Highly Suppressive Antiretroviral Therapy EA Berger1, PE Kennedy1, TK Bera2, M Gallo2, and I Pastan2 1 Laboratory of Viral Diseases, NIAID; 2 Laboratory of Molecular Biology, NCI, NIH, Bethesda, MD, USA Background: Highly sensitive assays have revealed the presence of extremely low levels of HIV-1 RNA in plasma despite highly suppressive HAART. This residual viremia is non-evolving, does not accumulate additional drug resistance mutations, and is non-responsive to intensification with new therapeutic agents targeting additional steps in the virus replication cycle. These findings have raised the notion that the residual viremia might come from long-lived productively infected cells, perhaps capable of proliferation. If true, this model would suggest the importance of therapeutic regimens aimed at selectively killing the HIV-producing cells that persist in the face of HAART. Methods: We have developed immunotoxins targeted to the HIV-1 Env as an approach to deplete HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies infected cell reservoirs persisting after HAART. Each immunotoxin is a recombinant single-chain chimeric protein containing the translocation and cytotoxic domains of Pseudomonas aeruginosa exotoxin A linked to a specific protein that binds Env. The first agent (CD4-PE40) employs sCD4 as the Env-targeting moiety; a newer, more potent agent (3B3-PE38) employs an SCFv of the high affinity 3B3 MAb against the highly conserved CD4 binding site. Results: Each protein killed chronically HIVinfected cells with very high potency (IC50 ≈ 2 nM for CD4-PE40, 0.03 nM for 3B3-PE38), while showing negligible activity against the uninfected parental cells. Each protein inhibited spreading infection by diverse primary HIV-1 isolates in both PBMCs and primary macrophages; again 3B3-PE38 proved more potent than CD4-PE40. Combined treatment of acutely infected T cell cultures with toxin plus an RT inhibitor completely eliminated infectious virus, a result not achieved with either agent alone. Similarly in collaborative studies using a murine model, HAART alone strongly suppressed HIV-1 replication, but the viral load rebounded after treatment was stopped; when an immunotoxin was included with HAART during the treatment phase, viral rebound was not observed after treatment cessation. These results highlight the particular value of combining HAART drugs that block HIV replication with immunotoxins that kill already-infected cells. Collaborative experiments have demonstrated immunotoxin efficacy in combination with activating agents that induce HIV expression from latently infected cells. In rhesus macaques, high levels of CD4-PE40 displayed some hepatotoxicity, in keeping with the dose-limiting hepatotoxicity observed in earlier Phase I trials in the pre-HAART era. By contrast, high levels of 3B3-PE38 displayed no hepatotoxicity in macaques, consistent with recent human clinical trial results with anticancer PE-based immunotoxins that have induced major remissions of leukemias and lymphomas without serious liver toxicity. Conclusion: These results suggest that Envtargeted immunotoxins might prove critical for depleting the HIV-infected cell reservoirs persisting in the face of HAART. Clinical trials are planned 13 to evaluate whether combining such agents with suppressive HAART might deplete infected reservoirs sufficiently to enable prolonged cessation of therapy without viral rebound. Abstract 10 Development of Pyrimidinedione NNRTIs with a High Genetic Barrier to Resistance RW Buckheit Jr., TL Hartman, L Yang, and KM Watson ImQuest BioSciences, Frederick, MD, USA With the increasing incidence of HIV drug-resistant viruses in the HIV-infected population, it is critical that a new generation of highly safe and potent drugs be developed to address this issue. Within the NNRTI class of inhibitors, the dual-acting pyrimidinediones offer an opportunity to provide a new alternative for both HAART and salvage therapy regimens. Among a SAR series of 68 pyrimidinedione compounds, a number were found to potently inhibit viruses with typical NNRT-resistance engendering mutations, such as Y181C, L100I, and K103N, in both cell-based and biochemical RT inhibition assays, suggesting that the molecules may interact with the binding pocket in a manner which would result in a higher genetic barrier to resistance. In addition, the series of compounds possess wild type levels of activity when tested against multi-drug resistant viruses obtained from patients failing prolonged courses of RT and PI therapies. In order to further evaluate this hypothesis, viruses resistant to the antiviral effects of the lead compounds were selected in cell culture using both serial dose escalation and fixed dose resistance selection methods, as well as through the evaluation of the activity of the pyrimidinediones against biologically selected and site-directed viruses with defined NNRTI-resistance mutations. These studies confirmed that the pyrimidinediones required the accumulation of multiple mutations in the RT in order to develop high level NNRTI resistance. Further mutations in envelope and/or core HIV-1 proteins 14 were required in order to achieve complete resistance. Antiviral assays with drug resistant and multi-drug resistant viruses indicated that the compounds were able to effectively inhibit viruses with NNRTIresistance mutations and exhibited enhanced sensitivity to multi-drug resistant viruses obtained from patients failing long courses of PI therapy as well as RT/PI therapy. Additional studies were performed with NNRTI- resistant viruses with the entire SAR series of molecules in an effort to define molecules with specific capability of inhibiting highly resistant viruses such as those with the Y181C, L100I, K103N (alone and in combination) as well as with MDRs with resistance phenotypes/genotypes to RT inhibitors, PI inhibitors and both RT and PI inhibitors. These molecules now represent leads for the continued development of a new SAR to identify clinical candidates with extremely high genetic barriers to resistance. The results of our in vitro studies would indicate that the pyrimidinediones possess a high genetic barrier to resistance based on both their dual mechanism of action as well as their low intrinsic level of resistance to individual RT amino changes. Their activity against MDRs suggests the compounds may be highly effective in primary or salvage therapy regimens. Abstract 11 Development of VS411, a Virostatic Drug Fixed Dose Combination (FDC) Designed to Inhibit both HIV and Immune Hyperactivation F Lori and M Stevens ViroStatics Srl, Sassari, Pavia, Italy, and Princeton, NJ, USA BACKGROUND: No available anti-HIV product is designed to down-modulate the immune system hyperactivation now recognized as a key component of HIV/AIDS pathogenesis and disease progression. Global Antiviral Journal Volume 4, Supplement 1 METHODS: VS411, a two-drug FDC, is designed to suppress HIV both directly and indirectly: one drug interferes with HIV reverse transcription, the other decreases availability of HIV natural targets (activated/proliferating T-cells) thus reducing the growth rates of wild-type viruses and drug-resistant escape mutants. This novel class of anti-HIV drugs has been named “virostatics” (antiviral+cytostatic). We have concluded a pilot Phase I (12 individuals), open label, randomized, single dose, 4-way crossover trial investigating the fasted and non-fasted oral bioavailability of didanosine (ddI) and hydroxyurea (HU), co-formulated and administered as two different FDC formulations, with the goal of decreasing ddI Cmax compared to commercially available ddI beadlets. An ongoing Phase II, multinational dosefinding study is exploring decreasing both HU and ddI dosages in 60 chronically infected, drug-naïve patients. RESULTS: VS411 has been designed as a QD capsule containing both an antiviral (ddI) and a cytostatic (HU) compound with synergistic HIV-suppressing activity. Phase I study results indicated 30% lower Cmax and similar AUC of ddI for both the VS4112 and VS411-4 test formulations compared to the commercial ddI formulation. The test formulations’ HU Cmax and AUC were virtually identical to commercial HU. As the absorption of ddI was less predictable for the VS411-4 formulation and intersubject variability of ddI concentrations was higher, the VS411-2 formulation was selected for further development. Food significantly decreased ddI and, unexpectedly, HU exposure, and increased intersubject variability. Since randomized, controlled clinical studies using the two components of VS411 in separate pills administered simultaneously have indicated that decreasing the hydroxyurea dose by 50% reduced the incidence of previously observed toxicity by 40% while preserving antiviral efficacy, the Phase II study explores decreasing the HU dosage to as little as 300 mg daily, 3- to 5 fold less than previously explored in HIV-infected individuals, and ddI dosage to as little as 200 mg daily, 2-fold less than routinely used. New experiments confirmed previous results identifying the G1/S (activation/commitment HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies to division) phase transition checkpoint as the cell cycle progression point that is critical for maximal HIV-1 replication. By limiting, without blocking, cell cycle progression through this checkpoint both cell proliferation and viral replication were significantly restricted, supporting the use of cytostatic drugs in HIV-infected individuals. CONCLUSIONS: VS411 represents the first drug combination targeting both components (HIV replication and immune system hyperactivation) responsible for disease progression. A VS411 capsule has been formulated for QD administration. A Phase I VS411 study supported QD administration and met the goal of lowering ddI Cmax while retaining total drug exposure. An ongoing Phase II, dose-finding VS411 study is exploring decreasing both HU and ddI dosages. Abstract 12 Inhibiting Vpu Function with the Novel Compound BIT225, Results in Inhibition of HIV-1 Release from Human Macrophage Reservoirs G Khoury1, G Ewart2, C Luscombe2, M Miller2 and J Wilkinson1 Biotron Limited, 1 Centre for Immunology, St Vincent’s Hospital; 2 Suite 1.9, 56 Delhi Road, North Ryde, Sydney, Australia Background: Biotron Limited is focused on the development of inhibitors for two viroporins; Vpu (HIV-1) and p7 (HCV). Our lead compound, BIT225, demonstrates good anti-HIV-1 activity (IC50=1.1±0.4 µM) with minimal cellular toxicity in macrophages (Mφ). Cells of the monocyte lineage are key reservoirs of HIV-1 and disseminate virus to the peripheral tissues. Here we further define the extent of BIT225’s novel antiviral activity that may represent a new class of antiviral therapeutics. 15 Methods: Monocytes isolated from seronegative donors were infected with HIV-1 or HIV-2 (No vpu) at day 14 of Mφ differentiation. Antiviral activity with BIT225 was determined in both ‘acute’ (HIV-1BaL at MOI of 0.05 for 3h) and ‘chronic’ (7d infection prior to treatment) infection assays. Virus was quantitated using real-time PCR, reverse transcriptase activity and the indicator cell line TZMb1. Additionally, samples were processed for confocal and electron microscopy and protein analysis by western blot. Abstract 13 Results: BIT225 at 10 uM resulted in >99% inhibition of HIV-1 integration and >95% inhibition of HIV-1 release in our acute assay and >92% inhibition of virus release in our chronic assay. Additionally, in co-culture BIT225 significantly inhibits HIV-1 transfer from Mφ to more permissive CD4+ T cells. Supporting our initial anti-vpu drug-screening program, BIT225 demonstrated no antiviral activity on two HIV-2 strains, HIV-2CBL-20 (rapid) and HIV-2CBL-23 (slow); further suggesting the activity is targeted against the vpu accessory protein of HIV-1. Infecting TZMbl’s in the presence of BIT225 confirmed that BIT225 activity occurs post-integration. Unlike RT inhibitors, infection was not inhibited by BIT225, which was comparable to protease inhibitor activity. Similarly, BIT225 had no direct affect upon the HIV-1 RT or protease enzymes further alluding to a novel mode of action. Visualisation of HIV-1 de novo synthesis by EM demonstrated that Mφ treated with BIT225 resulted in a morphological appearance suggestive of abnormal packaging compared to that of the control. Characterisation of these abnormalities has begun through preliminary protein analysis. Merck Research Labs, West Point, PA, USA Conclusions: This study shows that BIT225 is a late-phase inhibitor that significantly inhibits HIV1 release from human Mφ’s. BIT225’s activity is not directed at the HIV-1 enzymes RT or protease with evidence to date suggesting its antiviral activity is via vpu. The abnormal morphology observed by EM within the intracellular compartments hints toward a mechanism of action. 16 Integrase Inhibitors: New Insights into Mechanism of Action and Pharmacology with Implications for Treatment and Prevention Strategies D Hazuda for the HIV Drug Discovery Team With the approval of raltegravir in 2007, integrase inhibitors now offer an entirely new option for the treatment of HIV-1 infection. An increased understanding of this class is important to maximize their clinical utility and drive future drug discovery and development efforts. Studies by us as well as others have recently suggested that the mechanism of action inhibitors such a raltgravir which affect integrase strand transfer activity may offer a unique advantage over other ARVs. Because these inhibitors bind to the enzyme after reverse transciption and formation of the preintegration complex, they are fully effective in cell culture even when addition is delayed for as a long as eight to ten hours following infection. Moreover, raltegravir and “second generation” molecules which have been selected for their ability to inhibit resistant variants have a long residence time on the integrase/ DNA complex. While the off rate of different integrase inhibitors can range from several hours to days, in many cases this exceeds the half life of the pre-integration complex resulting in a functionally irreversible inhibition of integration and HIV-1 infection in cells. For raltegravir, in particular, these properties likely contribute to the observation that efficacy is not linked to trough concentrations in vivo. These insights into the mechanism of action and unique intracellular pharmacology of these agents have important implications for understanding the clinical pharmacology of this class and suggest integrase inhibitors could play an important role in eradication strategies as well as post-exposure Global Antiviral Journal Volume 4, Supplement 1 prophylaxis to prevent the acquisition of HIV-1 infection. Abstract 14 A Non-human Primate Model for Development of AIDS Eradication Strategies TW North, J Higgins, J Deere, TL Hayes, A Villalobos, LA Adamson, BL Shacklett, and PA Luciw University of California, Davis, CA, USA BACKGROUND: The long-term goal of this project is to develop and evaluate strategies for eradication of HIV-1 from infected individuals. These studies will be performed with an animal model that enables comprehensive analyses of viral reservoirs and replication dynamics during HAART. This model utilizes rhesus macaques infected by a chimeric virus of SIVmac239 containing the HIV-1 reverse transcriptase (RT) in place of the SIV RT (RT-SHIV). METHODS: In order to study eradication strategies in the RT-SHIV/macaque model, we have developed a sensitive virus load (VL) assay with a limit of detection of 1-2 copies of viral RNA (vRNA) per ml of plasma, and have also developed sensitive RT-PCR and PCR assays to measure vRNA and viral DNA (vDNA) in tissues. These methods have been used for a comprehensive analysis of RT-SHIV RNA and DNA in tissues, cells and fluids of infected macaques. For this study, nine HAART-treated macaques were used for analyses of cells and tissues collected at necropsy (5 macaques), and for rebound of VL after cessation of therapy (4 macaques). RESULTS: In this model a three-drug combination that is widely used in humans (efavirenz + FTC + PMPA) mimics HAART in HIV-1-infected individuals with respect to virus load (VL) suppression and rebound upon cessation of drug therapy. A comprehensive analysis of 28 different tissues collected at necropsy from a no-drug control and five HAART-treated, RTHIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies SHIV-infected macaques was completed. Viral DNA and RNA were detected in nearly all tissues of the control macaque examined including all lymphoid tissues, all regions of the gastrointestinal (GI) tract, several sites from brain and genital tissues. In the HAART-treated animals we detected RT-SHIV DNA and RNA in lymphoid tissues from all five of the animals. RT-SHIV DNA was detected in the thymus from all 5 HAART-treated animals, but vRNA was detected in only three. RT-SHIV DNA was detectable in most of the GI tissues from all HAART-treated animals, but vRNA was less prevalent (undetected in some samples) and was lower than in lymph nodes. In all other tissues, RT-SHIV RNA and DNA were undetectable or very low in HAART-treated macaques. Resting CD4+ T-lymphocytes obtained from spleen, lymph nodes, jejunum and PBMC had higher levels of RT-SHIV DNA than total cell suspensions from which they were obtained. CONCLUSIONS: These studies have identified potential sites of virus replication and latency during suppressive HAART. This model will be useful for determination of the relative contributions of residual replication and latency to residual viremia during HAART. It will also be valuable for testing eradication strategies to eliminate residual replication and/or reactivate latent virus during HAART. Abstract 15 Diminution of HIV Latency through Blockage of Arginine Methylation of Viral Proteins MA Wainberg, CF Invernizzi, S Rita, S Schildknecht, S Colby-Germinario, K Dahl, B Spira, and BG Brenner McGill University AIDS Centre, Jewish General Hospital, Montréal, Québec, Canada Background: We have shown that the HIV-1 proteins Tat, Rev, and NC are substrates for protein arginine methyltransferase 6 (PRMT6). Such arginine 17 methylation interferes with protein function, i.e. Tat transactivation activity is reduced and RNA export mediated by Rev is impaired. We have now further studied PRMT6 function in the HIV-1 life cycle: 1) involvement in establishing latency, 2) role as part of an antiviral host defense, and/or 3) participation in fine-tuning and optimization of localization patterns and functions of Tat and Rev. Conclusions: Intracellular PRMT6 levels primarily fine-tune the HIV-1 life cycle by subtly altering the localization patterns of Tat and Rev. This may slow down the switch from the early to the late phase of the viral life cycle in order to enhance overall viral replication. Abstract 16 Methods: Latency induction: We assessed RT activity in the supernatant of U1 cells treated with different drugs (NVP, PMA, Arginine Methyltransferase Inhibitors AMI1/AMI3.4) after 48 and 96 h. PRMT6 levels in PBMCs: Purified peripheral blood mononuclear cells (PBMCs) from individual donors were lysed with RIPA buffer. Cell lysates were analyzed for PRMT6 by western blotting and either normalized to total protein amounts or β-actin. Localization of Tat and Rev: HeLa cells were transfected with Tat (wt)/Tat (mut) or Rev (wt)/Rev (mut) and/or PRMT6 (wt or mut) or siRNA against PRMT6. Fixed cells were analyzed by confocal microscopy (Tat and Rev with fluorescent tag, PRMT6 with antibody). Results: U1 cells were readily inducible with PMA, which produced 10- to 40-fold increases in RT activity in the supernatants. In contrast, neither of the two PRMT6 inhibitors were able to significantly increase RT activity. Therefore, PRMT6 may not be fully involved in establishing latency. Intrinsic levels of PRMT6 in PBMCs were very low. Yet, PRMT6 may be able to play an important role in antiviral cellular host defense. Localization experiments showed an altered pattern of Tat and Rev when PRMT6 was cotransfected. While Tat alone mainly localized in the nucleoli, Tat was more evenly distributed within the nucleoplasm when PRMT6 was present. This was also true for mutated Tat that cannot be methylated. Rev, located in the nucleoli, was entirely relocalized to the cytoplasm in the presence of PRMT6, as also occurred with mutated non methylatable Rev. Furthermore, total amounts of Rev seemed reduced in the presence of PRMT6. 18 Combination Therapy for Treating HIV Latency TP Castor1, MA Munoz2, S Moreno2, E Munoz2 1 Aphios Corporation, Woburn, MA, USA; 2 University of Cordoba, Cordoba, Spain Background: HIV infects several cell types during the course of infection and progression to acquired immune deficiency syndrome (AIDS). The persistence of latent HIV-infected cellular reservoirs represents the major hurdle to virus eradication with highly active anti-retroviral therapy (HAART). Reactivation of the latent reservoirs could allow effective targeting and possible eradication of the virus. Experiments with relatively specific PKCs inhibitors suggest that bryostatin-1, a potent PKC modulator, re-activates HIV-1 latency thorough the PKC pathway. We thus investigated biochemical targets downstream of PKC. Methods: Bryostatin 1 was extracted and purified from Bugula neritina utilizing a supercritical fluid with a polar co-solvent followed by downstream chromatographic purification and crystallization. Jurkat-LAT-GFP cells were stimulated with increasing concentrations of bryostatin-1 and the phosphorylation and degradation of the NF-κB inhibitor IκBα. The phosphorylation (activation) of the MAPKs, ERK and JNK, were investigated by Western blot using specific mAbs. The percentage of GFP+ cells was analyzed by flow cytometry in an EPIC XL flow cytometer (Beckman-Coulter Inc. CA, USA). Jurkat LAT-GFP cells were pretreated with inhibitors for 30 min at the indicated dose and then stimulated with bryostatin-1 (10 nM) for 6h. Global Antiviral Journal Volume 4, Supplement 1 Results: Bryostatin-1 induced phosphorylation and degradation of IκBα, and also the activation of the MAPKs, ERK1+2 and JNK1+2 in a concentration dependent manner. Bryostatin-1 at the concentration of 10 nM does not induce IκBα phosphorylation and degradation and JNK activation but fully reactivates HIV-1 latency. Therefore, therapeutic activity of bryostatin-1 for HIV-1 latency can be achieved at concentrations that do not activate signal transduction pathways (i.e. NF-κB and AP-1) that may result in negative side effects. Abstract 17 In addition to its HIV-1-latency antagonizing activity, bryostatin-1 also down-regulates, at 10 nM concentration, the expression of the human HIV-1 receptors CD4 and CXCR4 and prevents de novo HIV1 infection as measured by virus-induced cytotoxicity assays (EC50 of 26 nM). 1 ImQuest Biosciences, Frederick, MD, USA; 2 Arisyn Therapeutics, Frederick, MD, USA Bryostatin-1 also synergizes with Histone Deacetylases (HDAC) inhibitors (valproic acid and TSA) to antagonise HIV-1 latency. HDAC inhibitors alone do not significantly reactivate HIV-1 latency but reduces the concentration of bryostatin-1 (at least one order of magnitude). Bryostatin-1 at 1 nM concentration can induce HIV-1 reactivation in the presence of therapeutically relevant concentrations of valproic acid. Thus, the therapeutic activity of bryostatin-1 can be drastically improved in humans by utilizing a HDAC inhibitor in combination therapy. Conclusions: Bryostatin-1 and their derivatives with HDAC inhibitors can be used for the treatment of HIV-1 latency. Combination therapy for the treatment of HIV-1 latency can employ bryostatin-1 (and derivatives) and one of the following HDAC inhibitors; valproic acid, butyrate derivatives, hydroxamic acids and benzamides. While HDACs can be used in continuous dosing protocol, bryostatins can be used following a cyclical dosing protocol. HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies Inhibition of Late Stage Events in HIV-1 Replication: Discovery and Development of Therapeutic Compounds with Effects on Viral RNA Synthesis Which Potentially Target Rev Function TL Hartman1 and RW Buckheit, Jr.1,2 Although the effective use of highly active antiretroviral therapy results in the suppression of virus production in infected individuals, it does not eliminate low level virus production in cells harboring virus in sanctuary sites or result in the elimination of infection. Thus, the continued search for new antiretroviral agents with unique and different mechanisms of HIV inhibition remains critical, and compounds that can reduce the level of virus production from cells already infected with HIV, as opposed to preventing de novo infection, would be of great benefit. We have discovered and evaluated a series of compounds that suppress HIV replication in cells which are chronically infected with and constitutively produce HIV. ATI0917 (formerly FB636 from The Proctor & Gamble Company) prevents virus replication by inhibiting the production of viral messenger RNA in HIV-1 infected cells. Mechanistic data suggests that the compound acts as a transcriptional inhibitor with a biochemical phenotype suggesting inhibition of Rev function in the infected cell. The compound may interfere with a cellular protein required for RNA synthesis but the antiviral effects of ATI-0917 can be clearly distinguished from any toxic effects on the target cells. ATI-0917 yields a reduction in the quantity of singly spliced and unspliced HIV RNA transcripts and a corresponding increase in the quantity of multiply spliced RNA species, resulting in a reduction in the production of virus proteins and progeny virus. ATI0917 interacted in an additive fashion with all other tested anti-HIV agents in both acute and chronic 19 in vitro antiviral assays. Resistant virus could not be selected even after three years of passage of virus in the presence of the compounds. Safety pharmacology and toxicology studies have been performed with ATI-0917 based on its original use as a systemic antifungal agent and these studies have demonstrated its safety for use in humans. ATI-0917 has been initially evaluated in a Phase 1 human clinical trial and shown to be safe. Based on the results obtained, additional formulation studies are in progress to improve the pharmacokinetic profile of ATI-0917 to allow once per day oral dosing. ATI-0917 is one of a series of molecules that have been discovered which target HIV transcription and which represent a novel new treatment strategy as an addition to existing HAART therapies and for the treatment of highly drug resistant viruses as a salvage therapy. Results: 42 patients had HIV/AIDS upon arrival, 14 were excluded secondary to lack of followup and information about CD4 counts; 73% were female, 58% were black, mean age was 40.5, mean initial CD4 count upon arrival was 483.7 and 54% of the people were started on ARTs initially. 