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51th ICAAC
Chicago, IL
September 17-20, 2011
Novel Boehringer-Ingelheim Integrase Inhibitor Retains Full Activity Against Raltegravir-Resistant Virus
51st Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), September 17-20, 2011, Chicago
Mark Mascolini
Four reports at this meeting spelled out early findings on BI 224436, an HIV integrase inhibitor with a mechanism different from that of the
"tegravir" strand-transfer integrase inhibitors. This novel agent, being developed by Boehringer Ingelheim, binds to a highly conserved
allosteric pocket of the integrase catalytic core--not to the catalytic site itself. LEDGF, a host cell factor important for HIV replication, binds
to the same site. Because of this novel mechanism, integrase inhibitors like 224436 are expected to have resistance profiles different from
strand-transfer integrase inhibitors. Complete data for these four studies is online at the NATAP links listed in the references below.
Phenotypic analysis of 40 recombinant viruses containing integrase from clinical isolates resistant to raltegravir determined that all 40
remained susceptible to 224436 with an average 50% effective concentration (EC50) of 26 +/- 16 nM and an average fold change in EC50
relative to wild-type (nonmutant) virus of 1.4 +/- 0.8 [1]. In contrast, raltegravir and elvitegravir had more than a 260-fold change in EC50
against these recombinant viruses.
These investigators also tested 224436 against HIV-1 isolates from 100 US and 100 European people naive to antiretroviral therapy [1].
Average EC50 and EC95 values for 224436 were 14 +/- 13 nM and 39 +/- 30 nM. Against these same viral isolates, average EC50 for
raltegravir was 6.7 +/- 1.4 nM and average EC95 78 +/- 18 nM.
A study in healthy male volunteers yielded results indicating that once-daily dosing with 224436 "may be feasible," the investigators
proposed [2]. This first-in-human double-blind placebo-controlled trial tested a single oral dose of 6.2, 12.5, 25, 50, 100, or 200 mg of
224436. In each dose group, 6 men took 224436 and 2 took placebo. All study participants were 21 to 50 years old and had a body mass
index between 18.5 and 29.9 kg/m(2).
Maximum concentration (Cmax) and three area under the concentration-time curve (AUC) measures increased in a dose-proportional
manner from 6.2 to 200 mg. Between-person variability for these parameters was low, with geometric coefficients of variation ranging from
13.1% to 31.5% for Cmax and from 20.2% to 31.7% for AUC 0-infinity. Absorption was rapid, with a median time to maximum
concentration of 0.5 h in each dose group (overall range 0.25 to 1.5 h). Arithmetic mean terminal elimination half life ranged from 6.38 to
7.85 h across the 6 doses.
The investigators determined that the 100-mg dose provided the geometric mean 24-hour concentration (496 nmol/L, range 314 to 867)
closest to the target minimum concentration of 500 nmol/L.
Mild adverse events included headache in 1 person, upper abdominal pain in 1, and oral paresthesia (tingling or numbness) in 2.
Boehringer Ingelheim scientists used a hollow-fiber infection model and pharmacokinetic modeling to project an optimal dose of 224436 in
humans [3]. In the hollow-fiber system, this novel integrase inhibitor had an EC50 of 12 nM. At 39 nM 224436 inhibited more than 90% of
virus. The investigators used these results and human PK data on the 100-mg dose [2] in Monte Carlo simulations to predict that the 100mg dose will suppress replication with more than 90% probability for 200 HIV-1 clinical isolates studied.
Preclinical analysis of 224436 determined that this agent has EC50s ranging from 4 to 15 nM against HIV-1 lab strains [4]. CC50 (cell
toxicity) values for 224436 exceeded 90 uM in peripheral blood mononuclear cells. Exposure to 50% human serum provoked only a 2.2-fold
shift in antiviral potency.
So-called ADME (absorption, distribution, metabolism, and excretion) properties included a favorable metabolic profile, low potential for
interaction with CYP3A4 and CYP2DG drug-metabolizing enzymes, and high permeability. Cell-based antiviral assays indicated that 224436
has an additive effect with agents in all current antiretroviral classes, including strand-transfer integrase inhibitors.
References
1. Fenwick C, Bethell R, Quinson A, et al. BI 224436, a non-catalytic site integrase inhibitor, is a potent inhibitor of the replication of
treatment-naive and raltegravir-resistant clinical isolates of HIV-1. 51st Interscience Conference on Antimicrobial Agents and
Chemotherapy (ICAAC). September 17-20, 2011. Abstract F1-1370. http://www.natap.org/2011/ICAAC/ICAAC_34.htm.