75% of the patients with initial CD4 counts less than 200 were started on antiretroviral therapy versus 50% of those with CD4 counts greater than 200 who were started on antiretroviral therapy. Of the 14 patients who were initially started on ARTs, 71% had an increase their CD4 counts at 3 months and 64% had an increase at 6 months. Although there was an increase at both intervals there was not a greater increase at 6 months than 3 months. This could be a factor of compliance as well as stress due to environmental changes. Conclusion: Our study shows CD4 cell counts are increased with the use of ART. Abstract 18 HIV Infected Refugees: ART Therapy and Its Affect on CD4 Counts at 3 and 6 Months M Patil1, A Oladele2, and S Cookson1 1 Emory University School of Medicine, GA, USA; 2 DeKalb County Refugee Clinic, GA, USA Objective: To determine if CD4 cell counts are affected by travel and antiretroviral therapy (ART) in HIV infected refugees. Methods: This study was a retrospective chart review conducted at a HIV refugee clinic from January 2004 to December 2005. Patient factors recorded included: age, sex, race, country of origin, date of arrival, initial CD4 counts and those at 3 and 6 months and the initiation of ART. The patients were also placed into categories based on whether or not their CD4 counts were greater or less than 200. The initiation of ART was compared to the initial CD4 counts and those at 3 and 6 month visits. Variable were compared by T-test and chi-square analysis. 20 Global Antiviral Journal Volume 4, Supplement 1 Prevention and Drug Resistance HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 21 Abstract 19 Milestones in HIV Drug Resistance: From In Vitro to In Silico B Larder HIV Resistance Response Database Initiative (RDI), Cambridge, UK This presentation will review over 20 years of HIV drug resistance research. Genetic characterisation of herpes simplex virus DNA polymerase genes with drug resistance mutations initially stimulated early exploration of HIV-1 reverse transcriptase (RT) functional domains. These in vitro site-directed mutagenesis studies, reported in 1987, highlighted potential RT residues that might mutate to confer drug resistance. Seminal studies in 1988 with HIV isolates from AZT-treated individuals led to the finding that AZT resistance developed progressively to high levels during monotherapy. Subsequently, a specific group of four RT mutations was identified as the genetic basis of this resistance. Further studies revealed that at least two additional RT mutations could combine with this group to confer varying degrees of AZT resistance. Resistance to other drugs was later discovered and characterised. In 1991, the surprise observation was reported that as a patient’s virus became resistant to ddI, pre-existing phenotypic AZT-resistance actually reversed. This was also the case for NNRTI-resistance conferred by RT mutation Y181C. By 1995, it was shown that when combined, AZT and 3TC significantly suppressed viral load even though the M184V signature 3TC-resistance mutation developed. An attractive explanation for the observed sustained response was that the M184V mutation suppressed the development of AZT resistance. In parallel to the mechanistic studies of drug resistance, new diagnostic tests and assays were being developed. Specific resistance mutations could be pinpointed using allele-specific PCR or automated DNA sequencing. The drug susceptibility phenotype could also be determined with accuracy using a new HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies standardised recombinant assay. Scale-up of these assays led to a rapid expansion of individualised drug resistance testing and the accumulation of large amounts of phenotype data linked to genotype. This culminated in the development of the ‘virtual phenotype’ in 2000. This was a new tool to predict phenotype from genotype by matching mutation patterns and deriving an average phenotype from the matched group. Computational modelling techniques, such as Artificial Neural Networks, were subsequently employed to refine the quantitative prediction of drug susceptibility from genotype. The HIV Resistance Response Database Initiative (RDI) was formed in 2003. This is a not-for-profit initiative that has collated large amounts of HIV patient clinical data and used computational modelling techniques to predict a patients’ quantitative response to drug combinations relative to viral resistance status. Results of a small prospective clinical pilot study, based on use of the latest models, will be presented. Abstract 20 STOP HIV/AIDS: Seek and Treat for Optimal Prevention of HIV/AIDS J Montaner British Columbia Centre for Excellence in HIV/AIDS (BCCfE), Vancouver, Canada and International AIDS Society Since 1996, widespread availability of HAART has dramatically decreased rates of AIDS-related morbidity and mortality among those engaged in care. The overall success of HAART, however, has been limited because of uneven access to therapy among various groups of HIV infected individuals and across geographical areas of the world. In British Columbia (BC), Canada, despite a universal health care system (which includes free provision of antiretrovirals) HAART coverage remains suboptimal, particularly among young men who have sex with men (MSM), Aboriginal individuals, the homeless, the poor, the mentally ill, and injection drug users (IDUs). As a 23 result, marginalized and hard-to-reach individuals continue to bear a disproportionate burden of HIV/AIDS related morbidity and mortality in the province. Over the last decade, increasing evidence has become available indicating that HAART can impact transmission of HIV (Montaner et al, Lancet, 2006;368:531-36). In brief, HAART rapidly and effectively renders HIV-1-RNA undetectable in blood and genital secretions and this is associated with decreased risk of transmission. We recently developed a mathematical model to predict the potential impact of expanding HAART coverage among those in medical need on the spread of HIV in BC (Lima et al, JID, July 1, 2008;198:59-67). The model used data on the natural history of HIV infection, risk factors, HIV-1-RNA and CD4 cell counts, and the sources of transmission to derive the probable incidence of HIV in the coming years. Based on the available BC data, the model indicates that the status quo (ie: initiation of HAART at CD4 counts of 200 cells/mm3 or less, with coverage levels of 50% among those in medical need, and current compliance levels of 79%) will result in a continued increase in new HIV infections somewhere between 400 and 600 per year. In contrast, the model predicts that an increase in HAART coverage to 75%, 90% and 100% among those in medical need would result in a decline in the annual new HIV cases in the range of 40%, 50% and 60%, respectively, resulting in very significant savings over the long term. Therefore, the BC-CfE proposes to expand HAART coverage in BC specifically among hard-to-reach HIV infected individuals. This program labelled “Seek and Treat for Optimal Prevention of HIV & AIDS” (STOP HIV & AIDS), will aim to further decrease AIDS-related morbidity and mortality among those already infected with HIV and to decrease new HIV infections. 24 Abstract 21 Transmission of Drug Resistant HIV: Patterns of Resistance to New Antiretroviral Agents and their Clinical Relevance CAB Boucher Department of Virology, Erasmus Medical Centre, Erasmus University Rotterdam and Department of Medical Microbiology, University Medical Centre Utrecht, the Netherlands Transmission of drug resistant HIV has been reported from all parts of the world. Most available data come from countries or region based surveillance studies and convenient sample testing. The variation in sampling strategies, technologies and definitions limits the possibility to perform meta-analysis to investigate the factors influencing transmission. Nevertheless, the incidence of transmission of drug resistant HIV ranges in areas where treatment is in place between 5-15%. The patterns of resistance vary from single mutations in one target gene to multiple mutations in two target genes (protease and RT). Initially it was believed that transmitted drug resistant variants (TDR) would revert over time to a fully sensitive wild type virus. This because frequently the replication capacity of drug resistant virus is reduced as compared to wild type virus. We have shown that frequently TDR can persist for years in the absence of therapy even when their replication capacity is lower than wild type. This is explained by compensatory fixation e.g. the virus cannot evolute to wild type viruses. Loosing compensatory mutations would drive the virus in fitness valleys, which represent dead end streets. No systemic prospective studies looking into the clinical relevance of TDR variants have been performed. However a couple of retrospective studies indicate that TDR mutations can affect outcome adversely. In this respect the drugs most sensitive to TDR are the one with a low genetic barrier specifically Global Antiviral Journal Volume 4, Supplement 1 the NNRTI class. Most regions with significant TDR now have base line screening introduced. Baseline resistant testing is most often done by population sequencing, which can miss minority population of TDR variants. In some retrospective studies minority populations resistant to NNRTI were shown to be clinically relevant. The implications of these findings for clinical practice remain to be determined. Abstract 22 Ninety-nine Is Not Enough: Kinetic, Structural and Thermodynamic Characterization of HIV Protease with Insertion in the Flap Region Isolated from an HIV-positive Patient M Kožíšek1,2, K Šašková1,2, P Řezáčová3, J Brynda3, NM van Maarseveen4, M Nijhuis4, and J Konvalinka1,2 1 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic; 2 Department of Biochemistry, School of Science, Charles University, Prague, Czech Republic; 3 Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; 4 EijkmanWinkler Institute, Department of Virology, University Medical Center, Utrecht, the Netherlands Background: HIV protease represents a prime target for rational drug design, and protease inhibitors are powerful antiviral drugs that significantly decrease patient mortality. However, they exert a powerful selection pressure on the virus, which results in appearance of viral strains less sensitive to the inhibitors. Resistance to virostatics is regarded a major obstacle in the treatment of HIV positive patients. In addition to resistant mutations, insertions in the genes encoding reverse transcriptase and protease were described. It is well known that the inserts in RT play an important role in development of drug resistance; their function in PR has been described recently (Kožíšek et al., J. Virol. 2008). Results: We identified an amino acid insertion at position 35 of HIV PR isolated from a patient HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies treated by protease inhibitors for a prolonged period of time, and we set out to characterize the contribution of this insertion to viral resistance on the molecular level. Resistant PR variants with or without the insertion at position 35 were cloned in E.coli, purified and enzymologically characterized using a chromogenic peptide substrate and a panel of inhibitors. We found that the E35EE insertion does not significantly decrease the catalytic activity of the enzyme but brings about an approximately tenfold increase in the relative inhibition constant for some protease inhibitors. X-ray structures of the resistant PR-inhibitor complexes were determined to 1.8 Å resolution with very good structural factors. Thermal analysis by differential scanning calorimetry (DSC) was applied to determine the thermal stability of proteases with and without insertion. Stabilization effect of insertion on structure was comparable to active site mutations. Conclusion: We conclude that amino acid insertions into the PR sequence in the vicinity of the binding cleft represent novel mechanism of HIV resistance development. Abstract 23 TOPO Cloning and Pyrosequencing Revealed a Novel S68 Deletion in HIV-1 Reverse Transcriptase Selected by Dexelvucitabine in Human Lymphocytes I Massud, M Ruckstuhl, K Rapp, M Detorio, and RF Schinazi Center for AIDS Research, Emory University School of Medicine/VA Med Center, Atlanta, GA, USA Introduction: Antiviral resistance is a major threat to successful anti-HIV treatment and the presence of low frequency mutations can facilitate viral escape. Resistance to multiple nucleoside RT 25 inhibitors (NRTI) has been associated with an amino acid substitution at the nucleoside binding site of the enzyme and with the insertions or deletions in the β3β4 hairpin loop in the finger subdomain of the HIV-1 RT. Our research has identified a novel deletion of the S68 codon in HIV-1 RT that reduces the effectiveness β-D-2’,3’-dideoxy-2’,3’-didehydro-5-fluorocytidine (Dexelvucitabine, DFC) and other NRTI in infected human PBM cells. Methods: Primary human PBM cells were treated with DFC at 0.1 µM for one hr prior to inoculation with HIV-1LAI (WT). Virus was passaged every 6 days with a fresh treatment of DFC, ranging from 0.1 µM to 6 µM over 52 weeks. RT activity was measured weekly and used to determine percent inhibition by DFC. HIV-1 from PBM cells supernatants was amplified and cloned using TOPO cloning (Invitrogen, Carlsbad, CA). Sequencing of HIV-1 RT amino acids 1-300 was performed in parallel between the control virus and DFC treated virus to uncover mutations selected from the applied drug pressure. Amplicons generated were pyrosequenced by Research and Testing Laboratory (RTL, Lubbock, TX) using the Roche FLX pyrosequencer (Roche Diagnostic Corporation, Indianapolis, IN). Pyrosequencing was performed using standard amplicon protocols as suggested by manufactures instruction. Each library was sequenced in both the forward and reverse direction. Results: In vitro testing of HIV-1 infected primary human lymphocytes (PBM cells) treated with DFC selected for a novel deletion of AGT at codon 68 (S68∆). Utilizing TOPO cloning, the S68∆ and K65R appeared at week 14 as 70 % and 10% of the total population, respectively and they continued to be present until week 52 were they appeared as a mixture with K65R (63%). Four of these samples were sequenced using the Roche FLX system. Pyrosequencing identified the S68∆ and 15 extra mutations of potential clinical significance with an occurrence higher that 1% and lower than 9%. At week 14, the frequency of S68∆ and K65R was 0.85% and 0.01%, respectively. By week 52, the frequency had increased to 22.3% and 24% respectively. For scoring drug resistance mutations, the HIV Drug Resistance Database at 26 Stanford University was used. The S68∆ selected virus was phenotyped against various NRTI and NNRTI to obtain a resistance profile by drug susceptibility and an enzymatic assay. Drug susceptibility results suggest that virus with the S68∆ displayed greater than 30-fold increase resistance to DFC, 3TC, (-)-FTC, TDF, ABC, and DAPD, but remained susceptible to ZDV, ddI, ddC, d4T, D-FDOC, or DOT. Conclusion: These studies revealed the significance of a novel deletion in the HIV-1 hairpin loop and demonstrated the feasibility of using pyrosequencing for efficient genotyping. The novel S68∆ may prove to be an important variable to be considered in NRTI multi-drug resistance (MDR) treatment strategies. Abstract 24 Operation Sweet Tooth: Effective Use of Social Marketing Campaigns in Non-Traditional Social Settings T McPhaul National AIDS Education & Services for Minorities, Inc., Atlanta, GA, USA OBJECTIVE: Operation “Sweet Tooth” is a social marketing campaign designed to capture the attention of African American MSMs. The campaign is designed to draw attention to some of the potential HIV/STD exposure risks associated with oral sex. The organization’s goal for this campaign is to distribute 10,000 condoms during the Black Gay Pride Labor Day Weekend, and to have quality education sessions regarding oral sex risks with at least 1000 eventgoers. METHODS: The subtheme for 2008 was “Team Survival” Your Mission: To Stay Safe. Staff and volunteers wore T-Shirts bearing the “Team Survival” slogan; also they wore hats, khaki shorts, boots, a canteen and dog tags. Candy is associated with candy flavored condoms (i.e. cherry candy with cherry Global Antiviral Journal Volume 4, Supplement 1 flavored condoms). Individuals are encouraged not to brush teeth just prior to performing oral sex, but instead to use mints, gum and candy as an alternative. Safer sex kits were packaged in 2.5” X 4.5” manila envelopes. There was a label on the exterior of the package displaying “Your Mission: To Stay Safe”. Condom kits contained condoms, lubrication, and a piece of candy. Social marketing campaigns can appear less threatening while adding clarity; often they provide brevity and sometimes levity. The advertisements are catchy even to non-gay identified men; this can lead to a diffusion of “guilt.” RESULTS: NAESM provided education in 1500 encounters in 2007. Even with the outreach efforts being lead by a different core group in 2008, more than 1000 education encounters was achieved. The message was visible at all of the larger and more popular gatherings from educational to merely social. Individuals were seeking out condoms and information on their own rather than being aggressively pursued; a likely result of more effective education. CONCLUSIONS: A significant amount of “health education” was provided during this opportunity. It was a chance to do more than just hand out condoms, which alone would have still proven beneficial. In total NAESM distributed over 10,000 condoms in 2007 and 9,000 condoms in 2008. The “Operation Sweet Tooth” images help reflect that healthier behavior is the “in” thing. This particular campaign is one whereby immediate and consistent positive feedback was provided from event-goers. A large number of those who came in direct contact with the images in “Operation Sweet Tooth” advertisements expressed that they enjoyed the images and the accompanying information which often prompted more STD/HIV related questions by guests. The questions even extended to medical care requests and social services inquiries beyond those provided by the NAESM. However, the staff was knowledgeable about the full range of resources available. HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies Abstract 25 Utilizing Mental Health Counseling in African American Men who have Sex with Men Minority Populations as a Primary and Secondary HIV Prevention Tool T McPhaul National AIDS Education & Services for Minorities (NAESM), Atlanta, GA, USA OBJECTIVE: Persons receive an HIV positive diagnosis, and often do not have adequate tools for processing this information in a healthy manner. Counseling can be extremely helpful as an HIV Prevention tool from the framework that often people engage in either risky or self-destructive behaviors due to a variety of other factors such as depression , low self-esteem, as well as a lifetime of neglect, deprivation and abuse. As a secondary prevention tool mental health counseling will address underlying causes for self-destructive behavior in HIV+ individuals and lead to less risk taking, and healthy disclosure. The U.S. Surgeon General has reported that overall only one-third of Americans with a mental illness or a mental health problem get care. Yet, the percentage of African Americans receiving needed care is only half that of non-Hispanic whites. METHODS: In this model counseling services will be provided by a Licensed Professional Counselor, or Licensed Clinical Social Worker. In addition to collaborations with various agencies providing services to at-risk populations, social marketing campaigns aimed at venues frequented by at risk populations will be employed; resulting in HIV+, and high risk HIV- individuals accessing mental health services. Individuals accepted into care will have a plan developed with a counselor, which will include a course of counseling ranging from 3 months to one year. 27 RESULTS: Limited data is available at this time because this program has been recently funded. Individuals clearly falling within critical values will then go on for an intake and assessment where depth and complexity of issues are examined. At the conclusion of the counseling term, an additional assessment will be provided to evaluate counseling effectiveness. Data will be analyzed and scored to chart success of the counseling term. CONCLUSIONS: African American Men who have Sex with Men, those hardest hit by the HIV/AIDS epidemic, have complex psychological underpinnings that are often augmented by racism, homophobia, familial and societal rejection, physical and emotional abuse, self-loathing and a struggle for identity. Culturally competent mental health counseling can help to prevent HIV transmission, and contribute to improved quality of life for HIV positive individuals. There exists research identifying that African Americans from various walks of life do not seek mental health treatment as often as is warranted, or as regular as their Caucasian counterparts. There may be no one cause defined for this phenomenon, however, historically African Americans and other people of color have had similar resistance and/or obstacles to receiving care for physical health as well. Studies show that counseling for African Americans can be effective when accessed in a timely pattern. Knowing at the outset certain factors that have influenced an individual’s mental health foundation, such as level of stability of the family structure that a person experienced as a child, can yield valuable insight into behavior patterns that if known by those providing HIV prevention services could yield quite different results. Thus, it is critical that the necessity for good mental health be realized and utilized as a tool to aid in both primary and secondary HIV prevention. Fully embracing this idea may be the most formidable approach to the eradication of HIV/AIDS. 28 Abstract 26 Sex is Good: An Investigation into the Quality of Life and Sexual Practices Among Individuals on HAART in British Columbia A Mtambo1, EK Brandson2, A Kaida2,4, KA Fernandes2, T Orchard2, JSG Montaner2,3, and RS Hogg1,2 1 Faculty of Health Sciences, Simon Fraser University, BC, Canada; 2 BC Center for Excellence in HIV/AIDS, BC, Canada; 3 Department of Medicine, University of British Columbia, BC, Canada; 4 School of Population and Public Health, University of British Columbia, BC, Canada Objective: We measured the prevalence of sexual activity among a cohort of HIV+ individuals on HAART to identify quality of life measures and socio-demographic predictors by gender and sexual orientation. Methods: Individuals enrolled in the longitudinal investigations into support and ancillary health services (LISA) cohort (N=457) answered questions about oral, anal or vaginal sexual intercourse and the HIV-targeted quality of life. Fifty five percent of participants (70% of gay and bisexual, 47% of heterosexual males and 49% of heterosexual women) indicated that they were sexually active in the six months prior to participating in the survey. Multivariate models were used to measure the associations. Results: The study found that individuals who were sexually active performed better on most of the quality of life measures than those who abstained. Consistent with other research, multivariate analyses for the whole cohort showed that being sexually active was associated with being in a relationship (OR: 0.26 (CI: 0.15,0.43)), having better body image (OR: 0.82 (CI: 0.70,0.95)), and higher scores on sexual function(OR: 1.39 (CI: 1.22, 1.58)). Among gay and bisexual men, being sexually active was also associated with higher life satisfaction (OR: 1.36 (CI: 1.02, 1.82)). In heterosexual females, being sexually Global Antiviral Journal Volume 4, Supplement 1 active was associated with being depressed and having less disclosure worries (OR: 1.28 (CI: 1.02, 1.61)). Conclusion: We need to further engage with people on HAART, their service providers, and other relevant community organizations to ensure that “sex” is on the agenda, supported in ways that are healthy and appropriate to these different groups and creatively incorporated into long-term HIV management programs and strategies in order to maximize function and wellbeing in individuals living with HIV. Abstract 27 Vertical Transmission of HIV Infection in Western Romania M Sandu, M Pop, R Costa, C Laudacescu, J John, and M Şerban “Louis Turcanu” Children’s Emergency Hospital, IIIrd Pediatrics Clinic, Timişoara, Romania Background: During the years 1995-1998 Romania became infamous making a sad European record - that of having the most number of HIV/AIDS children with horizontal transmission. Since the epidemic was discovered (1990), the number of cases with vertical transmission at the national level was 5%. During the course of time, the little girls infected with HIV in the late 80’s have become adolescents now and a part of them mothers to babies. Materials and methods: A retrospective analysis of vertical transmission of HIV infection in the western region of Romania. A total of 720 cases of HIV infection admitted during the period 19902007. Out of this lot, 41 cases (5.7%) transmitted from seropositive mothers. These cases have been analyzed based on their evolution, with or without HAART, clinically and immunologically. Results: Of the 41 children born to seropositive mothers, 6 are uninfected (14.63%). 23 cases were HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies diagnosed until the year 2000, while the mothers have been diagnosed after the child’s diagnosis. Only 5 mothers (since 2000) did the prophylaxis for vertical transmission. 6 mothers were infected during 88-90 while 3 (also born in 89) were diagnosed during their pregnancy. After 2000, 5 mothers were diagnosed post-child delivery. 