2. Aslanyan S, Ballow C, Sabo JP, et al. Safety and pharmacokinetics (PK) of single rising oral doses of a novel HIV integrase inhibitor in
healthy volunteers. 51st Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC). September 17-20, 2011. Abstract
AI-1725. http://www.natap.org/2011/ICAAC/ICAAC_35.htm.
3. Brown AN, McSharry J, Kulawy R, et al. Pharmacodynamics of BI 224436 for HIV-1 in an in vitro hollow fiber infection model system.
51st Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC). September 17-20, 2011. Abstract A1-1726.
http://www.natap.org/2011/ICAAC/ICAAC_33.htm.
4. Yoakim C , Amad M, Bailey MD, et al. Preclinical profile of BI 224436, a novel HIV-1 non-catalytic site integrase inhibitor. 51st
Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC). September 17-20, 2011. Abstract F1-1369.
http://www.natap.org/2011/ICAAC/ICAAC_32.htm.
Conference Reports for NATAP
51th ICAAC
Chicago, IL
September 17-20, 2011
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Safety and pharmacokinetics (PK) of single rising oral doses of a novel HIV integrase inhibitor in healthy volunteers
Reported by Jules Levin
ICAAC Sept 17-20 2011 Chicago
S. Aslanyan1, Ch. Ballow3, J. P. Sabo1, J. Habeck2, D. Roos1, T. R. MacGregor1, P. Robinson1, J. Kort1
1 Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT, US 2 Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany 3 Buffalo
Clinical Research Center, Buffalo, NY, US
ABSTRACT
Background: BI 224436 is a novel non-catalytic site HIV integrase inhibitor. Methods: Double-blind, placebo-controlled, first-in-human, single
rising dose trial of an oral solution formulation of BI 224436 in healthy male volunteers. Forty-eight subjects were randomized to 6 ascending
consecutive dose cohorts (BI 224436 6.2, 12.5, 25, 50, 100 and 200 mg); 6 subjects received BI 224436 and 2 received placebo within each
cohort.
Results: Geometric means (gmean) of selected PK parameters and dose cohorts are presented in the table.
Dose-proportional increases in plasma BI 224436 Cmax, AUC0-∞, AUC0-24, and AUC0-tz were observed. Inter-subject variability (geometric
CV%) was < 33% for these PK parameters. Oral solution of BI 224436 was rapidly absorbed with a median Tmax of 0.5 h (range, 0.25-1.5 h).
Arithmetic mean terminal elimination half-life (t1/2) was 7.11 h and consistent across all groups and variability (arithmetic CV%) was < 24%.
Four mild adverse events in 4 subjects were observed within 72 h of administration of BI 224436 without any dose related trend (headache [1]
and upper abdominal pain [1] in 25 mg cohort and oral paresthesia [2] in 50 mg cohort). No clinically relevant changes in vital signs and ECG and
laboratory parameters were noted.
Conclusion: Single doses of BI 224436 were safe and well-tolerated up to the highest dose tested. Based on the single-dose PK profile, the 100
mg dose provided the desirable therapeutic plasma drug concentration at 24 h after dosing (gmean 496 nmol/L [range 314 - 867 nmol/L]) relative
to a targeted therapeutic Cmin of 500 nmol/L. These results indicate that once-daily dosing with BI 224436 may be feasible.
INTRODUCTION
BI 224436, a novel allosteric (non-catalytic) site HIV-1 integrase inhibitor, provides an opportunity to further exploit inhibition of the HIV integrase
enzyme by a mechanism that is distinct and complementary to contemporary integrase strand transfer inhibitors.
- BI 224436 exerts this inhibition by binding to a highly conserved allosteric pocket on the catalytic core of integrase which also serves as binding
pocket for LEDGF, a host cell co-factor important for viral replication (See Poster F1-1369).
- Based on preclinical pharmacology data for BI 224436 (see Posters F1-1369 and A1-1726), a Cmin in humans of approximately 500 nM has been
selected. - Significant differences in the pharmacokinetics (PK) of BI 224436 among the various animal species tested were observed, which made
it not feasible to predict PK in humans, hence this first-in-human trial was advanced.
METHODS
- Single rising dose, randomized, double-blind, and placebo controlled within dose groups.
- First-in-human study conducted in healthy adult male volunteers at a single center.
- Tested sub-therapeutic doses and escalated up to doses in the therapeutic range.