41.2% of children infected through vertical transmission have died. HAART was introduced in 28 cases (starting since 1996). Currently, 7 patients are in Stage B, 9 in Stage C and 2 in Stage EB. Conclusions: 1. Low frequency of vertically transmitted HIV infection, 2. High mortality rate. Death occurred in smaller aged children and they did not benefit the HAART, 3. As the patients of the 1989-90 generations were known to be pregnant, prophylactic measures were initiated, while since 2000 majority of the newborns received prophylactic ARV’s (and were not breastfed). Abstract 28 Development of the Dual Acting Pyrimidinedione IQP-0528 as a Vaginal Topical Anti-HIV Microbicide KM Watson, L Yang, CE Buckheit, and RW Buckheit, Jr. ImQuest BioSciences, Inc., Frederick, MD, USA IQP-0528 is a highly potent dual-acting HIV inhibitor targeting both reverse transcription and virus entry. The compound is non-toxic to all tested cell lines in vitro, including primary human cells, and the normal vaginal flora Lactobacillus. In standard assays, IQP0528 was active against all clinical strains of virus in the nanomolar to sub-nanomolar concentration range in PBMCs, dendritic cells and monocytes macrophages with therapeutic indices greater than one million. Equivalent or greater activity was observed when IQP-0528 was evaluated in the presence of additives such as mucopolysaccharides or simulated vaginal 29 and seminal fluids. The activity of the compound was not affected in cell-based entry assays mimicking the transition from low to neutral pH that occurs at the time of ejaculation. IQP-0528 inhibited both cell-free and cell-associated virus transmission to CD4 expressing cells in virus transmission inhibition assays and was highly active in the microbicide transmission and sterilization assay (MTSA). IQP0528 was not active against several viral, bacterial or fungal STI-causing organisms. In pre-formulation studies using standard methodology, IQP-0528 was soluble in a variety of solvents and was stable at ambient temperature and at pH’s less than 8. Acute toxicology evaluations determined the compound to be non-toxic up to 1000 mg/kg/day when dosed intravenously. Genotoxicology evaluations were all negative. IQP-0528 is a novel candidate for a vaginal topical microbicide based on its dual mechanism of action, high level of potency, lack of toxicity, compatible formulation profile and toxicology profile. As a microbicide, IQP-0528 would potentially inhibit two steps in virus replication which occur prior to reverse transcription and could be effectively used in combination with other microbicide products. IQP0528 is currently being formulated in a gel and an intravaginal ring for delivery of the compound. both entry inhibitors in the central nervous system and potential development of resistance. We report low level penetration of both entry inhibitors and selection of enfuvirtide resistance in cerebrospinal fluid (CSF) of a patient with suppressed plasma HIVRNA levels. Abstract 29 Although adequate serum concentrations of enfuvirtide (3,74 µg/ml) could be detected in the plasma, only marginal enfuvirtide concentrations were observed in CSF (0,055 µg/ml). Furthermore, the maraviroc level in plasma was 0,146 µg/ml, while only traces below the limit of quantification were detected in CSF. Development of Resistance to Enfuvirtide in Cerebrospinal Fluid in a Patient with Suppressed Plasma HIV-1 RNA SFL van Lelyveld1, M Nijhuis1, I Wilting1, F Baatz2, M Kurowski3, WM van den Bergh1, D de Jong1, IM Hoepelman1, and AMJ Wensing1 1 University Medical Center Utrecht, Utrecht, the Netherlands; 2 Retrovirology Laboratory, Luxembourg, Luxembourg; 3 Therapia GmbH, Berlin, Germany Background: Currently, enfuvirtide and maraviroc are available for infections with multi-drug resistant HIV-1. Limited data are available on penetration of 30 Methods: Longitudinal plasma and CSF samples were analyzed. Population genotypic resistance analysis was performed (protease, RT and env). CSF and plasma drug levels were measured by liquid chromatography tandem mass spectrometry. Results: Salvage therapy with maraviroc, enfuvirtide, combivir and tenofovir was initiated in a 50 year old male with a long treatment history. After an initial slow decay, plasma HIV-RNA was suppressed below the limit of detection for 8 months. Then, lumbar punctions were performed because of neurological complaints. While plasma viremia was still suppressed, HIV RNA levels in CSF appeared to be 2780 and 1490 copies/ml. Subsequent addition of darunavir/r resulted in suppression of CSF HIV-RNA levels. Subacute combined degeneration of the spinal cord due to vitamin B12 deficiency was identified as the most probable cause of his symptoms and suppletion was started. Genotypic analysis of the virus population in CSF showed no changes in the viral protease and RT sequences compared to viral populations detected in plasma before initiation of salvage therapy. Also sequence analysis of the gp120 V3-loop of the CSF sample showed no differences compared with baseline, suggesting that no change in HIV-1 tropism had occurred. Interestingly, analysis of gp41 revealed the enfuvirtide-associated V38A mutation in CSF. Global Antiviral Journal Volume 4, Supplement 1 Conclusions: We observed detectable HIV-RNA levels in CSF in absence of plasma viremia. Addition of a boosted protease inhibitor suppressed HIVRNA levels in CSF, suggesting that the detected virus originated from viral replication and not from production of integrated virus only. Low enfuvirtide concentrations might have enabled low level viral replication in CSF and subsequent selection of drug-resistance. Alternatively, enfuvirtide-resistant virus could have been selected in the plasma population during the slow initial viral decay after start of the salvage regimen. In this scenario, replication of enfuvirtide-resistant virus was effectively inhibited by the other components of the antiviral regimen in the plasma but not in CSF. This case study stresses the potential risk of selection of resistant HIV-1 virus by low level penetration of antiretroviral drugs into the central nervous system. Further research on this topic is necessary to fully understand the clinical implications. HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 31 HIV Therapy in Developing Countries HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 33 Abstract 30 To Wean or Not to Wean: Prevention of Mother-to-child Transmission through Breast Milk C van der Horst University of North Carolina at Chapel Hill, USA In 2005, 360,000 infants were infected with HIV through breastfeeding. Breastfeeding confers nutritional, immunologic, developmental, psychologic, social, and economic benefits including overall lower infant morbidity and mortality, particularly in the first six months of life. The greatest benefits accrue to exclusively breastfed infants, who are less likely to become infected with HIV than infants who receive both breast milk and other liquids or solid. Breastfeeding also provides benefits to the mother including a delay in resumption of ovulation, resulting in increased child spacing. In addition to individual health benefits, there are economic and social benefits due to savings from formula purchases. Due to scarce or unsafe breast milk substitutes, the issue of HIV transmission to the infant via breastfeeding is unique to resource-poor countries. While formula feeding does decrease the risk of HIV transmission to the infant and may be associated with decreased maternal mortality, it is also associated with a higher number of early deaths among HIV-negative infants due to diarrheal disease and pneumonia although there is recent conflicting data on the latter. In those countries where breastfeeding is the norm, use of formula marks women as being HIV-positive. weight loss may place HIV-infected mothers at greater risk of succumbing to opportunistic infections, indirectly increasing their disease progression and risk of death. HIV-infected women in resource-limited settings are faced with a tragic dilemma. Either they breastfeed their infants with its associated risk of HIV transmission, or they protect their children from HIV by replacement feeding, which, while it may also be nutritionally less demanding for themselves increases the infant’s risk of malnutrition and death if replacement feeding is not affordable, feasible, and safe. Recent studies have begun to provide some possible solutions to this dilemma and will be reviewed. References: 1. Kilewo C, et al., J Acquir Immune Defic Syndr. 2008 Jul 1;48(3):315-23. 2. Kuhn L, et al., N Engl J Med. 2008 Jul 10;359(2): 130-41. 3. Kumwenda NI, et al., N Engl J Med. 2008 Jul 10;359(2):119-29. 4. Leroy V, et al., PLoS ONE. 2008 Feb 20;3(2):e1645. 5. Six Week Extended-Dose Nevirapine (SWEN) Study Team, et al., Lancet. 2008 Jul 26;372(9635):30013. 6.Thomas T, et al., 15th Conference on Retroviruses and Opportunistic Infections 2008. Abstract 45aLB. 7. van der Horst CV, et al., Contemp Clin Trials. 2008 Sep 7. Among breastfeeding populations, the transmission rates from 6 weeks to 6 months in the various non-formula arms ranged from 2.9% to 8.1%, and from 5.3% to 12.4% over the 6-week to 12-month. Producing breast milk of adequate quantity and quality is, however, nutritionally demanding for mothers, particularly for those who have chronic infections.. Maternal depletion leading to rapid HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 35 Abstract 31 Topical Microbicides: Moving Towards Topical Pre-exposure Prophylaxis (PrEP) S Hillier University of Pittsburgh School of Medicine, Department of Obstetrics, Gynecology and Reproductive Sciences, Pittsburgh, PA, USA Sexual intercourse is the primary mode of HIV transmission worldwide. Topical microbicides are new products being developed for the prevention of the sexual transmission of HIV, and are part of the HIV prevention toolbox. First generation microbicides were developed based on the premise that HIV could be inactivated in the vaginal lumen by acidifying the vagina (BufferGel), disrupting the viral membrane (Savvy) or by interrupting the fusion of HIV with host cells (Carraguard, PRO2000/5(P) and cellulose sulfate). Products which have completed effectiveness testing include cellulose sulfate, Carraguard and Savvy, but none were found to be effective in reducing the rate of male to female transmission of HIV. Two other nonspecific microbicides, BufferGel and 0.5% PRO2000/5 (P), are being evaluated for the prevention of HIV in women in studies which will be completed in 2008-9. Antiretroviral (ARV) agents having high potency against HIV are now being evaluted as topical microbicides. Tenofovir has been formulated for vaginal application in a water-based gel and has been evaluated for safety, pharmacokinetcs and acceptability in phase 1 and 2 studies. The results of these studies suggest that 1% tenofovir gel is safe and acceptable for use by women at the time of coitus or on a daily basis. Women who have applied 1% tenofovir gel vaginally have higher levels of tenofovir in the genital tissue compared to women using oral tenofovir, with a fraction of the systemic levels as compared with oral tenofovir. Thus, topically applied tenofovir may provide higher drug levels at the site of infection, with a lower risk of systemic toxicity, than oral PrEP. A phase 2B study of 1% tenofovir used before and after coitus for prevention of HIV 36 is underway (CAPRISA-004). A second study in 4200 women comparing oral tenofovir, Truvada, 1% tenofovir gel and placebo controls (the VOICE trial) is planned to start in 2009. This study will provide data on the relative effectiveness of orally and vaginally administered tenofovir for prevention of HIV. Nonnucleoside reverse transcriptase inhibitors, including UC781, dapivirine (TMC 120) and MIV 150, are also being developed for topical administration in the form of drug impregnated rings (which would be inserted into the vagina for 1-3 months), quickdissolve films, fast dissolving tablets and gels. A number of entry inhibitors, CCR5 analogs and combination approaches are currently in preclinical development as microbicides. Because receptive anal intercourse is common among both heterosexual women and MSM, the safety of vaginal microbicide products when applied rectally is being evaluated. In addition, some novel product formulations are being developed specifically for rectal application. The field of microbicides has transitioned from the use of nonspecific agents to the topical application of ARV’s for the prevention of HIV. Abstract 32 Glycan Deletions in the HIV-1 gp120 V1/V2 Domain Compromise Viral Infectivity, Sensitize the Mutant Virus Strains to Carbohydrate Binding Agents and Represent a Specific Target for Therapeutic Intervention J Auwerx1, KO François1, K Covens2, K Van Laethem2, and J Balzarini1 1 Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, K.U. Leuven, B-3000 Leuven, Belgium; 2 Laboratory of Clinical Virology and Epidemiology, Rega Institute for Medical Research, K.U. Leuven, B-3000 Leuven, Belgium Background: The glycoprotein gp120 of HIV1(IIIb) contains 24 utilised N-linked glycosylation sites Global Antiviral Journal Volume 4, Supplement 1 that contain oligosaccharides of the complex type, hybrid type and high-mannose type. Carbohydratebinding agents (CBAs), such as the mannose-specific Hippeastrum hybrid agglutinin (HHA) and the GlcNAcspecific Urtica dioica agglutinin (UDA), frequently select for glycan deletions in all different domains of HIV-1 gp120, except in the V1/V2 domain. To reveal the underlying mechanisms, a broad variety of 31 different virus strains containing one or several N-glycan deletions in V1/V2 of the gp120 of the X4tropic HIV-1NL4.3 were constructed by chimeric virus technology. Materials and methods: The NXS/T glycosylation recognition sites were converted into a QXS/T amino acid sequence by Multi Site-directed Mutagenesis (Stratagene). About 30 combinations of glycan deletions in V1/V2 were successfully introduced into pBlue-env (a plasmid containing the env-sequence) and amplified by PCR. After applying chimeric virus technology in 293T cells for this PCR product with a molecular HIV-1/NL4.3 clone containing the eGFP gene, we used the resulting recombinant virus strains to infect U87.CD4+.CXCR4+. CCR5+ cells and to prepare virus stocks. Afterwards, these virus stocks were used in co-receptor tropism determination, replication fitness and antiviral assays using HHA, UDA and pradimicin A (PRM-A) as CBAs. Results: A broad variety of virus strains containing one or several N-glycan deletions in V1/V2 of gp120 were obtained. The mutant virus strains analysed for co-receptor usage consistently used CXCR4. A coreceptor switch to CCR5 or dual co-receptor tropism was not observed. With a few exceptions, the more glycans were deleted in the gp120 V1/V2 domain, the more the replication capacity of the mutant viruses became compromised. As there was no difference between the mutant viruses in CD4 binding efficiency and their sensitivity to the inhibitory activity of soluble CD4 and the CXCR4 antagonist AMD3100, we concluded that the loss of replication capacity was not due to major structural envelope changes that would compromise CD4 or CXCR4 binding. HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies None of the mutant virus strains showed a markedly decreased sensitivity to the inhibitory activity of HHA and UDA. Instead, an up to 2- to 10-fold higher sensitivity to the inhibitory activity of these CBAs was consistently observed. Conclusions: Our data provide an explanation why glycan deletions in the gp120 V1/V2 domain rarely occur under CBA pressure and confirm the important functional role of the glycans in the HIV-1 gp120 V1/V2 domain. The detailed data are reported and discussed in J. Auwerx et al., Virology, in press (2008). Abstract 33 Gag NC/p1 Protease Resistance Mutations can cause Selection of Additional NC/p1 Changes to Optimize Cleavage Efficiency and Replicative Capacity NM van Maarseveen1, PJ Schipper1, D de Jong1, CAB Boucher1,2 and M Nijhuis1 1 Department of Medical Microbiology, University Medical Centre Utrecht, Utrecht, the Netherlands; 2 Department of Virology, Erasmus Medical Centre, Rotterdam, the Netherlands BACKGROUND: Substrate based protease inhibitor (PI) resistance due to mutations in NC/p1 is caused by an enhanced processing of the gag protein. We investigated the impact of enhanced gag processing due to NC/p1 resistance mutations on replicative capacity (RC) and the consequences for evolution in absence of protease inhibitor pressure. METHODS: A set of four recombinant viruses containing NC/p1 mutations conferring different levels of PI resistance were generated: HXB2431V, HXB2437V, HXB2437T and HXB2436E+437T. To investigate the effect of enhanced gag processing on RC, viral replication curves were generated. To investigate the potential evolutionary pathways in absence of 37 PI pressure, multiple individual in vitro evolution experiments were performed, after which complete Gag and protease were sequenced. From the viruses that were selected RC, gag processing (quantitative immunoblot analysis) and PI susceptibility (MTT assay) were assessed. RESULTS: Single NC/p1 mutants which displayed only a slight increase in PI resistance didn’t show an obvious change in RC compared to wild type. This was also reflected in the in vitro evolution experiments where the single NC/p1 mutants showed no signs of evolution, with the exception of the selection of A429K in 1/5 experiments for HXB2431V. In contrast, the double NC/p1 mutant (HXB2436E+437T) which displayed a clear increase in processing efficiency and PI resistance, also demonstrated a clear reduction in RC. Interestingly, in all evolution experiments, amino acid changes in/near the NC/p1 site were observed (2/5: -436E; 2/5: +435R; 1/5: +438R). These selected changes restored the processing efficiency and RC. Furthermore, it was observed that due to the normalisation of gag processing efficiency, in parallel PI susceptibility returned to wild type level. CONCLUSIONS: The results from this study clearly demonstrate that there is an optimum rate for HIV-1 gag cleavage. When enhanced gag processing due to PI resistance mutations in NC/p1 reduces RC, HIV-1 can modulate the NC/p1 sequence by selection additional changes to restore gag cleavage and RC. Abstract 34 Safety, Tolerability and Efficacy of Darunavir/ritonavir in Treatmentexperienced Women with HIV Infection: Interim Analysis of GRACE (Gender, Race, And Clinical Experience) C Zorrilla1, J Currier2, K Squires3, D Averitt Bridge4, R Ryan5, A Mazikewich6, and J Mrus6 on behalf of the GRACE Study Group 1 University of Puerto Rico School of Medicine, San Juan, PR, USA; 2 University of California, Los Angeles, School of Medicine, Los Angeles, CA, USA; 3 Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA, USA; 4 The Well Project, Inc., Atlanta, GA, USA; 5 Tibotec, Inc, Yardley, PA, USA; 6Tibotec Therapeutics, Bridgewater, NJ, USA Background: GRACE, the largest trial to date of antiretroviral treatment-experienced women in North America, is a multi-center, open-label phase IIIb trial designed to assess sex and race differences in efficacy, safety, and tolerability of darunavir/ritonavir (DRV/r) over 48 weeks. The study was designed to enroll a predominantly female, racially diverse population, with 65 GRACE study sites selected to meet this objective. We report a pre-planned interim analysis of the first 203 patients (of a total of 429 enrolled) to complete Week 24 or discontinue sooner. Methods: Adults with prior antiretroviral treatment experience and HIV-1 RNA ≥1000 copies/ mL were enrolled. All patients received DRV/r 600/100mg bid plus an investigator-selected optimized background regimen (OBR). Select NRTIs and etravirine were provided. This pre-planned interim analysis was conducted to assess safety and tolerability. We also present preliminary efficacy results using ITT-TLOVR (intention-to-treat-timeto-loss-of-virologic-response) and TLOVR-non-VF (non-virologic failure) censored algorithms; the latter excludes patients who discontinued for reasons other than virologic failure (VF). 38 Global Antiviral Journal Volume 4, Supplement 1 Results: 180 women and 23 men were included in the interim analysis; safety and efficacy are not presented separately for men due to small sample size. The mean age of women was 42 years (range 19-78). 65% of women were African American, and 22% were Hispanic/Latino. The mean baseline viral load and median baseline CD4 count for women were 4.67 log10 copies/mL (SD 0.86) and 206 cells/mm3 (range 1-868), respectively. More than half (59.4%) of women had prior treatment experience with ≥2 protease inhibitors at the time of study entry. Overall, 22.7% of patients (44 women and 2 men) discontinued the study; 7.4% of patients (14 women and 1 man) discontinued due to adverse events (AEs) and 0.5% of patients (1 woman and no men) discontinued for VF. Among all patients, the most common (≥2%) grade 2-4 AEs at least possibly related to study drug were nausea (5.9%), diarrhea (5.4%), rash (3.0%), weight gain (3.0%), increased transaminases (2.0%), dizziness (2.0%), and dyspepsia (2.0%). Serious AEs (SAEs) were reported in 18.2% of patients, and the most common SAE was pneumonia (3.9%). At 24 weeks, 50.6% (ITT-TLOVR) and 65.5% (TLOVR-nonVF censored) of women achieved HIV-1 RNA <50 copies/mL. The mean increase in CD4 count from baseline [ITT-non-completer = failure] for women was 86 cells/mm3. Conclusions: The GRACE study is fully enrolled (287 women, 142 men), demonstrating that women from North America, including women of color, can be successfully recruited to participate in clinical trials of antiretrovirals in HIV infection. No unexpected AEs were noted in this interim analysis. Approximately one-quarter of women discontinued the study prior to Week 24; reasons were generally unrelated to AEs or VF. Response rates were within the expected range from previous clinical trials of DRV/r in treatmentexperienced patients. HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies Abstract 35 96-Week (wk) Efficacy and Safety of Raltegravir (RAL) in Treatmentexperienced Patients J Gatell1, B-Y Nguyen2, B Grinsztejn3 , C Katlama4, J Eron5, A Lazzarin6, D Vittecoq7, C Gonzalez8, M Miller2, H Wan2, J Zhao2, C Harvey2, R Isaacs2, and The Protocol 005 Team 1 University of Barcelona, Barcelona, Spain; 2 Merck Research Labs, West Point, PA, USA; 3 Evandro Chagas Clin Res Inst/Oswaldo Cruz Fndn, Rio de Janerio, Brazil; 4 Hosp Pitie Salpetriere, Paris, France; 5 University of North Carolina, NC, USA; 6 San Raffaele Sci Inst, Milan, Italy; 7 Hosp Paul Brousse, Villejuif, France; 8 New York State Department of Health AIDS Institute, NY, USA Background: RAL, a HIV integrase inhibitor, demonstrated potent efficacy in combination with optimized background therapy (OBT) during the double-blind (DB) phase of Protocol 005, a phase II study in patient (pts) failing therapy with triple-class resistant virus. The study extension provides longterm data on RAL. Methods: After ≥ 24 wk of DB therapy (RAL 200, 400, or 600 mg bid or placebo (pbo)), all pts were offered open-label (OL) RAL 400 mg b.i.d. Efficacy measurements included % pts with HIV RNA (vRNA) <400 and <50 copies/mL, and change from baseline in CD4 cells. Results: 133 pts received RAL and 45 pbo. Baseline characteristics demonstrated heavily pretreated patients (~9 years prior ARTs); > 60% of pts had OBT with limited activity (GSS = 0). All RAL dose groups, including 94 pts (71%) who entered OL extension, were combined since all doses were efficacious during DB therapy; pbo groups data are not reported since only 6 pts (13%) entered OL. Efficacy is summarized: 39 Week 48 vRNA <400 copies/mL (95% CI) 2 vRNA <50 copies/mL (95% CI) 2 Mean ∆CD4 (cells/ mm3) ¶ 68% (59, 76) 55% (46, 64) 96 ( 71, 121) Week 96 55% (46, 64) 48% (40, 57) 104 ( 76, 131) 2 Non-Completer=Failure. N = 133 pts receiving RAL. ¶ Baseline values carried forward for virologic failures After wk 48, only 9 pts failed with vRNA >50 copies/ mL; 5 of 6 tested had signature integrase mutations at either Q148 or N155 in combination with other secondary mutations. RAL was generally well tolerated with few discontinuations (5 pts) due to AEs. Conclusions: In pts with limited treatment options, RAL in combination with OBT had potent and durable antiretroviral effect through wk 96, and was generally well tolerated. Abstract 36 Valacyclovir as Antiretroviral Therapy L Negruţiu1, O Roşca1, and M Pop2 1 University of Medicine and Pharmacy “V. Babeş” Timişoara, Romania, Department of Infectious Diseases I; 2 University of Medicine and Pharmacy “V. Babeş” Timişoara, Romania, Department of Pediatrics BACKGROUND: Acyclovir and its prodrug – valacyclovir – were recently tested in 3 clinical trials studying their effect on HIV, from the genital fluid of African persons coinfected with HIV and HSV-2. The results of these studies were contradictory; two of them confirming a decrease of HIV concentration in the seminal fluid, but not in the vaginal fluid; one of the studies confirmed the decrease of HIV viral load in plasma and rectal mucous. with cutaneo-mucous herpetic lesions. We followed the evolution of the HIV-1 VL in 8 patients (5 females, 3 males) with age between 21-36, HIV+, coinfected with cutaneo-mucous herpes (first episode): 5 genital and 3 labial. The patients were under clinic-biological monitoring for introduction of ART. We monitorised them repeatedly, before and after Valacyclovir therapy by: • quantification of TCD4+ cells by flow cytometry • VL assessment by PCR-Cobas-Amplicor • complete blood count: normal • Elisa HIV-1= +; Elisa HSV2= +; Western-Blot for HIV-1 +. The viral load and CD4+ count were repeated 2 months after the interruption of valacyclovir therapy. The valacyclovir dose: 2x1g/.zi, p.o., 7 days in genital herpes and 2g ,bid, for 2 days in labial herpes. RESULTS: Baseline: - CD4+ cells count: 420-512 cells/mm3 - plasma HIV-1 ARN was: 5000-8500 copies/ml Repetead: - CD4+ cells count: 620-840 cells/mm3 - plasma HIV-1 ARN: under 50 copies/ ml(undetectable) in 6 cases; 4450-5000 copies/ml in 2 cases with genital herpes that afterwards presented 3 recurences under TARV (instituted afterwards) CONCLUSION: It seems that in some patients coinfected with HIV-1 and HSV-2 the acyclovir administered as valinated prodrog can to induce a decrease in HIV replication, probably by the phosphorilation of acyclovir by HSV-2 that can directly inhibit the HIV reverse transcriptase. Our number of cases is small, representing only some preliminary results, but our data is in concordance with older and newer results from the literature. METHODS: The aim of our research is to study the reductive effect of valacyclovir on HIV plasmatic viral load (VL) in 8 HIV-1 positive patients, ARV-naïve, 40 Global Antiviral Journal Volume 4, Supplement 1 HIV/Hepatitis and Other Co-infections HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 41 Abstract 37 The Aging Liver in the HIV Population D Dieterich Mount Sinai School of Medicine, USA HIV was recognized more than 25 years ago. Since then, the survival of HIV-infected patients has increased due to highly active antiretroviral therapy (HAART) and we now face the long-term complications of HIV and its treatment. Cardiovascular disease, bone loss, renal disease, cancer development and liver disease are recognized more frequently in the HIV aging population and disease course is accelerated. (1) The usual causes of liver disease in HIVinfected individuals include chronic viral hepatitis, steatosis and steatohepatitis, alcohol abuse, drug hepatotoxicity and cryptogenic cirrhosis. Recently, a new entity in HIV has been identified: non-cirrhotic portal hypertension. (2-4) The more frequent clinical presentation is variceal bleeding and histology is characterized by hepatoportal sclerosis (HPS) and/ or nodular regenerative hyperplasia (NRH). Prior exposure to the antiretroviral drug didanosine (ddI) seems to be the common denominator among the majority of cases. Pathophysiology of the injury is still unclear but endothelial lesions of the portal system, mitochodrial toxicity and microbial translocation have been suggested. (4) We seek to characterize liver disease in our HIV aging population, specifically non-cirrhotic portal hypertension. We plan to thoroughly assess risk factors and the temporal relationship between ART exposure / removal and liver disease, describe clinical presentation, evaluate diagnostic approaches including liver biopsy and characterize evolution of liver disease. With the increasing population of aging HIV, we will encounter multiple and possibly severe complications of HIV itself or its treatment, such as non-cirrhotic portal hypertension. HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies References: 1. Effros RB, et al. Clin Infect Dis. 2008 Aug 15;47(4):542-53. 2. Maida I, et al. J Acquir Immune Defic Syndr. 2006 Jun;42(2):177-82. 3. Schiano TD, et al. Am J Gastroenterol. 2007 Nov;102(11):2536-40. 4. Maida I, et al. Antivir Ther. 2008;13(1):103-7. Abstract 38 Co-infection with HCV and HBV: When and How to Treat? B Polsky St. Luke’s-Roosevelt Hospital Center and Columbia University College of Physicians & Surgeons, New York, USA Liver disease has become a leading cause of serious morbidity and mortality in patients infected with HIV. The prevalence of HIV/HBV coinfection varies according to geography and risk category with global figures estimated in the 8-11% range. In contrast, the Centers for Disease Control estimates that at least 25% of HIV-infected individuals in the United States are coinfected with HCV. The occurrence of HCV coinfection is particularly high (>50%) among HIVinfected injection drug users. Previously, treatment of HBV and HCV in HIV-infected individuals was not recommended but with the advent of highly active antiretroviral therapy (HAART), the prognosis of HIV-related diseases has dramatically improved. Thus, the National Institutes of Health has issued a consensus statement that treatment of HCV infection in HIV-infected persons is not only possible but is recommended. Similarly, treatment of HBV infection in HIV-infected persons is recommended as progression to cirrhosis in the presence of HBV is hastened in the context of HIV infection. The availability of multiple approved agents with and without overlapping activity against HIV has increased options for physicians and patients and opened the way for combination therapy for HBV in the 43 context of HIV coinfection. In fact, the most common nucleos(t)ide backbone (tenofovir/emtricitabine) for initial HAART is now recommended when treating HIV/HBV coinfection. Likewise, new investigational agents against HCV, particularly genotype 1, hold similar promise and await clinical trial in coinfected patients. Abstract 39 Antimicrobial Molecules for Treatment of Multi-drug Resistant (MDR) and Extensively Drug Resistant (XDR) Strains of Mycobacterium Tuberculosis R Scott1, D Liu1, M Selbovitz, D Miller2, G Cruz, and W DeGrado3 1 PolyMedix, Inc., Radnor, PA, USA; 2 AIDS Institute, NY, USA; 3 University of Pennsylvania School of Medicine, Philadelphia, PA, USA BACKGROUND: Pandemic levels of multi-drug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB), with 90% mortality rate among HIV/MDR-/XDR-TB co-infected, as record levels of infection occurred in 2007 with 45 countries reporting at least one case of XDR-TB. A series of small, stable, synthetically-produced arylamide antimicrobials has been developed which mimic the activity of the host defense proteins and represent a novel class of therapeutics for the clinical treatment of MDR-/XRD-TB. These small molecules inhibit semidormant tubercule bacilli not inhibited by other TB drugs. The compounds work by directly lysing bacterial cell membranes, the same mechanism utilized by the host defense proteins. As a result, resistance to these compounds is highly unlikely to develop. Previous attempts to develop host defense proteins have failed due to inactivity in vivo, manufacturing problems, poor stability, and immunogenic responses; but these non-protein drugs avoid such limitations. 44 METHODS: Six molecules spanning several structural series were screened by the Tuberculosis Antimicrobial Acquisition and Coordinating Facility using in vitro assays to measure susceptibility against H37Rv strain of M. tuberculosis and cytotoxicity to monkey VERO cells. RESULTS: Three of the tested antimicrobial compounds exhibited high antimicrobial activity (IC90 < 5 µg/ml) against the H37Rv strain of M. tuberculosis, with selectivity greater than 30-120 fold for TB versus mammalian cells. The synthetic small molecules rapidly killed the M tuberculosis cells in vitro. These compounds are stable and not immunogenic. The results indicate they may be successful treatment for MDR and XDR strains of M. tuberculosis. CONCLUSIONS: Results indicate that synthetic smallmolecule mimics of the host defense proteins may be an effective treatment against strains of MDR-/ XDRTB. Existing knowledge of the mechanism of action through which these compounds work gives reason to believe that there is a lower risk of resistance developing to them. Further in vitro tests to measure susceptibility of isoniazid, rifampicin, fluoroquine, capreomycin, icanamycin, and amikacin- resistant strains to these compounds will be initiated. The lack of financial mechanisms committed to facilitating the development of new treatments is exacerbating the spread of MDR-/XDR-TB complicated by HIV infection. Global Antiviral Journal Volume 4, Supplement 1 Abstract 40 Progressive Multifocal Leukoencephalopathy – A Case Report O Roşca1, EC Roşca2, M Pop3, and L Negruţiu1 1 University of Medicine and Pharmacy “Victor Babeş” Timişoara, Romania, Department of Infectious Diseases I; 2 University of Medicine and Pharmacy “Victor Babeş” Timişoara, Romania, Department of Neurology; 3 University of Medicine and Pharmacy “Victor Babeş” Timişoara, Romania, Department of Pediatrics BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is a progressive demyelinating disease caused by JC virus, occurring in immunocompromised patients. In the HAART era, approximately 5% of the AIDS patients have been reported to develop PML. The clinical presentation of PML is quite variable because lesions may occur anywhere in the CNS white mater; the most common findings are motor weakness, visual defects (e.g. visual blurring, diplopia), and incoordination. The most frequently affected regions are the cerebral hemispheres, followed by subtentorial lesions. The diagnosis of PML in an immunocompromised patient with evocative clinical picture of focal neurologic deficits is made by demonstrating typical findings on brain imaging studies and detection of viral DNA in CSF by PCR examination. Brain biopsy should be reserved for cases with suspicious white matter lesions on CT or MRI in which JC virus is not detected in PCR. Differential diagnosis should consider other primary as well as opportunistic infections of CNS, other demyelinating diseases (such as multiple sclerosis), vascular lesions (e.g. ischemic stroke, HIV vasculopathy), tumors (e.g. lymphoma) and HIV encephalopathy with secondary changes in white matter. METHODS: The present paper describes the case of a HIV positive patient with clinical, biological and imaging findings highly suggestive for PML, but negative PCR for JC virus DNA. HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies RESULTS: The neurological examination revealed somnolence, confusion, left pyramidal signs, left hypotonia, incoordination of the left limbs, left oculomotor nerve palsy, left lower facial weakness and dysartria. The MRI scan revealed diffuse lesions up to 1,5 cm situated in the left internal capsula, left pons and midbrain, vermis and left cerebellar hemisphere with minimal gadolinium enhancement, no mass effect and no edema. The lesions were hypointense on T1-weighted images and hyperintense on T2weighted and Flair. The biological investigations were highly suggestive for PML, but JC virus DNA was not detected in CSF. The status of the patient improved under HAART therapy. CONCLUSION: Before the HAART era, JCV PCR had a high sensitivity and specificity for the diagnosis of PML, but over the past few years it has become more frequent to find negative CSF JCV PCR results in AIDS patients with clinical and radiological presentation indistinguishable from PML. However, a negative PCR test does not exclude this diagnosis. In these patients, a definitive diagnostic necessitates a brain biopsy, an invasive method not without risks, which sometimes might not be accepted by the patients, especially if their status is improving under therapy. The present data suggest that there is a need for the development of new diagnostic methods, with higher sensitivity, in concordance with the recent changes in the disease’s evolution. Abstract 41 Accelerated Approval and Postmarketing Commitments: A Delicate Balance T Swan Treatment Action Group, New York, NY, USA “Access to promising new drugs is a right one cannot deny patients with a fatal illness. However, this right carries with it the responsibility to provide information that will advance science and help future 45 generations of patients.” Carlton Hogan 1961-20031 Background: AIDS activists have focused on HIV drug development for more than two decades. In 1992, in part due to pressure from AIDS activists and pharmaceutical deregulators, FDA instituted Accelerated Approval regulation, allowing earlier approval of drugs for serious, or life-threatening diseases, based on unmet need and a surrogate endpoint. Clinical benefit and questions about optimal use are determined in subsequent trials, known as post-marketing commitments (PMCs). biologic agents likely to be used in children, these often lag far behind target completion dates. AIDS activists welcomed accelerated approval, but pressed for collection of data on pharmacokinetics, drug-drug interactions, dosing, efficacy and toxicity by disease stage, and in specific populations, during registration trials. They argued that post-marketing commitments are not always required or conducted expeditiously. Often, PMC results are not available until drugs have already been on the market for years. MedWatch data and medical literature were reviewed for instances when emergent safety and toxicity issues would have warranted studies in, or greater inclusion of, specific populations in clinical trials of novel antiretroviral agents or treatments for comorbidities prevalent among people living with HIV/AIDS. Valid scientific rationale, justice (as outlined by the Office of Human Subjects Research’s 1974 Belmont Report2), and HIV demographics in the United States argue for adequate enrollment of women, people of color, and current and former injection drug users in registration trials. Updated demographic data on HIV prevalence in the US indicate that ~25% women constitute ~25% of cases, African Americans a staggering 46%, Hispanics, ~17%, and injection drug users ~18%3. Methods: FDA’s post-marketing database was extensively reviewed to assess the status of ongoing and outstanding post-marketing commitments, when available (for tirpanavir, atripla, darunavir, didanosine EC, etravirine, emtricitibine/tenofovir, epivir/abacavir, epivir/retrovir/abacavir, invirase, mariviroc, nevirapine, and raltegravir), to assess instances of unacceptable delay. Conclusion: Specific examples are described, such as late characterization of sex-specific adverse events and life-threatening drug-drug interactions. Inadequate enrollment of women in HIV clinical trials has been a chronic—and dangerous— problem. Failure to perform drug-drug interaction studies on methadone and buprenorphine, agents used for opiate substitution treatment, and hormonal contraceptives, as well as using liver enzyme elevations as exclusion criteria in the absence of a compelling rationale, has resulted in the virtual exclusion of women and patients coinfected with viral hepatitis from many early-phase clinical trials of novel antiretroviral agents, when signals on differences in PK and toxicity might emerge. Although the 2003 Pediatric Research Equity Act (PREA) requires studies of drugs and 46 Global Antiviral Journal Volume 4, Supplement 1 Novel Therapeutic Approaches: Antiviral Mechanism and Predictive Toxicology HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 47 Abstract 42 Optimizing HAART Therapy in the Era of Integrase and Entry Inhibitors C Katlama Hopital Pitie-Salpétrière, Paris; COREVIH Paris; Pierre et Marie Curie University Paris VI, France Combined antiretroviral therapies have revolutionized the prognosis of HIV disease transforming an almost uniformly lethal disease in a chronic one with an estimated survival of several decades on effective antiretroviral therapy. However several issues had tempered this “idyllic” situation: cART cannot be stopped without damages such as an increases in number of deaths, comorbidities even at relatively high ratio of CD4 demonstrating that HIV is not only deleterious by its consequences on immune deficit but also in the fact that it promotes high immune activation that follows the rebound in HIV replication- cART at least NRTIs and PIs is associated with toxicities and comorbidities. This leads to investigate new strategies opening a new field of research to be able to maintain long-life therapy which will retain a full viral efficacy with a plasma viral load permanently below 50 copies /ml, a cut-off which has been associated to durability of the control of viral suppression , the absence of clinicallyrelevant development of viral resistance at least in plasma with a minimum induced toxicity, a mandatory issue for chronic administration over decades at all periods of life. These strategies may involve the existing classes of drugs trying to build drug-class sparing maintenance strategies. Maintenance strategies with protease inhibitor monotherapy is currently investigated mainly with lopinavir or darunavir under investigation in randomized studies. The development of new classes of drugs had already brought significant improvement in HIV disease management. Integrase inhibitors mainly the recently licensed raltegravir had lead to increase the rate of full viral suppression in salvage patients in combination HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies with new drugs from old classes such as darunavir or etravirine and allowed up to over 80% of plasma HIV RNA < 50 copies/ml. Inhibitors of coreceptors CCR5 mainly maraviroc are not only antiretroviral agents but appears also to have immune modulatory functions with specific action on CD4 lymphocytes, immune activation or potentially on apoptosis. Because these drugs act on different viral targets, because of their different resistance and toxicity profile from NRTIs or PIs, clinical research has to explore their potential role in the treatment gaps. Several important questions throughout remain to be explored: • What is the role of these new drugs in primary infection? • How can both raltegravir with its very rapid decrease in viral load and maraviroc with its action on CD4 decrease the delay needed time to control the virus and the time to restore CD4 above 200/mm3 in late HIV presenters? • How can these drugs be used in maintenance therapy in patients with undetectable viral load but a long history with subsequent toxicities of PIs or NNRTIs? Can we limit the number and the burden of drugs used to control viral replication? • Will these drugs in addition to classic cART be capable to decrease viral reservoirs and why not tends towards eradication? 49 Abstract 43 IDX899 - A Novel Once-a-day Second Generation NNRTI for the Treatment of HIV/AIDS D Mayers1, R Murphy2, C Zala3, XJ Zhou1, M. Seifer1, JJ Jakubik1, K Pietropaolo1, M-F Temam4, B Belanger1, J Sullivan-Bolyai1, and DN Standring1 1 Idenix Pharmaceuticals, Inc., Cambridge, USA; 2 Northwestern University Feinberg School of Medicine, Chicago, USA and Pierre et Marie Curie Université -Paris 6, Paris, France; 3 ACLIRES Argentina, Buenos Aires, Argentina; 4 Idenix Pharmaceuticals, Inc., Montpellier, France Background: IDX899, a second generation NNRTI, has potent in vitro antiviral activity against wild-type HIV-1 and remains active against many viruses bearing mutations selected by other first or second generation NNRTIs. In particular, IDX899 showed good activity against most HIV-1 isolates selected in vitro by efavirenz or etravirine, including isolates carrying as many as four or five resistance mutations conferring high level resistance to the respective selection agent. In vitro, IDX899 selected mutations in the reverse transcriptase gene including V90I, E138K, Y181C/I, S134I, I135R, G190E and M230L, and two primary pathways to IDX899 resistance were identified, starting with either E138K or Y181C changes. Compared to efavirenz, prolonged passaging and multiple mutations were required to confer high level resistance to IDX899. The favorable in vitro resistance profile of IDX899, together with its PK properties, suggests that IDX899 should display a high barrier to resistance in the clinical setting. Favorable clinical safety and pharmacokinetics in healthy volunteers supported a phase 1b/2a study which assessed antiviral activity, safety and pharmacokinetics of daily (QD) IDX899 in treatmentnaïve HIV-1-infected subjects. or placebo QD for 7 days. HIV-1 RNA levels were measured using the HIV-1 Roche COBAS Amplicor® 1.5 assay. IDX899 plasma levels were quantitated using a validated LC/MS-MS method. The protocol enrolled 40 subjects (37 men and 3 women) with mean baseline CD4+ of 479 cells/mL and baseline viral load of 4.71 log10 copies/mL. Subjects were infected with HIV-1 clades B or B/F. Results: The mean log10 decreases in HIV-1 plasma RNA from baseline to Day 8 were 1.78, 1.78, 1.84 and 1.87 in the 800, 400, 200 and 100 mg cohorts, respectively. The placebo group showed a 0.10 log10 increase. Steady-state trough concentrations well exceed the in vitro protein-binding adjusted EC50 and EC90 of IDX899 for all doses evaluated; consistent with the observed absence of a PK/PD relationship. There were no treatment discontinuations, treatment emergent SAEs or dose-limiting toxicities. No discernable patterns in adverse events or laboratory abnormalities were observed. Drug-drug interaction studies of IDX899 combined with either Truvada® or atazanavir in healthy volunteers have demonstrated no evidence of clinically significant interactions. Conclusion: Once daily oral IDX899 was well tolerated, demonstrated potent HIV-1 antiviral activity at low doses, and has shown no clinically significant drug-drug interactions to date. IDX899 offers the potential for treatment of both treatmentnaïve and treatment-experienced HIV-infected persons, along with the potential for co-formulation with other antiretroviral drugs. Methods: Enrolled subjects had HIV-1 RNA ≥ 5,000 copies/mL and CD4+ cell count ≥ 200 cells/mm3. Ten subjects per sequential dose cohort were randomized (8:2) to receive IDX899 (800, 400, 200 or 100 mg) 50 Global Antiviral Journal Volume 4, Supplement 1 Abstract 44 Vicriciroc: A Next-generation CCR5 Antagonist for Treatment of HIV LM Dunkle Schering-Plough Research Institute, USA Vicriviroc (VCV) is a specific antagonist of the CCR5 chemokine receptor and is a potent inhibitor of the entry of R5-tropic HIV into uninfected cells, thereby reducing replication of HIV. In vitro and in vivo studies have demonstrated potent additive-to-synergistic activity of VCV in combination with most other classes of antiretroviral agents (ARVs). In addition, VCV shares no cross-resistance with other classes of ARVs. As the envelope mutations that confer viral resistance to the CCR5 antagonist class have not been fully characterized, it is not clear whether crossresistance exists between VCV and other members of the class, e.g. maraviroc. It is clear that resistance to VCV develops slowly in vitro and very infrequently in vivo. As VCV has no activity against CXCR4-tropic HIV, clinical trials have focused largely on subjects with R5-tropic virus. When X4-tropic virus becomes detectable, usually as the result of variability in the tropism assay or “uncovering” of pre-existing X4 virus, viral suppression is maintained to the extent that adequate numbers of active ARVs, including a PI/r, are in the background regimen. VCV has been studied in three Phase 2 trials (2 in treatment-experienced and one in treatment-naïve individuals). The pharmacokinetics of VCV are very predictable, and no dose adjustment is required when used as currently recommended. A single dose of 30 mg once daily is appropriate in a regimen containing a PI/r in both first-line and treatment-experienced settings. Pharmacokinetic/pharmacodynamic analyses support 30 mg QD as the dose that most likely achieves the trough concentration of 100 ng/ mL, above which full and durable viral suppression is observed. This target concentration was not as HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies reliably achieved with lower dose levels of VCV. The safety profile in all recent studies has been promising, including absence of seizures and hepatotoxicity, malignancy rates comparable to background rates in advanced HIV disease and no increased infections, AIDS-defining events or ischemic cardiovascular events. Current Phase 3 studies, now fully enrolled globally, focus on the treatment-experienced population. Another Phase 2 study of VCV in a first-line regimen is on-going. All Phase 3 studies include a newly optimized background regimen that includes a PI/r as is standard in treatment-experienced subjects. The ongoing Phase 2 study explores a novel, class-sparing treatment paradigm combining VCV with boosted atazanavir in comparison with a standard first-line regimen of Truvada + boosted atazanavir. The studies and enrolled populations will be described fully. In conclusion, the clinical development program of VCV has progressed to full enrollment of Phase 3 in treatment-experienced subjects and of the first stage of the Phase 2 first-line therapy study. The pharmacokinetic, efficacy and safety profiles of VCV are robust and support a single 30 mg once daily dose in regimens containing a PI/r. Abstract 45 Amdoxovir Combined with Low Dose AZT for HIV-1 Therapy RL Murphy and the RFS-AMDX-203 Team Northwestern University, Chicago, IL, USA Background: Amdoxovir (DAPD) is a NRTI that is synergistic in human lymphocytes with zidovudine (ZDV), and in combination prevents selection of TAMs and K65R mutations. ZDV, the first effective NRTI developed for HIV has well characterized hematologic toxicities. In silico, ZDV (200 mg bid) produces reduced ZDV-monophosphate levels, associated with toxicity, while maintaining ZDV-triphosphate levels necessary 51 for antiviral effect. The objective was to evaluate the pharmacokinetics (PK) and anti-HIV activity of DAPD with and without ZDV at two doses, as a prelude to advanced phase 2 studies. Methods: Twenty-four subjects, not receiving antiretroviral therapy and with plasma HIV-1 RNA (viral load, VL) ≥ 5,000 copies/ml, were randomized to DAPD 500 mg bid, DAPD 500 mg plus ZDV 200 or plus ZDV 300 mg bid for 10 days. In each arm, subjects were randomized 3:1 to DAPD or placebo. VL was determined daily, and PK sampling was performed on Days 1 and 10. Analyses were performed using an intent-to-treat approach. The mean change in VL log10 from baseline at Day 10 and cumulative change in VL log10 from baseline to day 10 (AUCVL) were determined. Plasma DAPD, dioxolane guanosine (DXG) metabolite, and ZDV levels were measured by LC-MS/MS. Safety was evaluated by proportion of ≥ Grade 3 adverse events per treatment, using ACTG toxicity grading scale. ANOVA was performed to relate AUCDXG and AUCZDV with AUCVL. Results: On day 10, mean VL log10 change was +0.10 with placebo, -0.65 with ZDV 200, -0.45 with ZDV 300, -1.07 ± 0.80 with DAPD, -1.97 ± 0.16 with DAPD/ZDV 200, and -1.67 ± 0.21 with DAPD/ZDV 300. DAPD/ZDV 200 was significantly more potent than DAPD (p < 0.04), suggesting synergy, and there was markedly decreased VL variability with DAPD/ AZT compared with DAPD alone. VL decline was significantly improved with DAPD/ZDV 200 and 300 mg compared with ZDV monotherapy (p ≤ 0.0001). Adverse events were mild to moderate and transient. As anticipated, no statistically significant drug-drug interactions were noted (see also Asif et al, HIVDART, 2008). Conclusions: DAPD and DAPD/ZDV were well tolerated, and the combination produced a 2 log VL reduction after 10 days. The profound and consistent decrease in VL variability with DAPD/AZT compared with DAPD alone suggest strong in vivo synergy. The lack of correlation between variations in plasma AUCZDV with VL decline supports in silico findings (Antimicrob Agents Chemother, December 2008). 