- Dose escalation from each dose cohort to the next higher dose cohort was based on the assessment of safety and PK.
- The trial was conducted according to ICH GCP and was approved by research center IRB. All subjects have signed and dated written informed
consent prior to admission to the study.
- The primary objective of the study was to obtain single-dose PK of BI 224436 in healthy volunteers that may predict whether BI 224436 could
be administered once daily. Another objective was to obtain first data on the safety profile of the compound.
Bioanalytical
Concentrations of BI 224436 were determined in EDTA plasma by a validated HPLC-MS/MS assay (high performance liquid chromatography,
tandem mass spectrometry) with a lower limit of quantification (LLOQ) of 5.00 nmol/L.
PK end-points for BI 224436 were determined from the plasma BI 224436 concentration-time profiles using non-compartmental methods
(WinNonlin Professional v5.3; Pharsight Corporation, Sunnyvale, CA) (Table 1).
Safety was assessed descriptively based on:
- Physical examination
- Vital signs (BP, PR, RR, oral body temperature)
- 12-lead ECG and telemetry
- Clinical laboratory tests (hematology, clinical chemistry and urinalysis) - Adverse events
Subject Eligibility Criteria:
1. Healthy males
2. Age ≥ 21 and ≤ 50 years
3. Body Mass Index (BMI) ≥ 18.5 and BMI ≤ 29.9 kg/m2
RESULTS
Forty-eight subjects were randomized in this trial with 8 subjects within each of 6 dose cohorts (6.2, 12.5, 25, 50, 100 and 200 mg). Within each
dose cohort, 6 subjects received BI 224436 and 2 subjects received placebo, resulting in total 12 subjects receiving placebo. All subjects who were
randomized received the assigned study drug and completed all trial visits.
The demographic data and baseline characteristics of the subjects entered into this study (all male) were reasonably similar in all cohorts. There
were 28 White subjects, 17 African Americans and 3 Asians included. The overall median age (range) was 26.0 (21-50) years. The median BMI
(range) was 25.35 (18.9-29.8) kg/m2.
Pharmacokinetics:
- A summary of selected PK parameters following single-dose BI 224436 administration is presented in Table 1.
- Plasma BI 224436 Cmax, AUC0-∞, AUC0-24, and AUC0-tz increased in a dose proportional manner over the dose range of 6.2 to 200 mg
(Figure 2).
- Variability (geometric CV%) was low and less than 32% for these PK parameters (gCV% range: Cmax, 13.1 - 31.5%; AUC0-∞, 20.2 - 31.7%).
- BI 224436 was rapidly absorbed with Tmax occurring quickly after oral administration (median 0.5 h in each dose group; overall range 0.25 1.5 h).
- Arithmetic mean terminal elimination half-life (t1/2) was consistent across the 6 dose groups and ranged from 6.38 h to 7.85 h (arithmetic mean
7.11 h); variability (arithmetic CV%) was low and less than 24% (range 11.5 - 23.5%).
- Based on the single-dose pharmacokinetic profile, the 100 mg dose provided the desirable therapeutic plasma drug concentration (geometric
mean C24 of 496 nmol/L [range 314 - 867 nmol/L]) relative to the target therapeutic Cmin of 500 nmol/L.
Conference Reports for NATAP
51th ICAAC
Chicago, IL
September 17-20, 2011
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BI 224436, a Non-Catalytic Site Integrase Inhibitor, is a potent inhibitor of the replication of treatment-naïve and raltegravirresistant clinical isolates of HIV-1
Reported by Jules Levin
ICAAC Chicago Sept 17-20 2011
C. Fenwick1, R. Bethell1, M. Cordingley1, P. Edwards1, A-M. Quinson2, P. Robinson2, B. Simoneau1 and C. Yoakim1 1Boehringer Ingelheim Ltd., R & D,
Laval, Canada, 2 Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut, USA
Abstract
Background: Non-catalytic site integrase inhibitors (NCINIs) represent a novel class of HIV-1 antiretroviral. They bind to a conserved allosteric pocket on
integrase (IN) and have a distinct mechanism of action compared to raltegravir and elvitegravir which bind at the IN catalytic site.
Method: The in vitro antiviral activity of BI 224436 against 200 recombinant viruses encoding the IN from different clade B clinical isolates of HIV-1 was
assessed using the PhenoSense assay. Isolates were from 100 US and 100 European patients considered to be treatment naïve based on the absence of
NRTI and NNRTI associated resistance mutations. The antiviral activity of BI 224436 was also assessed against a panel of 40 recombinant viruses
containing IN from clinical isolates of HIV-1 resistant to raltegravir in an in vitro phenotyping assay performed by Virco.