52 The need of additional purine anti-HIV nucleosides, especially guanosine analogs to replace abacavir warrants further development of co-formulated DAPD/ZDV for therapy of HIV infections. Abstract 46 A New Era in HIV-antiretroviral Therapy: Darunavir, Etravirine and TMC278 E Lefebvre Tibotec, Amsterdam, the Netherlands Since the development of HAART, the prognosis for HIV-1-infected patients has improved dramatically. However, there remains a need for new antiretroviral agents with improved efficacy, convenience, tolerability and resistance profiles for both treatment-naïve and -experienced patients. J&J/Tibotec is currently developing innovative antiretroviral therapies to meet the needs of HIV-1-infected patients. The PI darunavir (DRV; PREZISTA™), administered with low-dose ritonavir (DRV/r) demonstrates efficacy and tolerability in both treatment-naïve and -experienced patients. In the Phase III ARTEMIS trial, treatment-naïve patients were randomised 1:1 to receive DRV/r 800/100mg qd (n=343) or lopinavir/r (LPV/r) 800/200mg total daily dose (n=346), both with TDF/FTC qd, for up to 96 weeks. At Week 96, significantly more patients receiving DRV/r than LPV/r achieved virological response <50 copies/ mL (ITT-TLOVR) (79% vs 71%), establishing both non-inferiority (p<0.001) and superiority (p=0.012) of DRV/r over LPV/r. DRV/r response rates were superior to LPV/r in patients with high baseline viral load (p=0.023) and low baseline CD4 count (p=0.009). Fewer patients receiving DRV/r than LPV/r discontinued due to an adverse event (AE) (4% vs 9%). Grade 2–4 treatment-related diarrhoea occurred less frequently with DRV/r than with LPV/r (4% vs 11%, p=0.0006). In addition, for treatment-experienced patients in TITAN at 96 weeks, approximately half Global Antiviral Journal Volume 4, Supplement 1 as many patients receiving DRV/r had virological failure than those receiving LPV/r (14% vs 26%) and less virological failures receiving DRV/r than LPV/r developed primary PI (18% vs 35%) or NRTI (10% vs 28%) mutations. Etravirine (ETR; INTELENCE™) is a next-generation NNRTI with activity against NNRTI-resistant HIV-1. In the ongoing Phase III DUET-1 and -2 trials, treatment-experienced patients with NNRTI resistance were randomised 1:1 to receive ETR (n=599) or placebo (n=604), with investigatorselected NRTIs and optional enfuvirtide. At Week 48, virological response (50 copies/mL; ITT-TLOVR) was significantly greater with ETR than with placebo (61% vs 40%, p<0.0001), and also for patients with ≥2 active agents (78% vs 67%, p=0.0022) or no active agents in their background regimen (46% vs 6%, p<0.0001). Fewer patients in the ETR arm experienced an AIDSdefining illness/death than those in the placebo arm (6% vs 10%, p=0.0408). The safety and tolerability profile of ETR was comparable to placebo with the exception of rash, which was mild-to-moderate in severity and resolved on continued treatment. The efficacy and safety of the investigational nextgeneration NNRTI, TMC278, is being investigated in TMC278-C204, a five-year Phase IIb dose-ranging study in treatment-naïve patients. Patients were randomised 1:1:1:1 to receive efavirenz 600mg qd (EFV; n=89) or TMC278 qd (25mg [n=93], 75mg [n=95] or 150mg [n=91]). Patients also received 2 NRTIs. TMC278 demonstrated a potent and sustained virological response (<50 copies/mL; ITT-TLOVR) to 96 weeks similar to that achieved with EFV (71–76% vs 71%). Mean CD4 cell count increased continuously from baseline during the trial and was similar across groups at Week 96 (146–172 vs 160 cells/mm3). TMC278 demonstrated tolerability benefits over EFV, with lower incidences of rash (TMC278 combined group: 9% vs EFV: 21%, p<0.01), nervous system (31% vs 48%, p<0.01) and psychiatric events of interest (16% vs 21%, p=0.26) and smaller lipid elevations. HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies With their efficacious and well-tolerated dosing regimens, DRV, ETR and TMC278 take antiretroviral therapy into a new era, further enhancing the lives of HIV-1-infected patients. An anti-tuberculosis drug, TMC207, is also in development, expanding the range of new anti-infective agents. Abstract 47 Low Potential for Class Related Toxicity of Next Generation Nucleotide Analog GS-9148 AS Ray1, G LaFlamme1, D Grant1, R Fisher2, AC Carey1, JE Vela1, RL Mackman1, and T Cihlar1 1 Gilead Sciences, Inc., Foster City, CA, USA; 2 Vitron, Inc., Tucson, AZ, USA BACKGROUND: Nucleoside and nucleotide reverse transcriptase inhibitors (N(t)RTI) are an important component of highly active antiretroviral therapy (HAART). However, the durability of N(t) RTI containing regimens can be limited by the emergence of drug resistance and side-effects. Two N(t)RTI class related mechanisms of toxicity include mitochondrial damage caused by inhibition of the mitochondrial DNA (mtDNA) polymerase gamma and nephrotoxicity resulting from transporter mediated renal accumulation. GS-9131 is a phosphonamidate prodrug of the 2’-deoxyadenosine monophosphate analog GS-9148. GS-9148 has been shown to have a favorable resistance profile against common N(t) RTI mutations (including M184V, L74V, K65R, and thymidine analog resistance mutations). In order to better understand the potential for side-effects, GS9148 was tested for N(t)RTI class related toxicities. METHODS: Effects on mtDNA were assessed in vitro in up to 21 day assays with the liver cell line HepG2. Depletion of mtDNA and chromosomal DNA were measured by sequential DNA hybridization with probes for the cytochrome b and 18S rRNA genes, respectively. Inhibition of mtDNA polymerase gamma by nucleoside triphosphate analogs was 53 measured using activated calf thymus DNA as a template. Accumulation of N(t)RTI were studied in cells overexpressing the human influx transporters organic anion transporter 1 and 3 (hOAT1 and hOAT3) and the efflux transporter multidrug resistance protein 4 (MRP4) as well as freshly isolated human renal cortex tissue. In order to determine the net effect of interactions with renal transports on kidney accumulation and renal clearance in vivo, beagle dogs were administered [14C] GS-9131 at a dose of 3 mg/ kg (10 µCi/kg) and kidney tissue and urine collected over 24 hours. RESULTS: Treatment of HepG2 cells with GS-9148 did not result in a selective depletion of mitochondrial DNA and GS-9148-diphosphate was found to have an inhibitory constant (IC50) of >300 µM for mtDNA polymerase gamma. GS-9148 was transported 60- to 100-fold less efficiently than acyclic nucleotides by the renal uptake transporter hOAT1. GS-9148 was also an inefficient substrate for hOAT3. In contrast, GS-9148 was effectively transported by the renal efflux transporter MRP4. Consistent with molecular transport studies, GS-9148 was found to have low net active tubular secretion and low accumulation in the kidneys of dogs administered [14C] GS-9131. GS9148 was also inefficiently taken up by human renal cortex tissue in vitro. CONCLUSIONS: The aging of the HIV infected population and the potential of treatment for the duration of a normal life span raise new challenges for HIV therapy. In addition to potency and durability, the long term safety profile of HAART combinations are of critical importance. GS-9131 is a promising next generation N(t)RTI currently in early clinical trials with a low potential for N(t)RTI class related toxicities caused by mitochondrial damage or renal accumulation. 54 Abstract 48 Mechanisms Associated with Delayed HIV RT Chaintermination E Tchesnokov1, A Obikhod2, RF Schinazi2, and M Götte1 1 McGill University, Montreal, Quebec, Canada; 2 Emory University School of Medicine/VA Medical Center, Atlanta, GA, USA BACKGROUND: Entecavir (ETV) is a potent antiviral drug to treat infection with the hepatitis B virus (HBV). Recent studies have shown that ETV has antiHIV activity and can select for the M184V mutation in HIV-1 reverse transcriptase (RT), which limits its clinical use in HIV/HBV co-infected individuals. However, the mechanism of drug action remains elusive. ETV is a guanosine nucleoside analogue that contains a 3’-hydroxyl group. Thus, the incorporated ETV-5’-monophosphate (ETV-MP) does not act as a classic chain-terminator. Here we utilized various biochemical tools to elucidate the anti-HIV mechanism of ETV and its implications with respect to major resistance pathways against established nucleoside analogue RT inhibitors (NRTIs) METHODS: We used a variety of biochemical approaches, including enzyme kinetics, binding studies, and high resolution footprinting experiments to assess the major contribution to ETV-mediated inhibition of DNA synthesis. RESULTS: Incorporation of ETV-MP at position n causes RT pausing at positions n and n+3. Increasing concentrations of natural dNTP pools at positions n+1 and n+4 can eventually overcome enzymatic pausing; however, incorporation of the natural nucleotide at position n+4 is severely compromised. Kinetic measurements revealed a subtle 8-fold decrease in efficiency of nucleotide incorporation at position n+1 when ETV-terminated primers are compared with the natural counterpart, while nucleotide incorporation at position n+4 is reduced by three orders of magnitude (1230-fold). High-resolution Global Antiviral Journal Volume 4, Supplement 1 footprinting experiments show that complexes with HIV-1 RT and a primer/template that mimics the latter situation are as stable as complexes that contain natural primers. Rather, ETV-MP forces the enzyme to slide away from the 3’-end of the primer at position n+3, which provides a plausible mechanism for such “delayed chain-termination”. RT enzymes with thymidine analogue associated mutations (TAMs) can efficiently excise the incorporated ETV-MP at position n, as demonstrated for several established NRTIs. However, ETV is fully sensitive against TAMs containing HIV variants, which shows that the additional nucleotides at positions n+1 to n+3 protect the inhibitor from excision. CONCLUSION: The results of this study demonstrate that “delayed chain-termination” at position n+3 is the dominant mechanism of action of ETV. The combined data provide a rationale for the development of ETV-like, delayed chain-terminators as anti-HIV compounds that can evade the excision mechanism. Abstract 49 Acyclovir as an HIV-RT Inhibitor in Herpesvirus-infected Human Tissues L Margolis National Institute of Child Health and Human Development, Bethesda, MD, USA Background: The emergence of drug-resistant viruses necessitates the discovery of new efficient anti-HIV drugs. that should also be inexpensive, in order to be available in limited-resource countries. Such an efficient and inexpensive drug exists for human herpesviruses (HHVs): acyclovir (ACV) is particularly active against α-HHVs but also inhibits, although with lower potency, the replication of the β- and γ-HHVs. Its exclusive anti-herpetic specificity is primarily based on the unique ability of herpesviral kinases to phosphorylate ACV into active ACVtriphosphate, a property that makes ACV inactive HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies against other viruses, including HIV-1. Here, we report on a newly found direct activity of ACV on HIV1 in human tonsillar, lymph node, rectal, and genital tissues coinfected with various HHVs, and we provide a molecular mechanism for this phenomenon. Methods: Tonsillar, lymph node, rectal and cervical tissues as well as isolated cells were infected ex vivo with HIV-1 and treated with ACV or ACV derivatives. Viral replication was evaluated with real-time PCR and ELISA, and HIV-1 RT activity was measured in a cell-free system using a gel-based assay. Results: ACV efficiently suppresses replication of HIV-1of various subtypes in human tonsillar, lymph node, vaginal, and rectosigmoid tissues ex vivo if and only if they carry one or several herpesviruses. ACV did not inhibit HIV replication in an HHV-free cell line. However, when HHV-6-infected cells were added to the HIV-1-infected cultures, ACV became an HIV1 suppressor. ACV-triphosphate (ACV TP; formed in HHV-infected tissues), but not ACV, suppresses RT; it is incorporated into the HIV DNA nascent chain, causing its obligate termination. Phosphorylated ACV-based prodrug suppresses HIV in HHV-free cells. Conclusions: In human tissues coinfected with various herpesviruses. including low-pathogenic HHV-6 and HHV-7, ACV is activated into an HIV RT inhibitor and suppresses HIV-1. ACV-phosphate prodrugs bypass the requirement for phosphorylation by herpesvirus kinases to become HIV-1 inhibitors. ACV may directly suppress HIV in humans already infected with one or several herpesviruses that can activate ACV. Our results suggest that ACV may be therapeutically beneficial for various HIV-1-infected patients, since the majority of humans are already infected with HHV-6, often together with other HHVs that activate ACV. However the use of ACV against HSV-2 in HIV-infected patients may select for HIV-1 variants resistant to other antiretrovirals. ACV and its prodrugs represent a new class of HIV RT inhibitor. In general, the use of one resident microbe to convert an inactive compound into an inhibitor of another pathogen constitutes a new therapeutic 55 principle against human pathogens, in particular HIV. In the case of ACV, its low toxicity and low cost make it potentially applicable for HIV treatment, possibly in combination with other drugs. Abstract 50 Comparison of Two Human Pancreatic Cell Lines for Predicting Mitochondrial Toxicity by Nucleoside Analogs L Bassit, J Cohen, A Obikhod, E Lee, and RF Schinazi Center for AIDS Research, Department of Pediatrics, Emory University School of Medicine, and Veterans Affairs Medical Center, Atlanta, GA, USA Background: Mitochondrial (mt) toxicity is a growing cause for concern in preclinical candidate failures, as well as post-market drug withdrawals. Several predictive cell-based models for cytotoxicity are currently available for experimental evaluations of novel antiviral agents, but different parameters can influence their performance and predictability in humans. For prediction of pancreatitis by NRTI, few studies have examined mt cytotoxicity in human pancreatic adenocarcinoma cellular systems. Therefore, in this study, PANC1 cells of pancreatic ductal adenocarcinoma origin were compared with the CAPAN1 cells of both ductal and acinar origin as well as the formation of NTP, to enable monitoring for early pancreatic cytotoxicity. Methods: Mt toxicity of D-D4FC (dexelvucitabine), tenofovir disoproxil fumarate (TDF), and tenofovir (TFV) was evaluated for their potential DNA polymerase γ-inhibiting capacity, using the two cell lines. Cells were seeded at 5,000 c/well and test compounds were added at various concentrations. Following 14 days incubation, total cellular DNA was isolated, and both mtDNA and nuclear DNA (nDNA) were amplified in parallel in a real-time PCR. 56 Results: There was a clear difference between the cell lines with respect to both mt- and nDNA polymerase γ-inhibition by NRTI. CAPAN1 cells appeared to be more sensitive since D-D4FC caused selective mtDNA toxicity with an IC50 of 4.3 µM; toxicity was ~8-fold higher as compared to PANC1 cells. In addition, TDF caused reduction in both mt- and nDNA, with IC50 in CAPAN-1 cells of 9.0 ± 1.8 µM and 8.0 ± 1.0 µM, respectively; it was slightly more toxic for mtDNA (~2-fold increase) and even more toxic for nDNA (~3fold increase). TFV at 10 µM did not show signs of toxicity for either mt- or nDNA in the two pancreatic cell lines. ddC used as a positive control, showed both mt and nDNA toxicity effects in CAPAN1 cells with an IC50 of 0.2 ± 0.2 µM and 5.8 ± 0.9 µM, respectively, whereas in PANC1 cells, ddC showed selective toxicity for mtDNA (9-fold lower as compared to CAPAN1). No apparent toxicity was observed for the negative control 3TC at 50 µM in the two cell lines. The NTP levels for 3TC, TDF, and D-D4FC at 10 µM after 4 hr incubation was overall higher in PANC1 than CAPAN1 cells. All three drugs were efficiently phosphorylated in PANC1 and CAPAN1 cells. The intracellular concentration of TDF-DP was on average 25 times higher than concentration of either 3TC-TP or D-D4FC-TP in both cell lines. These results suggest that in these cell lines, there was no direct relationship between drug phosphorylation and mitochondrial toxicity. Conclusions: CAPAN1 cells appear to provide the most reliable in vitro assessment of NTRI-associated mt toxicity for early prediction of pancreatitis. The results may help to explain cellular specific toxicity observed by nucleoside analogs used in HIV treatment. Based on these findings, we conclude that CAPAN1 is a more suitable cell line to study NRTI antiretroviral agents than PANC1 cells. Global Antiviral Journal Volume 4, Supplement 1 Abstract 51 ARTEMIS: Efficacy and Safety of Darunavir/ritonavir (DRV/r) 800/100 mg Once-daily vs Lopinavir/ritonavir (LPV/r) in Treatment-naïve, HIV-1-infected Patients at 96 Weeks D Jayaweera1, R Ortiz2, A Mills3, J Morales-Ramirez4, I Dierynck5, V Sekar6, C Vanden Abeele5, G De La Rosa7, and L Lavreys5 1 University of Miami, Miami, FL, USA; 2 Orlando Immunology Center, Orlando, FL, USA; 3 Los Angeles, CA, USA; 4 Clinical Research Puerto Rico, Inc., San Juan, PR, USA; 5 Tibotec BVBA, Mechelen, Belgium; 6 Tibotec Inc., Yardley, PA, USA; 7 Tibotec Therapeutics, Bridgewater, NJ, USA Background: ARTEMIS is a randomized, controlled, open-label, 192-week Phase III trial in HIV-1infected treatment-naïve patients, comparing oncedaily darunavir/r (DRV/r) to lopinavir/r (LPV/r), both given with fixed-dosed tenofovir/emtricitabine (TDF/ FTC). We report the efficacy, safety and tolerability of DRV/r and LPV/r at Week 96. Methods: Patients with HIV-1 RNA (VL) ≥5000 copies/mL were randomized to DRV/r 800/100mg once-daily or LPV/r 800/200mg total daily dose, plus TDF/FTC (300/200mg) and stratified by baseline viral load and CD4 count. The primary objective was to evaluate for non-inferiority of DRV/r to LPV/r (delta -12%) in confirmed VL <50 copies/mL (ITTTLOVR) at Week 48. DRV/r superiority (delta 0%) was assessed if non-inferiority was shown. Safety and tolerability were also assessed. 95% CI: 2, 15; P<.001) and demonstrating superiority (P=.012). Among patients with high baseline VL (≥100,000 copies/mL), virologic response was significantly higher for DRV/r (76%) than LPV/r (63%) (difference: 14%; 95% CI: 2, 25; P=.023). In patients with lower baseline VL, virologic response for DRV/r and LPV/r was 81% and 75%, respectively (difference: 5%; 95% CI: -2, 13; P=.174). Among patients with low baseline CD4 cell count (<200 cells/mm3), response rates were significantly higher for DRV/r (79%) than LPV/r (65%) (difference: 14%; 95% CI: 4, 24; P=.009). In patients with higher baseline CD4 cell count, 79% of DRV/r patients and 75% of LPV/r patients achieved VL <50 copies/mL (difference: 4%; 95% CI: -4, 12; P=.345). Fewer DRV/r than LPV/r patients (4% vs 9%) discontinued treatment due to adverse events. Fewer DRV/r than LPV/r patients (4% vs. 11%) had grade 2-4 treatment-related diarrhea. Grade 2-4 treatmentrelated rash occurred infrequently (3% DRV/r vs 1% LPV/r). DRV/r patients had smaller mean increases in triglycerides and total cholesterol (0.1 and 0.6 mmol/L) than LPV/r patients (0.8 and 0.9 mmol/L); levels remained below NCEP cut-offs for DRV/r for both parameters. Conclusions: In these treatment-naïve patients at 96 weeks, a significantly higher proportion of patients receiving DRV/r 800/100mg once-daily achieved VL <50 copies/mL compared with LPV/r, proving statistical non-inferiority and superiority of DRV/r. DRV/r was associated with lower rates of diarrhea and smaller mean increases in triglycerides and total cholesterol. Results: 343 and 346 patients were randomized to DRV/r and LPV/r, respectively (N=689). Overall, the mean baseline VL was 4.85 log10 copies/mL and the median baseline CD4 count was 225 cells/mm3. Significantly more patients on DRV/r (79%) than LPV/r (71%) achieved VL <50 copies/mL at Week 96, confirming DRV/r non-inferiority (difference: 8%; HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 57 Abstract 52 Etravirine (ETR; TMC125) Demonstrates Favorable Efficacy and Safety in the Phase III DUET Trials Regardless of Geographic Location C Katlama1, PM Girard2, P Junod3, M Peeters4 L Leopold5, B Baeten4, and G De Smedt4 1Hôpital Pitié-Salpêtrière, Paris, France; 2 Hôpital SaintAntoine, Paris, France; 3 Clinique Médicale du Quartier Latin, Montreal, Canada; 4 Tibotec BVBA, Mechelen, Belgium; 5 Tibotec Inc., Yardley, PA, USA BACKGROUND: Prevalence and incidence of HIV varies considerably across regions. HIV clade, ethnic influences on metabolism and comorbidities can vary according to region and may affect a drug’s efficacy and safety. It is therefore important to evaluate antiretrovirals in different regions. Etravirine (ETR) is a next-generation NNRTI which demonstrated potent, durable activity against HIV-1 in the multinational, cross-regional Phase III DUET trials over 48 weeks. We present pooled DUET 48-week efficacy and safety data according to geographic location. percentage of patients in Europe (69% for ETR and 44% for placebo) and Latin America (62% and 43%) achieved VL<50 copies/mL than patients in North America (55% and 35%). Importantly, the added benefit of ETR use versus placebo was similar across regions. The percentage of patients achieving VL<50 and <400 copies/mL was significantly higher for ETR versus placebo, irrespective of geographic location. Change in CD4 cell count from baseline was greater for ETR versus placebo in all regions. In general, adverse events (AEs) were comparable across groups and regions. However patients in North America reported a higher incidence of grade 3/4 AEs in both groups (40% each group). CONCLUSIONS: In DUET, while slight differences in efficacy and safety were observed across regions, patients receiving ETR+BR achieved statistically significantly higher virologic responses than those receiving placebo+BR, irrespective of geographic location. In addition, ETR was well tolerated, with incidence and severity of AEs comparable to placebo, regardless of location. METHODS: Patients with documented NNRTI resistance, ≥3 primary PI mutations and viral load (VL)>5000 copies/mL were randomized 1:1 to receive ETR 200mg or placebo, both twice-daily, with a background regimen (BR) of darunavir/low-dose ritonavir, investigator-selected NRTIs and optional enfuvirtide. The primary efficacy endpoint was the proportion of patients achieving VL<50 copies/mL. Safety and tolerability were also assessed. RESULTS: Patients were recruited from 185 centers across North America (n=524), Latin America (n=329), Australia (n=15), Europe (n=331) and Thailand (n=4). 599 and 604 patients received ETR+BR or placebo+BR, respectively. In general, baseline demographics were comparable across groups and locations, although a higher percentage of males were recruited in North America. A higher 58 Global Antiviral Journal Volume 4, Supplement 1 Parameter,% Efficacy VL<50 copies/mL VL<400 copies/mL Mean(SE) change in CD4 cell count from baseline Safety Any AEs Any Grade 3/4 AEs Any SAEs Discontinuations Europe ETR+BR Placebo+BR (n=162) (n=169) Geographic Location* Latin America ETR +BR Placebo+BR (n=162) (n=167) North America ETR+BR Placebo+BR (n=266) (n=258) 69‡ 77‡ 44 49 62§ 75‡ 43 54 55‡ 66‡ 35 41 105(7.5) 87(9.2) 121(10.8) 91(9.3) 79(6.3) 47(5.1) 96 29 20 6 96 33 23 5 95 25 20 6 97 31 22 4 97 40 20 9 95 40 25 8 Europe: France, Germany, Italy, Spain, Belgium, UK, Netherlands, Poland, Portugal; Latin America: Brazil, Argentina, Mexico, Panama, Chile, Puerto Rico; North America: United States, Canada. *Number patients in Thailand and Australia was too low to allow separate subgroup analyses; ‡p<0.0001;§p=0.0002. Abstract 53 Pharmacokinetics and Short-term Safety and Efficacy of Once-daily Etravirine Without and With Once-daily Darunavir/ritonavir in Antiretroviral-naïve HIV-1 Infected Adults J Lalezari1, E DeJesus2, O Osiyemi3, P Ruane4, R Ryan5, B Polsonetti6, TN Kakuda5, and J Witek6 1 Quest Clinical Research, San Francisco, CA, USA; 2 Orlando Immunology Center, Orlando, FL, USA; 3 Triple O Medical Services Inc., West Palm Beach, FL, USA; 4 Medical Practice of Peter Ruane, Los Angeles, CA, USA; 5 Tibotec, Inc, Yardley, PA, USA; 6 Tibotec Therapeutics, Bridgewater, NJ, USA; 6Yale-New Haven Hospital, New Haven, CT, USA; 7Tibotec, Therapeutics, Bridgewater, NJ, etravirine plus tenofovir/emtricitabine without and with darunavir/ritonavir in HIV-infected patients. Methods: HIV-1-infected antiretroviral-naïve adults without evidence of HBV/HCV-co-infection or resistance to study drugs were eligible. Patients received etravirine 400mg once-daily with tenofovir/ emtricitabine 300/200mg once-daily for 14 days, added darunavir/ritonavir 800/100mg oncedaily for Days 15–28, and discontinued etravirine for Days 29–42. Pharmacokinetic sampling was performed over 24 hours on Day 14 for etravirine, and Day 28 for etravirine, darunavir and ritonavir. All doses were administered following a meal. Pharmacokinetic parameters were determined using a noncompartmental model with extravascular input and evaluated by least squares mean (LSM) ratios Background: Etravirine has a terminal elimination half-life of 30–40 hours, and is a candidate for oncedaily dosing. In healthy volunteers, etravirine AUC was similar, Cmax was 44% higher and Cmin was 25% lower for once-daily versus twice-daily dosing. Oncedaily darunavir/ritonavir was shown to be effective and well-tolerated in antiretroviral-naïve patients. This multicenter, open-label Phase IIa trial evaluates pharmacokinetic and short-term safety and efficacy of HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 59 with 90% confidence intervals (CI). Fasting labs were evaluated in each phase. Results: Twenty-three patients (20 men) enrolled: 9 black, 9 white and 5 Hispanic; mean age 35.9 years; mean baseline viral load (VL) 4.2 log10 copies/ mL; median baseline CD4 403 cells/mm3. LSM and 90% CIs for etravirine with and without darunavir/ ritonavir demonstrated equivalence. Mean (SD) AUC24h, Cmax and Cmin for etravirine without and with darunavir/ritonavir were 10410 (4186) and 10720 (5459) ng*h/mL, 790 (287) and 801 (327) ng/mL, and 233 (130) and 236 (168) ng/mL, respectively. Mean (SD) AUC24h, Cmax and Cmin for darunavir were 76130 (22080) ng*h/mL, 7008 (1514) ng/mL, and 1049 (616) ng/mL, respectively. Mean (SD) AUC24h, Cmax and Cmin for ritonavir were 4128 (1854) ng*h/mL, 465 (231) ng/mL, and 27 (21) ng/mL, respectively. Darunavir pharmacokinetics were slightly higher and ritonavir pharmacokinetics slightly lower relative to historic controls (ARTEMIS week 4 PK substudy). Mean VL decline was 1.7 log10 copies/mL at Day 14 (n=21) and 2.0 log10 copies/mL at Day 42 (n=20). Median CD4 increase was 56 cells/mm3 (Day 42). Most common treatment-emergent AEs were nausea (n=4), headache (n=3), rash (n=3), and flatulence (n=2). No serious or grade 3/4 AEs were reported; no AEs led to discontinuation. Median (range) changes from baseline were 11 (-64, 58), -1 (-30, 8), 1.5 (-39, 49), and 32 (-88, 166) mg/dL for total cholesterol, HDL, LDL, and triglycerides, respectively, over 42 days. Conclusions: Addition of once-daily darunavir/ ritonavir to once-daily etravirine had no significant impact on etravirine pharmacokinetics, and exposure to etravirine was adequate with and without darunavir/ritonavir. The impact on metabolic parameters was small when etravirine was given with or without darunavir/ritonavir. Pharmacokinetic data and short-term safety and efficacy support further clinical investigation of etravirine 400mg once-daily in HIV-1-infected patients. 60 Abstract 54 Activation of Different Cell Death Pathways in Patients on Antiretroviral Therapy with Long-term Viral Suppression but Unfavorable Immunologic Response E Negredo1, M Massanella2, J Puig1, N Pérez-Álvarez1,3, B Clotet1,2, and J Blanco2 1 Lluita contra la SIDA Foundation, Germans Trias i Pujol University Hospital, Barcelona, Spain; 2 Irsicaixa Foundation, Germans Trias i Pujol University Hospital, Barcelona, Spain; 3 Statistics and Operations Research Department, Technical University of Catalonia, Barcelona, Spain Background: Apoptosis and other mechanisms of cell death play a determinant role in CD4+ cell loss in HIV-infected patients. Highly active antiretroviral treatment (HAART), by reducing viral replication, induces a decline in the level of apoptosis that contributes to immune recovery. However, some patients present a discordant response to treatment, maintaining or reducing CD4+ cell counts despite control of viral replication under detectable levels. Methods: A cross-sectional, descriptive, comparative study was performed to evaluate the role of apoptosis and other cell death pathways in CD4 T cell recovery. We included 168 patients on HAART with undetectable viral load (VL< 50 copies/mL) for more than 2 years; 50 of them were considered as “Discordant” (unfavorable immunologic response: increase <100 CD4+ cells/mm³ with respect to baseline or CD4+<350 cells/mm³) and 118 as “Concordant” (favourable immunologic response). Activation markers were measured in fresh blood, while cell death was analyzed in PBMC cultured for 0, 1 or 4 days in the absence or the presence of the pancaspase inhibitor ZVAD-fmk. Multicolor flow cytometry was used combining antibodies, the vital dye PI and the mitochondrial probe DIOC6. Global Antiviral Journal Volume 4, Supplement 1 Results: No baseline differences between discordant and concordant patients were observed regarding gender, mean age, mean time with HIV infection, current antiretroviral therapy (42.9% on PI; 57.1% on NNRTI) and percentage of HCV co-infection (33.9%). Levels of total death (defined as DIOC low cells) in CD4+ cells were statistically higher in discordant than in concordant patients at day 0 (p<0.001), day 1 (p<0.001) and day 4 (p<0.001). Significant differences were also found in a detailed analysis of cell death pathways. Necrosis (defined as DIOC low, PI+ cells) and global apoptosis (defined as DIOC low PI- cells) were higher (p<0.001) in discordant patients. Similarly increased levels of intrinsic (p<0.001) and extrinsic apoptosis (p<0.001), determined by the mitochondrial protection induced by ZVAD at day 1, were also higher in discordant patients. No significant differences were observed when we measured the same parameters in CD8+ cells except levels of necrosis at day 1 that were higher in discordant subjects (p=0.005). Consistently activation markers (CD95 or HLA-DR) showed more marked differences in the CD4 than in the CD8 T cell compartment. Conclusion: The higher levels of activation and CD4+ T cell death in discordant patients suggests a key role of the different mechanisms of cell death, including necrosis and apoptosis, in immune recuperation. Therapeutic interventions aimed to reduce cell death (by reducing toxicity or residual viral replication) may be required in discordant patients. Abstract 55 Safety and Tolerability of the Lopinavir/ritonavir (LPV/r) Tablet in Comparison to other Protease Inhibitors (PIs): The SAPIKAT trial UF Bredeek1, B Williams1, R Rode2, T Sparrow2, and R Stryker2 1 Special Immunology Associates, Tucson, AZ, USA; 2 Abbott Laboratories, Chicago, IL, USA Background: LPV/r tablet has improved characteristics compared to soft gel capsules (SGC). Side effects and tolerability of LPV/r tablets compared with other PIs have not been fully evaluated. Methods: Open-label, prospective evaluation of the tolerability and Quality of Life (QOL) of LPV/r tablets dosed once daily (QD) in virologically suppressed subjects switching the PI component of their antiretroviral therapy (ART). Patient-reported outcomes were evaluated at entry and 12 weeks using the Medical Outcomes Study-HIV (MOSHIV) Health Survey, the Center for Epidemiologic Studies–Depression (CES-D) questionnaire and a modification of the ACTG Symptoms Distress Module (SDM). CD4+, HIV-1 viral load (VL), fasting lipids, and chemistries were assessed at entry and 24 weeks. Analysis was done using paired t-tests and Wilcoxon signed rank tests. Results: A total of 60 subjects on atazanavir/ ritonavir (N=33), saquinavir/ritonavir (N=3), nelfinavir (N=2), fosamprenavir/ritonavir (N=11), or LPV/r SGC (N=11) containing regimens switched to LPV/r tablets QD. Reasons for switch were Grade 3-4 total bilirubin (N=33), desire for QD dosing (N=16), or withdrawal of the LPV/r SGC formulation (N=11). 7 subjects withdrew prior to week 24 for intolerance; 4 subjects were lost to follow-up. At week 12 or premature discontinuation, no statistically significant changes were observed in MOS-HIV domain scores or CES-D (P ≥ 0.188). However, mean symptom scores HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 61 were increased for diarrhea (0.6±1.55; P=0.010), bloating (0.5±1.53; P=0.011) and dizziness (0.4±20; P=0.013). Lack of a refrigeration requirement positively impacted QOL in 36% of subjects. 21% felt QOL was improved by lack of food requirements. 60% preferred LPV/r tablet, 25% preferred their previous PI, and 15% had equal preference. Reasons for premature discontinuations were not collected as part of the study. Bilirubin normalized in those subjects with baseline elevations. No significant changes from baseline to week 24 were observed for VL and CD4 count. Mean increases in total cholesterol (14.5±40.00 mg/dL; P=0.014), LDL (9.0±26.76 mg/dL; P=0.034) and triglycerides (99.4±370.98 mg/dL; P=0.067) were seen; however, HDL was not significantly changed. 2 patients were started on statin therapy during the study. with TDF/FTC. The TITAN trial (TMC114-C214) evaluated LPV/r versus DRV/r 600/100 mg BID in treatment-experienced patients with HIV-1 RNA >1000 copies/mL, in combination with an optimized background regimen consisting of nucleoside +/- nonnucleoside reverse transcriptase inhibitors. Conclusions: The change of the PI to LPV/r in otherwise unchanged ART allowed direct measure of LPV/r tablet tolerability without confounding therapy. HIV-1 suppression was maintained and despite an early increase in gastrointestinal related symptom scores, LPV/r was generally well tolerated, and preferred by most patients. RESULTS: The overall results from ARTEMIS and TITAN at Week 96 were as follows: Abstract 56 TITAN – Week 96, HIV RNA <400 ITT 66.4% 57.6% 8.8% ITT non-VF censored 88.4% 76.1% 12.4% Analysis of Virologic Endpoints Versus Standard Intent to Treat, in ARTEMIS and TITAN Trials A Hill1, L Chen2, F Tomaka2, H Huong3, and R DeMasi2 1 Liverpool University, Liverpool, UK and Tibotec, Mechelen, Belgium; 2 Tibotec R&D, Yardley, USA; 3 Janssen-Ortho, Toronto, Canada BACKGROUND: Phase 3 trials of new antiretrovirals are normally analyzed using Intent to Treat (ITT) methods, with any patient discontinuing randomized treatment counted as a failure. The ARTEMIS trial (TMC114-C211) evaluated lopinavir/ritonavir (LPV/r) versus darunavir/ritonavir (DRV/r) 800/100 mg QD in treatment naïve patients, in combination 62 METHODS: In the ARTEMIS and TITAN trials, the primary endpoint of “virologic failure” was HIV RNA >50 copies/mL (ARTEMIS) and >400 copies/mL (TITAN), as well as prior discontinuation for adverse events or other reasons. In this analysis, ITT analyses were repeated, excluding any discontinuation for non-virologic reasons (ITT non-VF censored). A MEDLINE search of other clinical trials was conducted to estimate the proportion of virologic endpoints in Intent to Treat analyses of other trials. Trial DRV/r LPV/r Difference ARTEMIS – Week 96, HIV RNA <50 ITT 79.0% 70.8% 8.2% ITT non-VF censored 92.8% 87.2% 5.6% p value <0.05 <0.05 <0.05 <0.05 In the ITT analyses of ARTEMIS and TITAN, 57/173 (33%) and 103/226 (46%) of the primary endpoints at Week 96, respectively, were virologic failures. Of the 57 virological endpoints in ARTEMIS, 13 (21%) were patients whose HIV RNA was never suppressed below 50 copies/mL (4 DRV/r, 9 LPV/r), while 44 (79%) were rebounds in HIV RNA after initial suppression below 50 copies/mL (17 DRV/r, 27 LPV/r). Of the 103 endpoints in TITAN, 68 (66%) were patients whose HIV RNA was never suppressed below 400 copies/mL (22 DRV/r, 42 LPV/r) while 39 (34%) were rebounds in HIV RNA after initial suppression below 400 copies/mL (13 DRV/r, 26 LPV/r). The MEDLINE search of 17 trials (n=8582) showed the percentage of Global Antiviral Journal Volume 4, Supplement 1 virologic endpoints in ITT analyses was 25% for trials of naïve patients and 74% for trials of experienced patients. CONCLUSIONS: In ARTEMIS and TITAN, there was a statistically significant increase in efficacy for DRV/r versus LPV/r, both in the primary ITT analysis, and when only observed virologic endpoints were included. If discontinuation rates for adverse events or other reasons are high in HIV clinical trials, additional analyses including only virologic endpoints are needed to confirm the efficacy conclusions. Abstract 57 Improvement of the Transglycosylation Reaction for the Synthesis of β-D-3’-Azido2’,3’-Dideoxypurine Nucleoside Analogs by Conventional and Microwave Assisted Heating SJ Coats1, H-W Zhang2, L Bondada2, M Detorio2, J Mellors3, and RF Schinazi2 1 RFS Pharma LLC, Tucker, GA, USA; 2 Emory Univ./VA Med. Ctr. Atlanta, GA USA; 3 Univ. of Pittsburgh/VA Med. Ctr., Pittsburgh, PA, USA synthetic approach starting with readily available 5’-protected AZT and its corresponding U-analog, AZU. Transglycosylation reaction conditions were modified using microwave-assisted synthesis and coupled with an analytical HPLC-MS method that allowed for rapid determination of yield of the desired beta isomer. Results: Treatment of 5’-protected AZT or AZU with an appropriate purine in the presence of Lewis acid produced a mixture of α and β isomers. Removal of the 5’-protecting group and subsequent purification provided the pure β-3’-azidopurine nucleosides in reasonable yield (30-40%). Reaction time, temperature, solvent, Lewis acid, and various additives were rapidly optimized using microwaveassisted synthesis. A series of novel 3’-azido-2’,3dideoxypurine analogs have been synthesized using this new method that have potent anti HIV-1 in human peripheral blood mononuclear (PBM) cells and low cytotoxicity in PBM, CEM and Vero cells. Conclusions: The transglycosylation reaction with conventional heating or microwave assisted synthesis proved to be an efficient means of synthesizing new, structurally diverse 3’-azido-2’,3’-dideoxypurine analogs, several of which have potent and selective anti-HIV activity and are undergoing further preclinical evaluation. Background: Recently we discovered that β-D-3’-azido-2’,3’-dideoxyguanosine has potent anti-HIV activity and a more favorable resistance profile compared to AZT. In contrast to 3’-azido pyrimidine nucleosides, 3’-azido-purine nucleosides are challenging to synthesize. Transglycosylation of 3’-azido pyrimidine nucleosides with purine bases offers an improved and facile synthetic approach, but yields of the desired beta isomer are low (4-27%). We sought to improve beta isomer yield from the transglycosylation approach. Methods: Traditionally, 3’-azidopurine analogs are prepared by low-yielding multistep nucleoside modification approaches. We investigated a simpler HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 63 Abstract 58 The Phtalocyanine Prototype Derivative Alcian Blue: The First Synthetic Agent with Selective Anti-human Immunodeficiency Virus Activity Due to Its Lectinlike Properties KO François, C Pannecouque, D Schols, and J Balzarini Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, Leuven, Belgium Background: The envelope of HIV consists of two subunits: the surface gp120, and the transmembrane gp41. Both units are highly glycosylated, which is essential for the virus to escape immune surveillance. Carbohydrate binding agents (CBAs), such as the plant lectins Hippeastrum hybrid agglutinin (HHA) and Urtica dioica agglutinin (UDA) bind to the glycans that are present on the envelope of HIV and inhibit the viral entry process. Recently, pradimicin A (PRM-A), an antifungal nonpeptidic antibiotic was described to possess lectin-like properties, to bind to HIV gp120 and to be able to efficiently prevent HIV infection. Another nonpeptidic compound, Alcian Blue (AB), a phtalocyanine derivative, also has antiviral activity against HIV. We investigated further its antiviral and lectin-like properties. Methods: A time-of-addition experiment was performed to determine the target of AB in the viral life cycle. An AB-resistant virus strain was selected in CEM T-cell cultures, under dose-escalating AB concentrations. The entire envelope region was sequenced after DNA extraction of the proviral DNA. Cross-resistance against other CBAs was determined in antiretroviral assays, based on syncytia formation. The antiviral activity was determined against several DNA and RNA viruses, and its ability to inhibit syncytium formation was observed using a co-cultivation assay between Sup-T1 cells and persistently infected Hut-78/HIV cells. Using a virusbinding-assay in the presence of mannan, the lectinlike properties of AB were investigated. 64 Results: Based on time-of-addition experiments, AB was determined to be an entry inhibitor. Its antiviral acitivity against HIV-1 in CEM T-cells was 4.8 ± 2.6 µg/ml, while the AB-resistant virus strain was about 10-fold more resistant to AB. AB was able to inhibit syncytium formation between persistently HIV-1infected Hut-78/HIV cells and Sup-T1 cells with an EC50 of 8.0 ± 2.8 µg/ml. AB-resistant virus, selected under increasing concentrations of AB, showed up to 4 mutations in N-glycosylation motifs (NXS/T, with X any amino acid, except P) of gp120, resulting in the deletion of 4 N-glycans. A similar mutational pattern was also observed in previous analogous experiments with the CBAs HHA, UDA, 2G12, cyanovirin and PRM-A. Virus-binding assays in the presence of mannan clearly indicated that AB, like the CBAs HHA and PRM-A, has a preference for high-mannose type sugars. Conclusions: Here we provide evidence for the antiviral and lectin-like properties of the nonpeptidic compound Alcian Blue. Its mode of action is comparable with the mode of action of the CBAs HHA, UDA and PRM-A. AB may be important as lead compound in the development of novel antiviral drugs targeting the envelope glycoproteins of HIV. Abstract 59 Long-time Virologic Efficacy of Boosted Double Protease Inhibitor Therapy H Knechten, C Höhn, J Vachta, and P Braun PZB Aachen, Germany Background: Double-protease inhibitor regimens have been shown to be a therapeutic option for patients with treatment failure or adverse events. In 2005 we presented at the IAS-conference following retrospective analysis “24 weeks follow-up with ATV and SQV/r in protease inhibitor (PI) experienced HIV1-infected patients”. The aim of this retrospective Global Antiviral Journal Volume 4, Supplement 1 evaluation is to examine the long-time efficacy of such double PI regimens. Methods: 144 weeks follow up of 12 PI-experienced patients starting with boosted ATV/SQV (IAS, Rio de Janeiro, 2005; Abstract: WePe12.9C11). Patients had a median HIV-RNA of 206 copies/ml and mean CD4 counts of 569 ± 390 cells/µl. The average number of therapies was 3.8 with a mean duration of 70.8 months. Furthermore we analysed virological and immunological parameter of 7 patients starting later on with a boosted double PI regimen (Lopinavir/ Saquinavir n=4; or Atazanavir/Saquinavir n= 3) (N=1 with 3TC). Therapy success was defined as a viral load ≤ 40 copies/ml. Results: 11 patients who started with boosted ATV/ SQV reached week 144. 1 patient was lost to follow up. 4 had an undetectable viral load, 1 was non-adherent, 2 changed therapy due to side effects (ikterus and buffalo hump) and 2 due to virological failure. 6 of 7 patients of the second group reached week 144 yet. 6 Patients had a viral load below the limit of detection, increasing CD4 counts and the regimens were well tolerated. 1 patient was non-adherent. Conclusion: Although double PI Regimens are no longer standard of care for patients with limited therapy options these regimens seem to be effective for a high a number of patients in this retrospective longtime evaluation. Nevertheless further exploration of these regimens with regard to metabolic side effects and quality of life is warranted, especially in the era of new drug classes. HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies Abstract 60 Etravirine Demonstrates a Favorable Safety and Tolerability Profile at Week 48 in the Pooled DUET Trials Irrespective of Gender, Age, Ethnicity and Weight JV Madruga1, EG Kallas2, J Sampson3, F Mazzotta4, M Peeters5, K Janssen5, E Lefebvre6, and B Woodfall5 1 Centro de Referência e Treinamento DST/AIDS, SãoPaulo, Brazil; 2 Federal University of São Paulo, São Paulo, Brazil; 3 Research & Education Group, Portland, Oregon, US; 4 Hosp. S M Annunziata, Firenze, Italy; 5 Tibotec BVBA, Mechelen, Belgium; 6 Tibotec, Amsterdam, the Netherlands BACKGROUND: Etravirine (ETR; TMC125) demonstrated favorable safety and tolerability in treatment-experienced, HIV-1-infected patients in the phase III DUET trials over 48 weeks. We present a sub analysis of safety data from pooled 48-week DUET subgroup analyses. METHODS: Patients were randomized 1:1 to ETR (200mg bid) or placebo, both in combination with a background regimen (BR) of darunavir/low-dose ritonavir, investigator-selected NRTIs and optional enfuvirtide. Pre-specified subgroup analyses of the effect of gender, age, ethnicity and weight on incidence/severity of adverse events (AEs; including laboratory AEs) were conducted on pooled 48-week data. RESULTS: 599 and 604 patients received ETR and placebo, respectively. Baseline characteristics were similar across treatment groups. The incidence of any AE and grade 3 or 4 AEs were generally comparable across subgroups and between treatments. For Hispanic patients, however, the incidence of grade 3 or 4 AEs, serious AEs (SAEs) and discontinuations due to AEs with ETR + BR was higher than with placebo + BR. Caucasian patients receiving ETR reported the lowest incidence of SAEs. Patients aged >55 years receiving placebo experienced a higher incidence 65 of SAEs compared with other age groups. A higher incidence of rash occurred in the ETR group in women (30%) than in men (18%). However, no differences in severity of rash or rate of discontinuation were observed in men versus women. No clinically relevant differences were observed for other AEs. CONCLUSIONS: In this 48-week subgroup analysis, the only clinically relevant difference in AEs was increased rash in women versus men. Findings in the small number of Hispanic patients in this analysis require further exploration in larger datasets. Overall ETR + BR demonstrated favorable safety and tolerability versus placebo + BR, irrespective of gender, age, ethnicity and weight. Any AE (%) Subgroup (N ETR, placebo) Gender Male (539, 535) Female (60, 69) Ethnicity Caucasian (373, 376) Black (70, 70) Hispanic (60, 66) Other* (29, 27) Age, years 18–40 (111, 116) >40–55 (412, 399) >55 (76, 89) Weight, kg‡ ≤62.8 (149, 151) >62.8–72.0 (151, 170) >72.0–80.3 (137, 145) >80.3 (162, 138) Grade 3/4 AEs (%) SAEs (%) ETR (N=599) Placebo (N=604) ETR (N=599) Placebo (N=604) ETR (N=599) Placebo (N=604) 95.9 96.7 96.3 94.2 33.6 30.0 35.0 34.8 20.0 16.7 24.3 15.9 96.2 94.3 95.0 96.6 95.5 95.7 97.0 96.3 31.4 37.1 41.7 24.1 35.4 44.3 22.7 40.7 16.6 24.3 28.3 17.2 23.4 25.7 16.7 33.3 96.4 95.6 97.4 98.3 94.7 98.9 33.3 33.5 31.6 35.3 34.3 37.1 22.5 18.7 21.0 21.6 21.8 32.6 97.3 96.7 97.1 93.2 95.4 97.6 95.9 94.9 36.2 34.4 37.2 25.9 38.4 34.7 31.0 35.5 25.5 19.2 21.9 13.0 26.5 23.5 19.3 23.9 *Including Asian patients; ‡Categories were done by weight quartiles. 66 Global Antiviral Journal Volume 4, Supplement 1 Abstract 61 Adherence to Darunavir/ritonavir (DRV/r) and Lopinavir/r (LPV/r) in Treatment-naïve HIV Patients in ARTEMIS TM Mills1, P Chetchotisakd2, R DeMasi3, L Chen3, E Smets4, V Sekar3, S Spinosa-Guzman4, and E Lefebvre5 1 Los Angeles, CA, USA; 2 Khon Kaen University, Khon Kaen, Thailand; 3 Tibotec Inc, Yardley, PA, USA; 4 Tibotec BVBA, Mechelen, Belgium; 5 Tibotec, Amsterdam, the Netherlands Background: ARTEMIS is a controlled Phase III trial examining efficacy and safety of DRV/r qd vs LPV/r in treatment-naïve HIV patients. This analysis examined patient-reported adherence and its association with other data collected to week 48. Methods: The M-MASRI questionnaire assessed self-rated adherence by percentage of doses of DRV/r and LPV/r taken during the past 30 days. Rates were transformed into a binary variable (adherent [>95%] and non-adherent [≤95%]). At week 48, confirmed virologic responses and AEs were tabulated over time by adherence. Results: Overall adherence was high; however, 48 (15%) of DRV/r patients and 58 (18%) LPV/r patients had week-4 to week-48 mean adherence ≤95%. Response rates (<50 copies/mL, ITT-TLOVR) were higher in the adherent vs non-adherent groups (OR: 0.5 (0.3, 0.8)), with a smaller difference in response for DRV/r (DRV/r: 87% vs 79%; LPV/r: 85% vs 69%). Non-adherent patients reported more AEs. At week 12, 28% of DRV/r and 23% of LPV/r non-adherent patients reported GI AEs, compared with 7% and 13%, respectively of adherent patients. Reported GI events dropped steeply after week 12, making it difficult to assess impact on adherence after this time. Total distress scores from a list of 39 symptoms were higher in non-adherent versus adherent patients in both arms. Adherence to both DRV/r and LPV/r differed by race (Black patients, DRV/r: 23% non-adherent; HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies LPV/r: 38% non-adherent; non-Blacks, 12% and 13% non-adherent, respectively). Self-reported missed doses due to symptoms (P<0.01), and plasma drug concentrations (P<0.01) correlated with adherence, suggesting validity of this assessment. Conclusion: Non-adherence to LPV/r compromised virologic response more than non-adherence to DRV/r. Non-adherence was correlated with AEs (particularly GI), and symptoms or symptom-related perceived distress associated with ARV therapy, suggesting factors other than convenience are also significant drivers of adherence. Abstract 62 Irreversible Pepsin Fraction (IPF) Displays Significant Antiretroviral Activity via Specific Novel Cytokine Stimulation In Vitro Investigation of Activity on Human Lymphocytes H Zhabilov1, M Selbovitz, D Miller2, and J Gibbs 1 Immunotech Laboratories, CA, USA; 2 AIDS Institute, NY, USA Background: Resistance to all commercially available antiretroviral (ARV) agents within all classes has been reported. The occurrence of multiclass resistance remains high, with 20% of infected individuals developing resistance to two or more classes within six years of initiating treatment, and 10% of newly diagnosed infections already resistant to at least one class in the U.S. Multi-class resistance is even more prevalent in disenfranchised patient populations, whose rates of successful adherence to even the most simplified regimens available remains prohibitively low. Other mechanisms to treat HIV infection are sorely needed. IPF, like other natural autoantibody based fractionated proteins, has an affinity to pathogenic binding and simultaneously produces effects of immune homeostasis in the 67 presence of replacativly competent HIV. IPF has shown significant antiretroviral activity via immune stimulatory pathways in vitro, notably helper T1 cells elaborate cytokines INFy, IL-2. These cells selectively promote cell-mediated immune responses that are disadvantageous to viral replication with selection for the pathogenesis of resistant profiles of minority subspecies. Methods: Flow cytometric analysis of these cells was conducted using DC monoclonal antibodies and Annexin-V. A Biacore assay system that measures changes in the surface mass concentration was used to determine interactions between IPF molecules and CD4+ cells. Changes were expressed in resonance units (RU), with one RU representing a change in concentration of 1 pg/ mm. T cells were purified from peripheral blood mononuclear (PBMC) cells using anti-body coated magnetic beads. Results: Laboratory analysis indicates that IPF is able to mediate maturation of dendrites cells in vitro, as determined by up-regulation of MHC class-ll, CD86 and CD83 molecules, regulate pro-inflammatory cytokines IL-12 and INFy, and enhanced T-cells stimulatory capacity. Observable characteristics include modulation of complement activation, saturation of Fc receptors on macrophages, and suppression of various inflammatory mediators, including cytokines and chemokines. IPF demonstrated increased synthesis of Th-1 cells. IPF displayed spontaneous binding with gp41 when prepared for gel electrophoresis, and subsequent fusion inhibition of HIV with CD4+ cells and increased gp41 and gp120 antigenic activity. Virus-specific CD8 cells were stimulated. Flow cytometric analysis revealed apoptosis in CD4+ cells and stimulation of virus-specific CD8 cells. Conclusions: IPF appears to modulate helper T1 cells’ expression of elaborate cytokines INFy, IL-2, which selectively promote cell-mediated immune response and subsequently stimulate cytotoxic lymphocytes. These lymphocytes have a prominent role in the host’s immunologic response to HIV infection. Proteins encoded by these pathogens enter 68 the endogenous pathway for antigen presentation and are expressed on the surface of the infected cell as a complex with class l MHC- proteins. IPF appears to present a novel mechanism to reduce viral burden and stimulate innate immune responses to the virus for patients with significant antiretroviral resistance. ABStract 63 Successful Use of Dual Therapy with Etravirine and Raltegravir in Patients with HIV Infection G Pierone1, JA Mieras1, DE Bulgin2, AJ Balconis2, and CD Kantor1 1 Treasure Coast Infectious Disease Consultants, FL, USA; 2 AIDS Research & Treatment Center of the Treasure Coast, FL, USA Background: New classes of antiretroviral agents and advancements in existing medication classes have greatly expanded the strategic options for the selection of antiretroviral cocktails. Some patients have side effects and or viral resistance to both nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs). For these patients, a NRTI-sparing and PI-sparing regimen might be useful. Since etravirine and raltegravir have become available, we have used dual therapy with this combination in patients with resistance and or side effects from PIs and NRTIs. Methods: We reviewed the medical records of all patients in our clinic who had been prescribed etravirine and raltegravir as dual therapy. Chart reviews included collection of data that included previous antiretroviral regimens, resistance assays, CD4+ lymphocyte counts and HIV RNA levels, lipid profiles, and the reasons for the selection of this regimen. Results: We identified 20 patients who were prescribed dual therapy with etravirine and raltegravir and had follow up viral load and CD4+ lymphocyte Global Antiviral Journal Volume 4, Supplement 1 count testing. Antiretroviral medication intolerance was the primary reason for change in 17 patients and multi-drug resistant virus in the other three patients. Eight of these 20 patients had undetectable viral load at time they started this regimen. Two patients stopped therapy because of side effects. Of the remaining 18 patients, two experienced an early rebound in viral load on this regimen and had treatment intensified. In retrospect, these two patients had diminished etravirine activity based on archived resistance testing and prior treatment history. Of the remaining 16 patients, two had detectable viremia with HIV RNA readings of 71 and 128 copies/ mL respectively and continue dual therapy. The remaining 14 patients have viral load <48 copies/ mL at a mean follow up of 16.6 weeks (range 4 to 60 weeks). Conclusion: These retrospective short-term results suggest that the use of dual therapy with etravirine and raltegravir may be useful in the management of HIV infection. This regimen deserves further study in patients with NRTI or PI associated side effects and/or viral resistance. Abstract 64 HIV-1 Co-receptor Use in Heavily Treatment-experienced Spanish Patients JR Arribas1, S Moreno2, A Rivero3, F RodriguezArrondo4, M Leal5, and R Sanchez-de la Rosa6 patients infected with only CCR5-tropic (R5) HIV1 detectable. Information on the epidemiology of HIV-1 co-receptor use has been provided by clinical trials of entry inhibitors (TORO [Whitcomb et al. CROI 2007], MOTIVATE [Coakley et al. Targeting HIV Entry Workshop 2006], and ACTG 5211 [Wilkin et al. Clin Infect Dis 2007]) and a limited number of observational studies. However, little is known about the different patterns of tropism distribution in specific countries. The objective of our study was to evaluate the distribution of R5, dual/mixed-tropic (D/M), and CXCR4-tropic (X4) HIV-1 in heavily treatment-experienced patients in Spain. Methods: All treatment-experienced Spanish patients who participated in the maraviroc expanded access program (A4001050), the named patient program in Spain (July 2007 to January 2008), or the ALLEGRO Study (A4001077; January to June 2008) and who were screened for HIV tropism were included. Tropism was determined using the original/standard Trofile™ assay (Monogram Biosciences, South San Francisco, CA, USA). Results: Co-receptor use was evaluated for 865 treatment-experienced patients. In 125 cases (14%), tropism results were non-determinable or nonreportable owing to various reasons, including one sample found to be HIV-2. Of the remaining samples, 491 (66%) had an R5 tropism result, 23 (3%) were X4, and 226 (31%) had a D/M tropism result. Conclusions: The distribution of HIV-1 tropism in heavily treatment-experienced patients in Spain was similar to that reported for heavily treatmentexperienced patients in other regions where subtype B predominates. 