Results: BI 224436 had a mean EC50 value of 14 ± 13 nM for the 200 clinical isolates tested in the PhenoSense assay. Consistent with BI 224436 having
a steep dose-response curve slope of ~4.0, the mean EC95 value against these 200 viruses was 39 ± 30 nM. By comparison, raltegravir had an EC50
value of 6.7 ± 1.4 nM and EC95 of 78 ± 18 nM against the same panel. BI 224436 also retained antiviral potency against 40 recombinant viruses with IN
from raltegravir resistant clinical isolates. BI 224436 had a mean EC50 value of 26 nM ± 16 nM and mean fold change (FC) in EC50 values of 1.4 ± 0.8
relative to a wild type control virus. Raltegravir and elvitegravir profiled in parallel both displayed a > 260 FC in EC50.
Conclusion: BI 224436 had a potent antiviral profile in IN phenotyping assays against treatment naive clade B isolates of HIV-1 and raltegravir-resistant
clinical isolates. Based on its excellent biological and PK profile, and an absence of cross resistance to all other classes of approved antiretroviral agents
including IN catalytic site inhibitors such as raltegravir, BI 224436 has been advanced into Phase I clinical trials.
Introduction
The primary function of HIV integrase is to integrate newly synthesized viral DNA into the host genome, which is an obligate process for subsequent
expression of viral genes. Integrase performs two related reactions: the enzyme-catalyzed hydrolysis of a dinucleotide from the 3'-end of each viral LTR
DNA and the strand transfer reaction that integrates the viral DNA into the host cell's DNA.
In a high throughput screening campaigns at Boehringer Ingelheim (BI), selective hits were identified using an integrase LTR DNA 3'-processing assay and
were sequentially optimized through a combination of medicinal chemistry, parallel synthesis and structure guided drug design.
This novel class of integrase inhibitors, referred to as Non-Catalytic site Integrase Inhibitors (NCINI), bind to a conserved allosteric pocket on integrase
that is also targeted by LEDGF.1 Compounds from the NCINI class have been shown to maintain antiviral activity against recombinant viruses encoding
INSTI or NNRTI susceptibility mutations (Table 1).2 In addition, raltegravir and elvitegravir have been shown to maintain antiviral activity against
recombinant viruses encoding substitutions in integrase that result in reduced susceptibility to the NCINI series.
BI 224436 is a potent NCINI antiretroviral that is closely related to BI-B and has been advanced into Phase 1 clinical trials. Further details on BI 224436
are presented in ICAAC posters A1-1725, A1-1726 and F1-1369.
Methods
Phenotyping of HIV-1 integrase from treatment naïve clinical isolates: The integrase PhenoSense assay3 was performed by Monogram Biosciences with
100 US and 100 European treatment naïve clade B clinical isolates selected from a wide geographic distribution throughout the US (patients from: 2 AL,
19 CA, 1 CT, 3 DC, 5 FL, 2 GA, 18 IL, 2 MA, 1MD, 1ME, 1 MI, 2 NC, 3 NJ, 27 NY, 1 TN, 9 TX, 2 VA and 1 WA) and Europe (patients from : 4 Belgium, 13
France, 21 Germany, 1 Ireland, 5 Italy, 2 Netherland, 8 Portugal, 21 Spain, 6 Switzerland and 19 United Kingdom). Fold change in EC50 was determined
relative to a NL4.3 control.
Phenotyping of HIV-1 integrase from patients that failed antiviral therapy with raltegravir: Phenotyping was performed by Virco with a panel of 40 viruses
selected with resistance mutations in integrase that represent the most prevalent INSTI escape pathways including E92Q, G140S/A, Y143C/R, Q148H/K/R,
N155S amino acid substitutions.4 Fold change in EC50 was determined relative to an HXB2 control. In addition, recombinant viruses were generated by
introducing single and multiple amino acid substitution in the integrase gene by site directed mutagenesis in the HIV-1 IIIB laboratory strain.
1- Cherepanov, P. et al PNAS 2005 102(48):17308
2- Fenwick, C et. al International Workshop for HIV and Hepatitis Drug Resistance, 2011, Poster #1
3- Petropoulos CJ et al. Antimicrob Agents Chemother. 2000;44(4):920
4- Hertogs K, et al. Antimicrob Agents Chemother 1998;42(2):269.