1 Hosp. Universitario La Paz, Madrid, Spain; 2 Hosp. Universitario Ramón y Cajal, Madrid, Spain; 3 Hosp. Universitario Reina Sofía, Córdoba, Spain; 4 Hosp. de Donostia, Donostia, Spain; 5 Hosp. Universitario Virgen del Rocío, Seville, Spain; 6 Pfizer Medical Unit, Madrid, Spain Background: Maraviroc (MVC) has been approved for use in treatment-experienced adult HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 69 Abstract 65 Cost-effectiveness of Maraviroc plus Optimized Background Therapy in Treatment-experienced Patients with R5 HIV-1 in Spain S Moreno1, JM Llibre2, I Lekander3, B Martí4, R Sánchez-de la Rosa4, and MA Casado5 1 Hospital Ramón y Cajal, Madrid, Spain; 2 Fundació Lluita contra el SIDA, Hospital Germans Trial y Pujol, Barcelona, Spain; 3 i3 Innovus, Stockholm, Sweden; 4 Pfizer Medical Unit, Madrid, Spain; 5 Pharmacoeconomics & Outcomes Research Iberia, Madrid, Spain Background: Maraviroc (MVC) is a first-inclass oral CCR5 antagonist, which prevents CCR5tropic (R5) HIV-1 from entering CD4+ cells. Phase 3 pivotal studies (MOTIVATE 1 & 2) of MVC 300mg administered twice daily (BID) added to optimized background therapy (OBT) in viremic, treatmentexperienced patients with CCR5-tropic (R5) HIV1 showed a clinically and statistically significant reduction in viral load and increase in CD4+ cell count compared to placebo (PBO) plus OBT at 48 weeks. MVC was well tolerated, resulting in a low rate of discontinuation due to adverse events (AEs). The objective of this study was to evaluate the costeffectiveness in the Spanish setting of MVC + OBT vs PBO + OBT in a treatment-experienced patient population similar to that recruited to the MOTIVATE studies. (ARV) regimen costs were derived from local official sources. Non-ARV drug costs were obtained from published literature and a national cost database. All costs were expressed in 2007 euros (€). The annual discount rate for both costs and effects was set to 3.0%. The main outcomes were cost per life years gained (LYG) and cost per quality-adjusted life years (QALY) gained. To assess the uncertainty of the results, oneway sensitivity analyses and a probabilistic sensitivity analysis (PSA) were performed. Results: The results of the economic analysis showed that adding MVC to OBT increases LYG by 0.952 years and QALY by 0.909. Total cost were €291,663 for maraviroc plus OBT and €268,012for OBT alone with an incremental cost of €23,651. The resulting cost per LYG was €24,852 and the cost per QALY gained was €26,026. The model seemed robust for variation in key parameters, ranging from €-3,500 (cost saving) to €35,000 per QALY in the deterministic sensitivity analyses. The results from the PSA indicate that the probability of the cost per QALY falling below €30,000 is 95%. Conclusions: Based on the superior clinical efficacy results from the combined analysis of MOTIVATE 1 & 2 trials, our analysis indicates that maraviroc 300mg BID in combination with OBT is a clinically valuable and cost-effective option for the treatment of ARV-experienced patients infected with R5 HIV-1 in Spain. Methods: A lifetime deterministic cohort model from the hospital perspective was developed based on combined data from MOTIVATE 1 & 2. In the base case, treatment duration reflects the clinical trial follow-up—ie, a lifetime effect of 1 year’s treatment is assessed. Clinical data, cohort characteristics, probability of treatment success, rate of CD4+ cell increase, and the link to disease states and probability of AEs (both opportunistic infections and drug associated) were taken from the trials and published literature, as appropriate. Other input parameters were taken from published sources. Antiretroviral 70 Global Antiviral Journal Volume 4, Supplement 1 Abstract 66 Once Daily Darunavir/ritonavir: A Single Centre Cohort Experience C Scott, A Teague, M Bower, B Gazzard, M Nelson Chelsea & Westminster Hospital NHS Foundation Trust, London, UK BACKGROUND: Darunavir (DRV) is a novel nonpeptidic protease inhibitor (PI) with activity against wild type and protease inhibitor resistant HIV-1 when boosted with low dose ritonavir. We describe our experience of using once daily darunavir/ritonavir (900/100mg) in both treatment naïve and treatment experienced patients with no baseline darunavir resistance within the Chelsea & Westminster Hospital cohort. METHODS: This prospective observational study followed a cohort of patients who received DRV/r (900/100mg) od plus reverse transcriptase inhibitors (RTIs). RTIs consisted of nucelos(t)ide analogues. CD4 count, viral load (VL), and routine safety bloods where measured at baseline, 0, 12, 24, 36 & 48 week intervals. with DRV/r (900/100) was 78% at week 12, 94% at week 24, 100% at week 36 and 100% at week 48. The proportion (number) of patients achieving HIV RNA <50 copies/mL in the treatment naïve group on treatment was 53% at week 12 (16/30), 68% at week 24 (13/19), 88% at week 36 (7/8) and 100% at week 48 (2/2). In the treatment experienced group the proportion of patients with HIV RNA <50 copies/mL was at 89% at week 12, 91% at week 24, 86% at week 36 and 100% at week 48. The overall percentage of patients in our cohort with VL<50% receiving DRV (900/100) at weeks 12,24, 36 and 48 was 83%, 87%, 87% and 100%. 25 (13%) patients discontinued therapy and 5 patients were lost to follow up. Main reasons for discontinuation were GI intolerance (8), elevated liver function tests (5) and patient discontinuation (5). CONCLUSIONS: In this cohort of treatment naïve and experience patients with no background PI resistance, once daily darunavir/ritonavir (900/100mg) is both effective in terms of virological suppression and well tolerated. RESULTS: 187 patients commenced RTI + DRVr (900/100). All patients had a phenotypic resistance test confirming no baseline resistance to DRV. 32/187 patients were naïve to anti-retroviral therapy. 155/187 patients were anti-retroviral treatment experienced and were switched to DRVr (900/100). Reasons for DRV use in these patients were virological failure on current regimen, toxicity on current therapy or physician decision to switch. 108/155 patients switched with VL<50 copies/mL. Data were available for 187, 140, 72 and 32 patients up to weeks 12, 24, 36 and 48 respectively: In the treatment naïve group receiving DRVr (900/100) the median CD4 cell count was 106 and VL 29564 copies/mL. The proportion of patients on treatment achieving HIV RNA <500 copies/mL HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 71 Pharmacology and Drug Metabolism HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 73 Abstract 67 Etravirine (ETV, TMC-125) Plasma Levels in Decompensated Liver Disease: a Case Report M Aboud, S Castelino, and R Kulasegaram Harrison Wing, Department of GU/HIV Medicine, Guy’s and St Thomas’ Hospitals NHS Foundation Trust, London, UK Introduction: Etravirine is a 2nd generation non-nucleoside reverse transcriptase inhibitor (NNRTI). In combination with other active agents, it has been shown to be effective in reducing HIV1 viral load in triple-class resistant individuals (DUET). Data exists on ETV plasma levels in mild to moderate liver impairment- manufacturer comment: ‘caution is advised in patients with moderate hepatic impairment’. But no data exists for severe liver impairment or decompensated liver cirrhosis- here the manufacturer says: ‘its use is therefore not recommended’. Case: A 50 year old African lady was diagnosed HIVpositive in 1996, and has been on various antiretroviral combinations since 1998; various switches were made for side effects, toxicity, virological failure and drug resistance. In January 2008, she developed ascitis; investigation for this revealed liver cirrhosis and portal vein thrombosis. Biopsy was not performed initially because of anticoagulation. The aetiology was uncertain, but presumed to be related to prior antiretroviral therapy. In January 2008, her regimen of Tenofovir, ddI and boosted FosAmprenavir was switched to Tenofovir, ddI, and ETV initially, and then as planned ddI was switched to boosted Darunavir. As soon as ETV TDM became available (7 months later) the levels were measured, and her ETV trough level was found to be 60 times the recommended. The patient did not experience any worsening of symptomatology or liver function, nor indeed any ETV-related side effects over this period. ETV was discontinued; 2 weeks later her ETV trough level was 17.9 times the target. HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies Discussion: Etravirine levels in severe liver impairment or decompensated liver cirrhosis have not previously been reported. This case showed excessive plasma levels of ETV at standard dosing; high levels persisted even after 2 weeks of discontinuation. This was not associated with any clinical adverse outcomes. ETV levels were very high in this patient with severe liver impairment; this calls for extreme caution with its use in such circumstances. We will continue to measure her ETV levels off ETV therapy. Abstract 68 Virologic Efficacy of Dual-boosted Once-daily (QD) Atazanavir, Fosamprenavir, and Ritonavir (ATV/FPV/RTV): 48 Week Results UF Bredeek1, B Williams1, R Feldman1, and T Lancaster2 1 Special Immunology Associates, Tucson, AZ, USA; 2 GlaxoSmithKline, RTP, NC, USA BACKGROUND: Dual-boosted PI regimens have been evaluated primarily in HIV-infected subjects with limited treatment options, and most combinations studied include LPV/RTV or SQV as one of the components. The use of dual-boosted PI regimens in subjects with limited PI resistance is not well studied. METHODS: This was an open-label, prospective study evaluating ATV 400 mg, FPV 1400 mg and RTV 100 mg QD in subjects who were PI naïve or PI experienced and with HIV RNA >1,000 c/mL. Baseline genotyping of HIV-1 protease and RT were performed on stored plasma samples (HIV GenoSURETM, Labcorp). RESULTS: 19 of 22 subjects completed the study through 48 weeks (2 withdrew consent; 1 was lost-tofollow-up). At baseline (BL), most subjects (91%) were Caucasian males with median HIV-1 RNA and CD4+ 75 of 4.8 log10 c/mL and 253 cells/mm3, respectively; 11 subjects had baseline resistance mutations, 6 of whom had 1 to 8 PI mutations (1 of whom had a major PI mutation [L90M]). The duration of prior PI therapy ranged from 1 to 8.5 years, and the PIs used were NFV, SQV, LPV/RTV, or IDV. After 48 weeks of ATV/FPV/RTV, 55% (11/20) and 85% (17/20) maintained HIV-1 RNA <50 c/mL and <400 c/mL (M=F), respectively. The median increase in CD4+ cell counts was 221 cells/mm3. Of the 3 subjects with HIV-1 RNA >400 copies/mL between weeks 24 and 48, none had developed resistance to PIs. The median fasting lipid values (% change from BL) at 48 weeks were 171 mg/dL (+28%) for triglycerides, 201 mg/dL (+34%) for cholesterol, 42 mg/dL (+30%) for HDL, 134 mg/dL (+34%) for LDL, and 4.6 (-3%) for total cholesterol:HDL ratio. instantaneous inhibitory potential is clinically very relevant and depends on the Hill slope. Here we use viral dynamics simulations to investigate the effect of multi-round signal amplification on the estimation of Hill slopes. CONCLUSIONS: In this study, ATV/FPV/RTV QD provides virologic suppression and had a moderate impact on lipids in subjects with limited PI resistance through 48 weeks of therapy. Larger studies are needed to further evaluate this regimen as a QD option for subjects who have developed RT or NNRTI resistance. • No net influx of uninfected cells (only proliferation); • free virions are depleted exclusively by primary or secondary infection; • cellular proliferation and death rates for uninfected cells are combined in a single parameter r. For infected cells, this combined rate gradually declines to reflect the increasing cytopathicity during the experiment; • infection is read out by counting the relative number of infected cells (rather than viral RNA count) Abstract 69 A Kinetic Simulation of In Vitro Pharmacodynamics for HIV Drugs E Gustin, H Ceulemans, K Cao-Van, K Van Acker, and HL De Bondt Tibotec BVBA, Generaal De Wittelaan L11B3, 2800 Mechelen, Belgium BACKGROUND: The study of HIV viral dynamics has progressed and yielded insights into the pharmacodynamics of HIV inhibitors. Ferguson (2001) predicted that the steepness of the concentration-response curve for instantaneous inhibition is overestimated by cumulative viral inhibition assays. As shown by Shen (2008), the 76 METHODS: An in vitro viral dynamics model was constructed using a set of 3 differential equations. The simulated dose-response curves were compared to experimental data obtained from an antiviral assay derived from Hertogs (1998) with experimental dose-response curves collected at day 2, 3 and 4 after infection. The in vitro model differs from the simplest in vivo viral dynamics model (Funk, 2001) in the following points: Modeling was done in MathCAD and Berkeley Madonna. Readouts were simulated at 1, 2, 3 and 4 days. RESULTS: The model below is consistent with the experimental data d/dt U(t) = r × U(t) - kinf × U(t) × V(t) d/dt I(t) = r × (1-V/ Vmax) × I(t) + kinf × U(t) × V(t) d/dt V(t) = P(C) × I(t) - kinf × (U(t)+I(t)) × V(t) with U, uninfected cells; I, infected cells; V, free virions; r, combined growth-death rate of uninfected cells; P(C), viral production rate at drug concentration C as calculated from the Hill equation; Vmax = P(C)/kinf. Other parameters are r = 0.6931/day, kinf = 0.00025 Global Antiviral Journal Volume 4, Supplement 1 µL/day and P(no inhibitor) = 250 virions/(cell×day). All used parameters values are in agreement with Funk (2001). These simulations are also confirmed by experimental observations such as possible apparent virulence stimulation by the inhibitor (seen as slightly negative % inhibition values). The multiple viral lifecycle rounds yield an increased steepness of the concentration-response curve (compared to instantaneous inhibition). This sharpens the signal and requires a transformation to derive Hill slopes. CONCLUSION: A thorough understanding of the kinetics of the multi-round assay through viral dynamics modeling elucidates the complex relationship between the amplified readout of the in vitro multi-round assay and the underlying inhibition of viral replication, which can be modeled as a Hill equation. REFERENCES: Funk et al., JAIDS 2001; 26(5):397. Ferguson et al., TiPS 2001; 22(2):97. Shen et al., Nat Med 2008; 14(7):762. Hertogs et al., AAC 1998; 42(2):269. Abstract 70 Lower Levels of Nucleoside Analog Triphosphates in Primary Human Macrophages Compared to Human Lymphocytes Could Impair Potency of Antiretroviral Drugs in Human Viral Reservoirs C Gavegnano, E Fromentin, and RF Schinazi Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, GA, USA Background: A significant obstacle in eradication of human immunodeficiency virus (HIV-1) are latently infected viral reservoirs. Macrophages are a latently infected viral reservoir, and cellular pharmacology of antiretroviral therapy (ART) in macrophages directly impacts viral loads, resistance, eradication of systemic virus, and long-term survival. We sought to determine the intracellular bioavailability of nucleoside reverse transcriptase inhibitors (NRTI) in primary human macrophages and lymphocytes. Our plan is to eventually correlate the level of phosphorylated nucleoside analogs (active form of drug) with their known antiviral activity in lymphocytes and macrophages. Methods: Human macrophages and PBM cells were isolated using a Histopaque technique from buffy coats derived from healthy donors. Macrophages were stained with CD11b-APC and purity was greater than 99% as determined with FACS. Cells were exposed to 10 µM AZT, ABC, (-)-FTC, or TDF. Drugs and their phosphorylated derivatives were extracted from the cells and quantified with LC-MS/MS. Results: CBV-TP (active form of ABC) was quantified at 0.415 ± 0.182 µM in PBM cells and 0.010 ± 0.004 µM in macrophages. TFV-DP (active form of TDF) levels in PBM cells were 394.5 ± 86.6 µM in PBM cells versus 142.7 ± 121 µM in macrophages. (-)-FTC-TP HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 77 was detected at 35.3 ± 7.4 µM in PBM cells and 0.667 ± 0.116 µM in macrophages. AZT-TP was below the limit of detection, but AZT-MP was detected (79.6 µM in PBM cells versus 13.8 µM in macrophages). Conclusions: Levels of (-)-FTC-TP, CBV-TP, and TFV-DP were significantly lower in macrophages than human PBM cells (p < 0.05), suggesting that these nucleosides may be less potent in macrophages. Quantification of NRTI in both lymphocytes and macrophages as demonstrated herein provide the foundation for a comprehensive study to determine NRTI-TP levels across multiple donors and cell types. Abstract 71 Lack of Pharmacokinetic Interaction for Low and Normal Dose Zidovudine with Amdoxovir in HIV-1 Infected Individuals G Asif1, SJ Hurwitz1, PM Tharnish1, RL Murphy2, and RF Schinazi1 1 Center for AIDS Research, Emory University School of Medicine/VA Medical Center, Atlanta, GA, USA; 2 Northwestern University, Chicago, IL, USA BACKGROUND: Amdoxovir (DAPD) inhibits HIV-1 containing the M184V/I mutation in the pol region, and is rapidly absorbed and deaminated to the active metabolite, β-D-dioxolane guanosine (DXG). A recent in silico pharmacokinetic (PK)/enzyme kinetic study suggested that ZDV 200 mg bid may reduce toxicity without compromising efficacy relative to the standard 300 mg bid regimen (Hurwitz et al., AAC, in press). Therefore, an intense PK clinical study was conducted using DAPD 500 mg bid alone or with ZDV 200 or 300 mg bid. 3:1 to DAPD or placebo. Full plasma pharmacokinetic profiles were collected on days 1 and 10, and steady Cmin was collected on day 5. Complete urine sampling was performed on Days 9 (0-4 h, 4-8 h, 8-12 h), and during the 12 h dose interval following the next dose. A validated LC/MS/MS method was developed to measure DAPD, DXG, ZDV in plasma and ZDV-5’-Oglucuronide (GZDV) in the urine. Data were analyzed using non-compartment methods. RESULTS: Co-administration of DAPD with ZDV did not affect the systemic clearance (CL/F) (1.0 ± 0.4 and 2.2 ± 0.7 L.h-1.kg-1, respectively) and the renal clearance (0.26 ± 0.1 and 1.49 ± 0.5 (ZDV+GZDV) L.h-1.kg-1) of DXG or ZDV, respectively. There were no statistically significant differences (p > 0.05 using non-adjusted Student t-test) in the urine elimination of DXG + DAPD in the “DAPD only” treatment arm versus “DAPD and ZDV200” (p = 0.44); “DAPD only” treatment arm versus “DAPD and ZDV300” (p = 0.26); and “DAPD and ZDV200” versus “DAPD and ZDV300” (p = 0.7). There were no significant differences in the elimination of ZDV and its metabolite (GZDV) in the different treatment arms: “200 ZDV only” versus “DAPD and ZDV 200” (p = 0.20); “300 ZDV only” versus “DAPD and ZDV300” (p = 0.07); and “DAPD and ZDV200” versus “DAPD and ZDV300” (p = 0.79). CONCLUSIONS: Elimination pattern suggested no PK interactions between DAPD and ZDV in any treatment arm. Levels of GZDV in urine were not affected by co-administered DAPD. Prolonged studies with DAPD/ZDV (200 mg) are planned with larger cohorts. METHODS: Twenty-four subjects, not receiving antiretroviral therapy (12m, 12f; viral load, VL ≥ 5,000 copies/ml), were randomized to oral DAPD 500 mg bid, DAPD 500 mg plus ZDV 200 or plus ZDV 300 mg bid for 10 days. In each arm, subjects were randomized 78 Global Antiviral Journal Volume 4, Supplement 1 Abstract 72 Metabolism of Highly Active and Selective 3’-Azido-2’,3’Dideoxypurine Nucleosides in Primary Human Lymphocytes and MT-2 Cells A Obikhod1, E Fromentin1, M Detorio1, SJ Coats2, N Sluis-Cremer3, JW Mellors3, and RF Schinazi1 1Emory University/VA Medical Center Atlanta, GA, USA; 2 RFS Pharma LLC, Tucker, GA, USA; 3 University of Pittsburgh, PA, USA Background: Nucleoside reverse transcriptase inhibitors (NRTI) are the backbone of combination anti-HIV-1 therapy. Understanding the metabolism of these therapeutics in different cell systems is critical to predicting their potency. NRTI can compete with endogenous nucleosides for cellular kinases which are required for conversion to the HIV reverse transcriptase active triphosphate form. In addition, variation in dNTP levels could lead to selective mutations and influence excision activity. We evaluated the effects of highly potent NRTI such as 3’-azido-2’,3’-dideoxyguanosine (AZG) and its 6-chloro prodrug (2-amino-6-chloro-3’-azido-2’,3’dideoxypurine-riboside; 6-Cl-AZG) on the dNTP pool in PBM cells and MT-2 cells to better understand the difference in anti-HIV activity in these cellular systems (EC50 = 0.36 µM and 1.64 µM, for AZG and 0.19 µM and 2.8 µM for 6-Cl-AZG, in PBM and MT-2 cells respectively). versus PBM cells (p = 0.1). However, the levels of dATP and dGTP were 26 and 100-fold higher in MT-2 cells than in PBM cells (p < 0.005) and the levels of dCTP and TTP were 10 and 20-fold higher in MT-2 cells than in PBM cells (p < 0.05). Increasing the concentration of AZG from 0 to 50 µM did not effect NTP levels. The chlorinated prodrug 6-ClAZG crossed the cell membrane in MT-2 cells, but its phosphorylation metabolites were not found in either cell line, suggesting its fast bioconversion to AZG-TP. The amount of intracellular 6-Cl-AZG was 15-fold higher than the amount of AZG itself after incubation for 20 min in MT-2 cells, as well as the amount of AZG-TP was 4 fold higher after incubation with 6-Cl-AZG than with AZG itself. Conclusion: AZG was successfully converted to its active metabolite AZG-TP in both cell types. Our study shows the chlorinated prodrug is transported faster through the cell membrane, however after 4 h the levels of AZG-TP formed were not significantly different in both cell type suggesting no major advantages of 6-Cl-AZG. Incubation of AZG at four different concentrations suggested that phosphorylation reaches steady state at 30 mM. The higher levels of dNTP in MT-2 decreases the ratio of AZG/dGTP and provides an explaination for the weaker antiviral potency of AZG in MT-2 cells. Method: AZG and 6-Cl-AZG were incubated for 4 h in human PBM and MT-2 cells at 1, 10, 30 and 50 µM. Cells were then washed with 1x PBS and metabolites were extracted with 60% methanol in water and analyzed by ion-pair chromatography coupled with positive mode MS/MS (Hypersil GOLD column; 2 mM ammonium phosphate buffer, 3 mM hexylamine (HMA) and acetonitrile). Results: The levels of AZG-TP were slightly lower when the incubation was performed in MT-2 cells HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 79 Author Index Author Abstract Page Author Abstract Page Aboud, M. . . . . . . . . . . . . . . . . . . . . 67. . . . . . . . . . . . . . . . . 75 Mtambo, A. . . . . . . . . . . . . . . . . . . . 26. . . . . . . . . . . . . . . . . 28 Arnold, E . . . . . . . . . . . . . . . . . . . . . . 6. . . . . . . . . . . . . . . . . 11 Murphy, RL. . . . . . . . . . . . . . . . . . . 45. . . . . . . . . . . . . . . . . 51 Auwerx, J. . . . . . . . . . . . . . . . . . . . . 32. . . . . . . . . . . . . . . . . 36 Nijhuis, M . . . . . . . . . . . . . . . . . . . . 33. . . . . . . . . . . . . . . . . 37 Bassit, L. . . . . . . . . . . . . . . . . . . . . . 50. . . . . . . . . . . . . . . . . 56 North, TW. . . . . . . . . . . . . . . . . . . . 14. . . . . . . . . . . . . . . . . 17 Berger, EA . . . . . . . . . . . . . . . . . . . . . 9. . . . . . . . . . . . . . . . . 13 Obikhod, A. . . . . . . . . . . . . . . . . . . 72. . . . . . . . . . . . . . . . . 79 Blanco, J. . . . . . . . . . . . . . . . . . . . . . 54. . . . . . . . . . . . . . . . . 60 Pakes, G. . . . . . . . . . . . . . . . . . . . . . . 5. . . . . . . . . . . . . . . . . . . 6 Boucher, CAB . . . . . . . . . . . . . . . . . 21. . . . . . . . . . . . . . . . . 24 Patil, M. . . . . . . . . . . . . . . . . . . . . . . 18. . . . . . . . . . . . . . . . . 20 Bredeek, UF. . . . . . . . . . . . . . . 55, 68. . . . . . . . . . . . . . 61, 75 Pierone, G . . . . . . . . . . . . . . . . . . . . 63. . . . . . . . . . . . . . . . . 68 Buckheit, Jr., RW. . . . . . . . . . . . . . 10. . . . . . . . . . . . . . . . . 14 Polsky, B. . . . . . . . . . . . . . . . . . . . . . 38. . . . . . . . . . . . . . . . . 43 Castor, TP . . . . . . . . . . . . . . . . . . . . 16. . . . . . . . . . . . . . . . . 18 Pop, M . . . . . . . . . . . . . . . . . . . 36, 40. . . . . . . . . . . . . . 40, 45 Chen, L. . . . . . . . . . . . . . . . . . . . . . . 56. . . . . . . . . . . . . . . . . 