Other scientists who supported this work include: Murray Bailey, Lee Fader, Anne-Marie Faucher, Stephen Kawai, Marc- Andre Poupart, & Youla Tsantrizos
in Chemistry; Pierre Bonneau, Rene Coulombe, Araz Jakalian & Steven LaPlante in Structural Research; Ma'an Amad, Mireille Cartier, Jianmin Duan, Rob
Elston, Louie Lamorte, Laibin Luo, Michel Garneau, Steve Mason, Nathalie Rioux & Myriam Witvrouw in Biological Sciences.
Conference Reports for NATAP
51th ICAAC
Chicago, IL
September 17-20, 2011
Pharmacodynamics of BI 224436 for HIV-1 in
an in vitro hollow fiber infection model system
Reported by Jules Levin
ICAAC Chicago 2011 Sept 17-20
A.N. Brown1, J. McSharry1, R. Kulawy1, C. Fenwick2, R. Bethell2, J. Duan2, J. Kort3, P Robinson3, B. Simoneau2, C. Yoakim2 and
G. Drusano1 1University of Florida, Albany, NY, USA , 2Boehringer Ingelheim Ltd., R & D, Laval, Canada, and 3 Boehringer
Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut, USA
Background
BI 224436 is a novel integrase inhibitor with potent antiviral activity against HIV-11. This compound belongs to a new class of
integrase inhibitors, referred to as Non- Catalytic site Integrase inhibitor, that bind to a conserved allosteric pocket on the viral
integrase (Figure 1) and acts as a LTR-DNA 3' processing inhibitor.2
Our goal was to identify optimal dosing regimens for BI 224436 that prevent HIV-1 replication using the hollow fiber infection
model (HFIM) system.
Figure 1. Crystal structure of the HIV Integrase catalytic core dimer with the NCINI BI-B. BI-B is a structurally related compound
to BI 224436 that binds to the LEDGF binding pocket on the integrase catalytic core inhibiting LTR-DNA 3' processing, integraseLEDGF interaction, and integrase-LTR DNA interaction.
Objectives
1) Evaluate the efficacy of BI 224436 against the IIIB strain of HIV-1 in the hollow fiber system.
2) Determine the most predictive PK/PD index for BI 224436 by a dose fractionation experiment.
3) Predict efficacy of BI 224436 in the clinic using Monte Carlo simulations.
Figure 2. The HFIM System. The HFIM is a dynamic in vitro model that can simulate concentration-time profiles for any dosage
regimen. The system contains two compartments, the central reservoir (CR) as the central compartment and the hollow fiber (HF)
cartridge as the peripheral compartment. Cells and virus are maintained within the peripheral compartment.
Methods
Protein Binding Assay. Tissue culture flasks were inoculated with 104 HIV-infected H9IIIB cells and 106 uninfected CEM-SS cells in
in the presence or absence of 4 mg/dl human serum albumin (HSA) and 1.5 mg/ml of α-1 acid glycoprotein (AGP). Flasks were
incubated with various amounts of drug at 37 C, 5% CO2 for 4 days. Antiviral activity was determined by measuring the
percentage of cells positive for the p24 antigen using flow cytometry. EC50 values were calculated by fitting a sigmoid Emax
model to the data.
HFIM Studies. HF cartridges were inoculated with 108 uninfected CEM-SS cells and 106 HIVIIIB-infected H9 cells.
(A) Dose Ranging Studies: Various concentrations of BI 224436 were added directly to the central reservoir and pumped into the
extracapillary space (ECS) via a continuous loop. Fresh medium with or without BI 224436 was added to the system daily. The
EC50 value was determined in the HFIM system using FACS analysis values on day 8.
(B) Dose Fractionation Studies: An area under the concentration time curve (AUC) exposures equivalent to 13 nM continuous
infusion (312 nM*h over 24 hrs) and 19 nM continuous infusion (456 nM*h over 24 hrs) for BI 224436 was evaluated at three
different schedules of administration.
Schedule of Administration: Continuous Infusion
Q24h as a 1-h infusion, or
Q12h as a 1-h infusion
A half-life of 7h was simulated for BI 224436 based on single dose data in humans3.
For dose range and dose fractionation studies, serial drug concentrations were determined in the central reservoir over the first
48h and quantified by LC-MS/MS. Drug effect was determined from samples (~ 0.5 mls) harvested from the ECS as depicted in
Figure 3.