62 Ray, AS. . . . . . . . . . . . . . . . . . . . . . . 47. . . . . . . . . . . . . . . . . 53 Coats, S. . . . . . . . . . . . . . . . . . . . . . 57. . . . . . . . . . . . . . . . . 63 Sanchez-de la Rosa, R. . . . . . . 64, 65. . . . . . . . . . . . . . 69, 70 De Bondt, HL . . . . . . . . . . . . . . . . . 69. . . . . . . . . . . . . . . . . 76 Sandu, M. . . . . . . . . . . . . . . . . . . . . 27. . . . . . . . . . . . . . . . . 29 Dieterich, D. . . . . . . . . . . . . . . . . . . 37. . . . . . . . . . . . . . . . . 43 Scott, C. . . . . . . . . . . . . . . . . . . . . . . 66. . . . . . . . . . . . . . . . . 71 Divita, G. . . . . . . . . . . . . . . . . . . . . . . 4. . . . . . . . . . . . . . . . . . . 5 Siliciano, RF. . . . . . . . . . . . . . . . . . . . 7. . . . . . . . . . . . . . . . . 11 Dunkle, LM. . . . . . . . . . . . . . . . . . . 44. . . . . . . . . . . . . . . . . 51 Spearman, P . . . . . . . . . . . . . . . . . . . 3. . . . . . . . . . . . . . . . . . . 4 François, KO. . . . . . . . . . . . . . . . . . 58. . . . . . . . . . . . . . . . . 64 Stevenson, M. . . . . . . . . . . . . . . . . . . 1. . . . . . . . . . . . . . . . . . . 3 Gatell, J . . . . . . . . . . . . . . . . . . . . . . 35. . . . . . . . . . . . . . . . . 39 Swan, T. . . . . . . . . . . . . . . . . . . . . . . 41. . . . . . . . . . . . . . . . . 45 Gavegnano, C . . . . . . . . . . . . . . . . . 70. . . . . . . . . . . . . . . . . 77 van der Horst, C. . . . . . . . . . . . . . . 30. . . . . . . . . . . . . . . . . 35 Götte, M. . . . . . . . . . . . . . . . . . . . . . 48. . . . . . . . . . . . . . . . . 54 Wainberg, MA. . . . . . . . . . . . . . . . . 15. . . . . . . . . . . . . . . . . 17 Hartman, TL. . . . . . . . . . . . . . . . . . 17. . . . . . . . . . . . . . . . . 19 Watson, KM. . . . . . . . . . . . . . . . . . . 28. . . . . . . . . . . . . . . . . 29 Hazuda, D . . . . . . . . . . . . . . . . . . . . 13. . . . . . . . . . . . . . . . . 16 Wensing, AMJ. . . . . . . . . . . . . . . . . 29. . . . . . . . . . . . . . . . . 30 Hillier, S. . . . . . . . . . . . . . . . . . . . . . 31. . . . . . . . . . . . . . . . . 36 Wilkinson, J . . . . . . . . . . . . . . . . . . 12. . . . . . . . . . . . . . . . . 15 Hurwitz, SJ. . . . . . . . . . . . . . . . . . . 71. . . . . . . . . . . . . . . . . 78 Witek, J. . . . . . . . . . . . . . . . . . . . . . 53. . . . . . . . . . . . . . . . . 59 Jayaweera, D. . . . . . . . . . . . . . . . . . 51. . . . . . . . . . . . . . . . . 57 Wright, ER. . . . . . . . . . . . . . . . . . . . . 2. . . . . . . . . . . . . . . . . . . 3 Katlama, C. . . . . . . . . . . . . . . . 42, 52. . . . . . . . . . . . . . 49, 58 Zorrilla, C. . . . . . . . . . . . . . . . . . . . . 34. . . . . . . . . . . . . . . . . 38 Khoo, S. . . . . . . . . . . . . . . . . . . . . . . . 8. . . . . . . . . . . . . . . . . 12 Knechten, H . . . . . . . . . . . . . . . . . . 59. . . . . . . . . . . . . . . . . 64 Kožíšek, M. . . . . . . . . . . . . . . . . . . . 22. . . . . . . . . . . . . . . . . 25 Larder, B. . . . . . . . . . . . . . . . . . . . . . 19. . . . . . . . . . . . . . . . . 23 Lefebvre, E. . . . . . . . . . . . . 46, 60, 61. . . . . . . . . . . 52, 65, 67 Lori, F. . . . . . . . . . . . . . . . . . . . . . . . 11. . . . . . . . . . . . . . . . . 14 Margolis, L. . . . . . . . . . . . . . . . . . . . 49. . . . . . . . . . . . . . . . . 55 Massud, I. . . . . . . . . . . . . . . . . . . . . 23. . . . . . . . . . . . . . . . . 25 Mayers, D. . . . . . . . . . . . . . . . . . . . . 43. . . . . . . . . . . . . . . . . 50 McPhaul, T. . . . . . . . . . . . . . . . 24, 25. . . . . . . . . . . . . . 26, 27 Miller, D. . . . . . . . . . . . . . . . . . 39, 62. . . . . . . . . . . . . . 44, 67 Montaner, J. . . . . . . . . . . . . . . . . . . 20. . . . . . . . . . . . . . . . . 23 HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 81 Appendix HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 83 Late Breaker Abstracts HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 85 HIV DART 2008 Frontiers in Drug Development for Antiretroviral Therapies Late Breaker Abstracts Abstract 73 Genome-scale RNAi Screen for Host Factors Required for HIV Replication H Zhou1, M Xu1, Q Huang1, AT Gates1, XD Zhang2, JC Castle3, E Stec4, M Ferrer4, B Strulovici4, DJ Hazuda1, and AS Espeseth1 Department of 1 Virus and Cell Biology, 2 Biometrics Research, and 4 Automated Biotechnology, Merck & Co., Inc., West Point, PA USA; 3 Rosetta Inpharmatics LLC, a wholly owned subsidiary of Merck & Co., Inc., Seattle, WA , USA BACKGROUND: Human immunodeficiency virus (HIV)-1 depends on the host cell machinery to support its replication. The identification of host factors required for HIV infection may lead to the discovery of host targets for antiviral drug discovery efforts. The advent of genome scale siRNA screening technologies has made it possible to identify previously unknown genes involved in retroviral replication. METHODS: To discover cellular factors required for HIV-1 replication we conducted a genome-scale siRNA screen. In this screen, we transfected HeLa P4/R5 cells, which contain an integrated LTR-bGAL reporter to indicate successful HIV infection, with siRNAs targeting 19,709 genes. 24h following transfection, the cells were infected with HIV. B-GAL activity was then assayed 24h following infection, to identify host factors associated in early events in HIV replication (entry through tat-transcription), and at 96h following infection, to identify host factors associated with late events in viral replication. RESULTS: Analysis of the siRNAs that effectively reduced HIV infection revealed more than 311 host factors, including 259 that were not previously linked to HIV. Surprisingly, there was little overlap between these genes and the HIV Dependency HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies Factors described following two other siRNA screens. However, an analysis of the genes identified in both screens revealed overlaps in several of the associated pathways or protein complexes, including the SP1/ mediator complex and the NF-kB signaling pathway. cDNAs for a subset of the identified genes were used to rescue HIV replication following knockdown of the cellular mRNA providing strong evidence that the following 6 genes are previously uncharacterized host factors for HIV: AKT1, PRKAA1, CD97, NEIL3, BMP2K, and SERPINB6. CONCLUSIONS: This study highlights both the power and shortcomings of large scale loss-of-function screens in discovering host-pathogen interactions. The identification of genes with well characterized roles in HIV infection in this screen validates the overall approach. Despite this, off target silencing or failure to silence by a given siRNA pool can lead to false positives and false negatives in any siRNA screen. Although further validation of the role played in HIV replication will be required, the druggable genes identified in this screen and confirmed by cDNA rescue, including AKT1, PRKAA1, CD97, NEIL3, BMP2K, and SERPINB6, all have the potential to become drug discovery targets. 87 Abstract 74 Quantification of HIV Tropism by "Deep" Sequencing Shows a Broad Distribution of Prevalence of X4 Variants in Clinical Samples Associated with Virological Outcome LC Swenson1, WDong1, T Mo1, C Woods1, A Thielen2, M Jensen3, C Glascock1, JS Montaner1,4, and PR Harrigan1,4 1 BC Centre for Excellence in HIV/AIDS, Vancouver, Canada; 2 Max-Planck Institute for Informatics, Saarbrucken, Germany; 3 Fortinbras Research, Buford, GA, USA; 4 Faculty of Medicine, University of BC, Vancouver, Canada BACKGROUND: “Deep” sequencing (Roche GS-FLX, or “454”) detects minority HIV variants in clinical samples. Here, we performed both deep and standard sequence analyses of the HIV V3 region from individuals entering a clinical trial of maraviroc, all of whom had evidence of X4/DM virus and would not be expected to show a virological response. forward and reverse sequencing directions, with <4% coefficient of variation. The percentage of X4 determined was generally similar whether the PSSM or geno2pheno interpretations were used, (72% of samples fell within 4% of each other), despite some large outliers. Standard sequence analysis failed to detect X4 in 28% of samples. This was primarily driven by the low prevalence of X4 – samples which standard sequence detected as having X4 virus had a much higher proportion of X4 (median 78% X4; IQR 38-99% by “deep” analysis) compared to those missed by standard sequencing (median 9% X4; IQR 2.5-21%); p<<0.05. Preliminary analysis of viral load reductions in the BID maraviroc arm showed greater response for those with <10% X4 by deep sequencing/ PSSM (-1.8; -2.2; and -2.6 mean log changes at weeks 2, 4, and 8 respectively) compared to either those with >10% X4 or to those who received placebo. All of these showed less than -1.75 log reductions at these timepoints. CONCLUSIONS: Deep sequence analyses detect and quantify low prevalences of X4 HIV within clinical isolates that are not detected by standard sequence analysis. A low prevalence of X4 was associated with improved virological response to maraviroc, even where the standard Trofile reported DM virus. METHODS: Screening samples from the Pfizer A4001029 study were PCR amplified in triplicate (N=202). Tropism phenotype of all samples was X4 or DM by the standard Monogram Trofile assay as defined by the study entry criteria. Conventional (N=153) and “deep” sequencing using the GSFLX was performed using a “barcoding” approach, allowing the simultaneous analysis of 48 samples in both directions (N=202) in a blinded manner. V3 genotypes were interpreted using the PSSM and/or geno2pheno algorithms. RESULTS: An average of >4000 V3 sequences were obtained from each sample. All samples had detectable levels of X4 HIV by deep sequencing, with 4% of patients having <1% inferred X4 by PSSM, 14% having 1-10% X4, 50% having 10-90% X4 and 31% of patients having more than 90% X4. Tight correlations were generally observed between the 88 Global Antiviral Journal Volume 4, Supplement 1 Abstract 75 Antiviral Activity and Tolerability of PRO 140, a Humanized Monoclonal Antibody to CCR5 JM Jacobson1, MA Thompson2, MA Fischl3, MS Saag4, BS Zingman5, R Liporace 6, JP Lalezari7, CJ Fichtenbaum8, DS Berger9, N Stambler10, Y Rotshteyn10, P D'Ambrosio10, PJ Maddon10, WC Olson10, and SA Morris10 1 Drexel U. Coll. of Med., Philadelphia, PA, USA; 2 ARCA, Atlanta, GA, USA; 3 U. Miami, Miami, FL, USA; 4 U. Alabama, Birmingham, AL, USA; 5 Montefiore Med. Ctr., Bronx, NY, USA; 6 Albany Med. Ctr., Albany, NY, USA; 7 Quest Clinical Res., San Francisco, CA, USA; 8 U. Cincinnati, Cincinnati, OH, USA; 9 NorthStar Med. Ctr., Chicago, IL, USA; 10 Progenics Pharmaceuticals, Tarrytown, NY, USA were +0.06, -1.88 (p<0.0001), and -2.01 (p<0.0001) for placebo, 5mg/kg and 10mg/kg, respectively. The mean viral load reduction was >1.5 log10 through Day 22 at 10mg/kg. PRO 140 was generally well tolerated. Trial enrollment has completed, and updated data will be presented. Conclusions: The data confirm the antiviral activity reported previously for 5mg/kg PRO 140. A 10mg/kg dose increased the duration of antiviral effect. The findings indicate the potential for infrequent IV dosing. SC dosing regimens are also being evaluated. Background: PRO 140 is a humanized monoclonal antibody that binds CCR5 and potently inhibits CCR5tropic (R5) HIV-1 replication. In a prior study, single 5mg/kg IV doses reduced HIV RNA by 1.83 log10 in subjects with early-stage disease and R5 virus only. The present study compared 5mg/kg and 10mg/kg IV doses for antiviral activity and tolerability. Methods: After IRB approval, nine clinical sites contributed HIV positive subjects to a randomized, double-blind, placebo-controlled trial of IV PRO 140. Entry criteria included HIV RNA >5,000 copies/mL, R5 virus only, CD4 >300/μL, and no antiretroviral therapy for 12 weeks. Subjects were randomized to receive placebo, 5mg/kg PRO 140 or 10mg/kg PRO 140. They were followed for 58 days post-treatment. An interim analysis was performed on data from the first 15 subjects. Results: Interim enrollment was equally distributed across the treatment groups. Baseline HIV RNA and CD4 averaged 35,480 cps/mL and 403/μL, respectively. Mean maximum log10 reductions in HIV RNA were 0.48 (range 0.15-0.73) for placebo, 1.90 (range 1.44-2.17, p<0.0001) for 5mg/kg PRO 140 and 2.17 (range 2.09-2.26, p<0.0001) for 10mg/kg PRO 140. At Day 12, mean log10 changes in HIV RNA HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 89 Abstract 76 Discordance between the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Assay and the Roche COBAS Amplicor HIV1 MONITOR Version 1.5 Assay in a Proof-of-concept Trial in Treatment-naïve HIV-1-infected Patients RL Murphy1, C Zala2, F Fay3, V Calvez4, M Wirden4, DB DuBois5, K Pietropaolo6, J Molles6, B Belanger6, and JZ Sullivan-Bolyai6 1 Northwestern University, Chicago, IL, USA and Pierre et Marie Curie Université Paris 6, Paris, France; 2 ACLIRES Argentina SRL, Buenos Aires, Argentina; 3 CIBIC Argentina, Rosario, Argentina; 4 Hôpital Pitié-Salpêtière, Laboratoire de Virologie, Paris, France; 5 Cenetron, Austin, TX, USA; 6 Idenix Pharmaceuticals, Cambridge, MA, USA BACKGROUND: Accurate and reproducible quantitation of plasma HIV-1 RNA in subjects enrolled in clinical trials is critical for the success of drug development programs. Plasma HIV-1 RNA is essential for determination of study subject eligibility and for measurement of early and late virologic efficacy of drugs in development. During the course of a Phase Ib/IIa clinical trial of IDX899, a novel nonnucleoside reverse transcriptase inhibitor, it was observed that the Roche COBAS AmpliPrep/COBAS Taqman HIV-1 assay (TaqMan) and the Roche COBAS Amplicor HIV-1 Monitor version 1.5, Ultrasensitive version (Amplicor) produced markedly discordant results. the TaqMan assay. Patients were referred to the study by area clinicians who had already done plasma HIV1 RNA (Amplicor) and CD4 counts as part of routine care. When the discordance was noted, all prior and subsequent samples were tested by both TaqMan and Amplicor assays at the certified labs. RESULTS: 40 patients enrolled into the study, 10 (8 active:2 placebo) each in dosing cohorts of 800 mg, 400 mg, 200 mg and 100 mg administered once daily. A total of 394 paired samples were tested with both the TaqMan and Amplicor assays. During the screening period, 42% of samples had >0.5 log HIV RNA discordance, Amplicor assay results being higher. This discordance contributed to the overall virologic screen failure rate of 29% when the TaqMan was used compared to 8% with Amplicor. In samples taken throughout the trial, TaqMan underquantitated by >0.3 log HIV-1 RNA in 44%, with a mean discordance of -0.3 log HIV-1 RNA lower overall. Longitudinal analysis of viral load patterns revealed that assay discordances were not randomly distributed but related to specific subjects. In a discrete subgroup of 8 patients (20%), the TaqMan significantly underestimated HIV-1 RNA at all 9 study time points. CONCLUSIONS: TaqMan compared to Amplicor, underestimated the prevalence of HIV-1 quasispecies which resulted in the exclusion of a high percentage of patients and underestimated the change in HIV-1 RNA during the trial. METHODS: In a single site, double-blind placebocontrolled study, treatment (ART)-naïve patients with plasma HIV-1 RNA >5000 copies/ml and CD4 count >200 cells/mm3 were enrolled in a 7 day study with IDX899 monotherapy. At day 8, patients either started combination ART or were treated with 1 month of lopinavir/ritonavir. Baseline eligibility and monitoring of HIV-1 RNA were originally done with 90 Global Antiviral Journal Volume 4, Supplement 1 CME Program Information HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 91 HIV DART 2008 Frontiers in Drug Development for Antiretroviral Therapies Program Information Sponsored by Emory University School of Medicine, Office of Continuing Medical Education Learning Objectives Upon completion of this activity, participants will be able to: 1. Understand the role of viral targets in the drug discovery and development process 2. Identify novel therapeutic agents and viral targets 3. Develop improved strategies to reduce or eliminate drug-resistance and toxicity 4. Provide insights on novel immunological approaches compatible with clinical drug development 5. Develop approaches to eliminate or eradicate HIV from viral reservoirs 6. Understand the consequences of co-infection with hepatitis B & C on the management of subjects with HIV infection Accreditation Statement Emory University School of Medicine is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. Designation Emory University School of Medicine designates this educational activity for a maximum of 17 AMA PRA Category 1 Credit(s)TM. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Certificates In order to obtain your CME certificate for this activity, please complete the Credit Hour Form available at the registration desk. Forms should be completed in full and returned to the registration desk at the end of the meeting. Certificates will be mailed 3 - 4 weeks following the activity to the address provided on your registration form. Educational Support This educational activity is supported by unrestricted educational grants from the following: •Abbott Laboratories •ACLIRES International Ltd •American Academy of HIV Medicine •Boehringer Ingelheim International GmbH •Bristol-Myers Squibb •Department of Veterans Affairs •Gilead Sciences, Inc. •Idenix Pharmaceuticals •Merck & Co., Inc. •National Institutes of Health •Presidio Pharmaceuticals, Inc. •RFS Pharma, LLC •Roche Laboratories •Samchully Pharm. Co., Ltd •Schering-Plough Corporation •Tibotec Therapeutics HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 93 Faculty/Speaker Disclosure Statement and Disclosure for Discussions of Off-Label/Investigational Use of Pharmaceutical Products In accordance with ACCME Standards for Commercial Support of Continuing Medical Education and Emory University School of Medicine’s disclosure policy for CME activities, faculty members have been asked to disclose any relationship they may have with commercial supporters of this CME activity or with companies providing products or medical equipment that may have relevance to the content of their presentations. Such disclosure is intended to provide participants with sufficient information to evaluate whether any given presentation has been influence by the faculty’s relationships(s) or financial interest with said companies. This activity may include information regarding the off-label and/or investigational use(s) or various pharmacologic agents. The faculty have disclosed below if they will be discussing a product which is still investigational or not labeled for the use under discussion Note: If the disclosure information was not received before the production of the syllabus, “refused to disclose” is listed below and the moderator will state the faculty’s disclosure information during the introduction of their presentation. Course Directors and Planning Committee Members Disclosure Information: Organizing Committee David Cooper GSK, Gilead Sciences, BMS, Roche, Pfizer, Merck, Abbott, Boehringer-Ingelheim, Johnson & Johnson (investigator, advisor and speaker). Joep Lange Nothing to disclose. Robert Murphy Idenix, Genetic Immunity, Viryxsys (consultant); Idenix (stockholder); Bristol-Myers Squibb, Abbott Laboratories (grant support). José RodriguezOrengo Nothing to disclose. Raymond F. Schinazi Founder and major shareholder: Pharmasset, Inc., Idenix Pharmaceuticals, RFS Pharma LLC; Will discuss any of the drugs from these companies that might be presented. Scientific Advisory Committee 94 Françoise Brun-Vezinet Nothing to disclose. Sal Butera Nothing to disclose. Pedro Cahn Nothing to disclose. Bonaventura Clotet Boehringer Ingelheim, Bristol Myers Squibb, Gilead, GlaxoSmithKline, Janssen, Merck, Pfizer, Roche, Siemens (consulting fees or paid advisory board). Steven Deeks Pfizer, Merck, Gilead, BMS (research support); GSK, Roche, Schering-Plough, Panacos, Argos (ad hoc consulting). Courtney Fletcher Nothing to disclose. Global Antiviral Journal Volume 4, Supplement 1 Joel Gallant Gilead Sciences, Merck, Pfizer, Roche Pharmaceuticals, Tibotec (research support); Abbott Laboratories, Gilead Sciences, Koronis (DSMB member); Bristol-Myers Squibb, Gilead Sciences, Merck, Pfizer, RAPID Pharmaceuticals, Schering-Plough, Tibotec (Advisory Board); Abbott Laboratories, GlaxoSmithKline, Vertex (consultant); Abbott Laboratories, Gilead Sciences, GlaxoSmithKline, Monogram Biosciences, Tibotec (honoraria). José Gatell Have received research grants and honoraria for speaking or advisory board. Brian Gazzard Nothing to disclose. Matthias Götte Nothing to disclose. Eric Hunter Nothing to disclose. John Idoko Christine Katlama Nothing to disclose. Nothing to disclose. Paolo La Colla Nothing to disclose. Alain Lafeuillade Nothing to disclose. Hiroaki Mitsuya Julio Montaner Alan Perelson Nothing to disclose. Nothing to disclose. Nothing to disclose. Praphan Phanuphak Nothing to disclose. Richard Pollard Nothing to disclose. Bruce Polsky Idenix Pharmaceuticals (stockholder); Gilead Pharmaceuticals (consultant, Speaker Bureau member); Bristol-Myers Squibb (spouse employed, stockholder). Francois Raffi Nothing to disclose. Douglas Richman Nothing to disclose. Ian Sanne Nothing to disclose. Charles van der Horst GSK, Abbott, BMS and Boehringer provide medications for my CDC funded research study in Malawi. GSK and Abbott have also both contributed funds for specific lab tests as part of that study. All four companies and Gilead support my HIV continuing education meeting. Gilead provides funding for residency training for Malawian physicians sponsored by UNC. I have less than $10,000 in stock in Roche and BMS each. Stefano Vella Nothing to disclose. Mark Wainberg Nothing to disclose. Speakers, Moderators and Panelists’ Disclosure Information: Joeri Auwerx We have a long term collaboration with Gilead Sciences on structural studies of HIV-1 RT with tenofovir (Gilead markets Viread). I visit and consult periodically with scientists at Gilead, but not within the last 12 months. Nothing to disclose. Leda Bassit Edward Berger Charles Boucher Robert Buckheit Nothing to disclose. Nothing to disclose. Nothing to disclose. Nothing to disclose. Edward Arnold HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 95 Douglas Dieterich Gilles Divita Lisa Dunkle Amy Espeseth Matthias Götte P. Richard Harrigan Daria Hazuda Sharon Hillier Schering-Plough Corporation (employee). Nothing to disclose. Nothing to disclose. I have received grants or honoraria and/or consulted for a variety of pharma and diagnostic companies. The research presented here was supported by an investigator initiated research grant by Pfizer. Merck & Co. (employee). Christine Katlama Nothing to disclose. BMS, Gilead, Tibotec, GSK, Roche (grants/research support, consultant, honorarium, speaker bureau); Virco, Vertex (consultant, honorarium, speaker’s bureau); Abbott, BIPI (honorarium, speaker bureau). Nothing to disclose. Saye Khoo Nothing to disclose. Joep Lange Nothing to disclose. Brendan Larder Nothing to disclose. Eric Lefebvre Employee of Tibotec. Franco Lori Employee as CEO and President of Virostatics. Leonid Margolis Douglas Mayers Julio Montaner Stephen Morris Monique Nijhuis Thomas North Nothing to disclose. Chief Medical Officer for Idenix Pharmaceuticals. Nothing to disclose. Senior Director Clinical Research, Progenics, Inc. Idenix, Genetic Immunity, Viryxsys (consultant); Idenix (stockholder); Bristol-Myers Squibb, Abbott Laboratories (grant support). Nothing to disclose. Nothing to disclose. Richard Pollard Nothing to disclose. Dushyantha Jayaweera Robert Murphy Bruce Polsky Adrian Ray José RodriguezOrengo Ian Sanne Idenix Pharmaceuticals (stockholder); Gilead Pharmaceuticals (consultant, Speaker Bureau member); Bristol-Myers Squibb (spouse employed, stockholder). I am employed by and hold stock in Gilead Sciences, Inc., the marketer of HIV drugs (tenofovir and emtricitabine) and a company involved in studying clinical candidates for the treatment of HIV (including GS-9131/GS-9148 and GS-9137). My talks will present balanced discussions of drugs and drug candidates from Gilead and other companies. Nothing to disclose. Robert Siliciano Nothing to disclose. Founder and major shareholder: Pharmasset, Inc., Idenix Pharmaceuticals, RFS Pharma LLC; Will discuss any of the drugs from these companies that might be presented. Nothing to disclose. Paul Spearman Nothing to disclose. Mario Stevenson Nothing to disclose. Tracy Swan Nothing to disclose. Raymond F. Schinazi 96 Roche, Gilead, BMS, Novartis, Boehringer Ingelheim (grants); Roche, Gilead, BMS, Novartis (consulting and lecturing). Nothing to disclose. Global Antiviral Journal Volume 4, Supplement 1 Charles van der Horst GSK, Abbott, BMS and Boehringer provide medications for my CDC funded research study in Malawi. GSK and Abbott have also both contributed funds for specific lab tests as part of that study. All four companies and Gilead support my HIV continuing education meeting. Gilead provides funding for residency training for Malawian physicians sponsored by UNC. I have less than $10,000 in stock in Roche and BMS each. Mark Wainberg Nothing to disclose. John Wilkinson Nothing to disclose. James Witek Employee and stockholder of Johnson & Johnson. Elizabeth Wright Nothing to disclose. Carmen Zorrilla Tibotec (clinical trial sponsorship, Advisory Committee). This activity is being sponsored by Emory University School of Medicine. The medical school has no significant relationship with the commercial companies whose products are services are being discussed in this educational activity. HIV DART 2008 - Frontiers in Drug Development for Antiretroviral Therapies 97 Informed Horizons presents... Upcoming Meetings HIV DRUG RESISTANCE workshop basic principles & clinical implications *$-%'),'("%' 2009 XVIII International HIV Drug Resistance Workshop: Basic Principles & Clinical Applications June 9-13, 2009 Fort Myers, Florida HEP DART 2009: Frontiers in Drug Development for Viral Hepatitis December 6-10, 2009 Kohala Coast, Hawaii This workshop is renowned for the quality of the data presented and the depth of the scientific interaction and debate. The focus of HEP DART 2009 is to assemble clinicians, researchers, and basic scientists together to advance our knowledge of the ongoing drug development processes in the treatment of hepatitis B and hepatitis C. For more information visit our website at www.informedhorizons.com Coming in 2009... From the American Academy of HIV Medicine: Fundamentals of Global HIV Medicine Relevant and practical core HIV knowledge for global HIV medical care providers For more information, contact IHL Press at [email protected]