Conference Reports for NATAP
51th ICAAC
Chicago, IL
September 17-20, 2011
Back
Preclinical Profile of BI 224436, a Novel HIV-1 Non-Catalytic Site Integrase Inhibitor
Reported by Jules Levin
ICAAC Chicago 2011 Sept 17-20
Christiane Yoakim , Ma'an Amad, Murray D. Bailey, Richard Bethell, Michael Bos, Pierre Bonneau, Michael Cordingley, Rene Coulombe,
Jianmin Duan, Paul Edwards, Lee Fader, Anne-Marie Faucher, Michel Garneau, Araz Jakalian, Stephen Kawai, Louie Lamorte, Steven
LaPlante, Laibin Luo, Steve Mason, Marc-Andre Poupart , Nathalie Rioux, Bruno Simoneau, YoulaTsantrizos#, Myriam Witvrouw and
Craig Fenwick Boehringer Ingelheim (Canada) Ltd., Research and Development
Abstract
Background: Non-catalytic site integrase inhibitors (NCINIs) represent a novel class of HIV-1 antiretroviral that bind to a conserved
allosteric pocket on integrase (IN) and specifically inhibit the IN LTR DNA 3' processing activity. In contrast, marketed and late stage
clinical drugs targeting IN bind at the catalytic site and are primarily integrase strand transfer inhibitors (INSTIs). Consistent with a
novel mechanism of action, NCINIs have a non-overlapping resistance profile with INSTIs such as raltegravir.
Methods: Using an enzymatic IN LTR DNA 3' processing assay a 3-quinolineacetic acid derivative was identified as an interesting lead
structure. A combination of medicinal chemistry and structure-guided drug design has led to the identification of BI 224436 as a
candidate for pre-clinical profiling.
Results: BI 224436 has antiviral EC50 values ranging between 4 and 15 nM against different HIV-1 laboratory strains and CC50
values >90 μM in different cells, including peripheral blood mononuclear cells. BI 224436 also has a low, 2.2-fold shift in antiviral
potency in the presence of 50% human serum and by virtue of a steep dose-response curve slope, BI 224436 exhibits serum-shifted
EC95 values ranging between 22 and 75 nM. Drug combination studies performed in cell-based antiviral assays have shown that BI
224436 displays, at the least, an additive effect in combination with any of the marketed antiviral classes including INSTIs. BI 224436
has drug-like ADME properties including a Caco-2 cell permeability of 14 .10-6 cm/sec, solubility > 24 mg/ml in the pH range 2.0-6.8
and low cytochrome P450 inhibition. Moreover BI 224436 shows excellent PK profiles in rat (CL=0.7% QH; F= 54%), monkey (CL=
23% QH; F= 82%) and dog (CL= 8%QH; F= 81%).
Conclusion: BI 224436 is a potent HIV-1 integrase inhibitor. Based on the excellent biological and PK profile, BI 224436 has been
advanced into Phase 1 clinical trials.
Introduction
It has become increasingly challenging to identify compounds within the existing classes of HIV-1 inhibitors due to cross-resistance and
lack of differentiation in their profiles. Moreover new candidates should possess suitable properties for once daily dosing and inclusion in
fixed dose combinations. As a result, research focus has been shifting in recent years towards the identification of new mechanisms of
inhibition in order to provide novel regimen combinations for the treatment of HIV-1 infection. HIV-1 integrase (IN) has two related
enzymatic activities that are essential for viral replication; the LTR DNA 3' processing reaction and the strand-transfer reaction which
integrates viral DNA into the host genomic DNA (Figure 1). Marketed and late stage clinical drugs targeting IN bind at the catalytic site
and are primarily IN strand-transfer inhibitors (INSTIs). Using an IN LTR DNA 3' processing assay in a high throughput format, followed
by hit to lead optimization led to the identification of a 3-quinolineacetic acid series. This series generated inhibitors with high potency in
antiviral assays and excellent ADMETproperties.
Methods
-The LTR DNA 3'- processing assay used a DNA probe consisting of two annealed oligonucleotides with a 3'-end Black Hole Quencher
and a 5'-end Rhodamine Red fluorophore (Figure 2). The E. coli DNase cleavage counter screen assay was conducted using the same
oligonucleotide probe.
- Apparent KD values were determined with a biochemical displacement assay using a biotinylated inhibitor probe. The probe used has
an estimated KD of 0.2 μM.
-Antiviral assays: EC50 values were determined using C8166 HIV-1 Luciferase assay (WO 2007 / 131350). Cells were infected with
either HXB2 or NL4-3 lab HIV strains. A standard p24 ELISA assay was also used for key compounds for comparison purposes.