Download scientific approaches Alan Stone (London, UK)

Non-clinical evaluation of
microbicides:
scientific approaches
Alan Stone (London, UK)
Regional meeting on regulatory issues
in microbicide research
28-31 October 2007, New Delhi, India
Aims of non-clinical evaluation
To provide a sufficient body of evidence about anti-infective
potency, toxicity and formulability to justify the clinical
evaluation of the product under investigation.
BRIEFLY:
(a) Product active against HIV and, ideally, against other STIs,
either by destroying or damaging the pathogen directly, or by
preventing attachment/fusion/entry to the host cell, or by
preventing the pathogen’s replication. Multiple mechanisms?
(b) Low or no toxicity in cell and tissue systems, and in
relevant animal models.
(c) Can be formulated to give a stable preparation suitable for
self-administration by users.
Screening a large number of compounds
to identify antiviral activity
Cell-based in vitro assays
• Standard lab strains of HIV.
• R5 and X4 strains (use respectively CCR5 and CXCR4 coreceptors on surface of human lymphocytes).
• Cell-free and cell-associated virus.
• 1 or 2 dilutions of compound.
More detailed in vitro evaluation
Dose-response relationship and mechanism of action
• Virus inactivation assays.
• Binding and fusion assays.
• Virus replication assays.
• Broad range of relevant cell types (PBMCs, macrophages,
?dendritic cells; ex-vivo vaginal, cervical and penile explants.
• Wide range of clinical HIV isolates derived from lower
reproductive tract (and lab strains for comparison); R5 and
X4. [Eventually: regionally prevalent strains]
• Exposure of virus to microbicide before, during or after
infection.
• Impact of increasing dose of virus.
• Effect of pH.
Potency versus cytotoxicity
Compare anti-HIV activity and cytotoxicity simultaneously in vitro.
AIM: identify lowest effective concentration, and the highest
concentration where level of cytotoxicity still acceptable.
Therapeutic Index: drug concentration giving 50% reduction of
cell viability : drug concentration giving 50% reduction of infectivity
[Relationship to drug levels needed (and achievable) in human
vaginal environment?]
Also need to investigate:
•
effects of excipients in the formulated product, eg a gel.
•
effects of semen and cervico-vaginal secretions.
In vitro data from laboratory of Professor D J Jeffries
(London, UK)
Nonoxynol-9
PRO 2000
PRO 2000
In vitro data from laboratory of
Professor D J Jeffries (London, UK)
Effects on other microorganisms and sperm
Normal vaginal microflora
Lactobacilli: effects of drug in vitro; effects in pig-tailed macaque
vagina.
STI pathogens
In vitro assays to establish inhibitory concentration against
N. gonorrhoeae, C. trachomatis, HSV-2, HPV, T. vaginalis, H.
ducreyi, T. pallidum.
Also to rule out possibility that drug might potentiate these
infections.
Pigtailed macaque model for C. trachomatis.
Mouse rectal model for assessing product’s activity against
HSV-2.
Human sperm
In vitro spermicidal activity.
Drug resistance
• In principle could apply to any class of microbicide, but it applies
especially to certain antiretroviral drugs such as reverse
transcriptase inhibitors and possibly also to co-receptor blockers.
• Genotypic and phenotypic basis of resistance (mutations,
proteins, fitness etc).
• Implications of resistance to same drug, and cross-resistance to
other drugs, being used for treatment in populations where
microbicide will be made available.
• Drug combinations could provide a way of getting around the
resistance problem.
Combination microbicides: advantages
A combination of two or more microbicides which
work at different stages of the HIV life-cycle could:
A reverse transcriptase inhibitor combined with eg an
attachment/fusion blocker such as PRO 2000 or a
co-receptor blocker could reduce the likelihood of
transmitting, or being infected by, drug-resistant HIV.
Evaluation of mutual compatibility of the APIs, drug
interactions etc.
In addition, combinations could:
• provide a greater degree of protection against infection and
unwanted pregnancy.
• exhibit a broader spectrum of activity against a variety of pathogens
and sperm.
• minimise any adverse effects by permitting lower doses of each
component.
Animal models
Rabbit vagina: 10-day irritation study to detect adverse effects on vaginal
mucosa. Test formulated product. Negative and positive controls: formulation
lacking drug and nonoxynol-9 formulation. Examine for signs of
irritation/inflammation/ulceration.
Pigtailed macaque vagina: assessment of product’s effects on mucosa
and on normal vaginal flora and pH.
Rat rectum: 4-month toxicity study. [inclg microbicides intended for vagina.]
Mouse vagina and rectum: good model for assessing impact on HSV
infection.
Rhesus macaque vagina and rectum: efficacy in preventing infection
by SIV or SHIV. Various protocols. Often carried out, but significance in terms of
the microbicide’s ability to protect in human sex is unknown.
Hu-SCID mouse model (immuno-compromised mouse reconstituted with
elements of human immune system): can be infected vaginally with HIV, but
model still at experimental stage.
Pharmacokinetic and toxicology studies
Pharmacokinetics: Administer formulated product vaginally and measure
drug’s absorption, distribution, metabolism and excretion.
General toxicology: Administer at low, intermediate and maximum
tolerated doses in one rodent species and one non-rodent species and monitor
general health, weight, eating, haematology, serum, urine, organs. Acute study
(dosing for 1 day), use API via oral or parenteral routes. Longer-term studies
use formulated product via vaginal or rectal route.
Genetic toxicology: ICH-recommended tests (mutagenicity in bacteria;
chromosomal damage in vitro; chromosomal damage in vivo in rodent
haematopoietic cells).
Reproductive toxicology: Segment I (fertility and early embryonic
development) and Segment II (embryo-foetal development) preferably prior to
human exposure. Segment III (peri- and post-natal development) later, eg
during Phase III trial.
Carcinogenicity studies: 2-year dosing in mice/rats, or 6-month dosing
in Tg.AC transgenic mice, prior to product’s market approval.
Chemistry, manufacturing and controls
Identity, strength, purity and stability studies to assure adequate
quality of the API and the formulated product.
Formulation studies
To select dosage form suitable for vaginal application (adequate
dose volume, retention, distribution, and other important attributes
eg colour, odour, taste, viscosity).
Intravaginal rings for slow release of eg reverse transcriptase
inhibitors. Stability, drug release characteristics etc……
Compatibility with physical barriers
To ensure no adverse effects on condoms, latex diaphragms etc,
under various conditions designed to mimic real-life usage.
IWGM non-clinical recommendations
J Acquir Immune Defic Syndr 2004, 36: 541-552
RECOMMENDATIONS FOR THE NONCLINICAL DEVELOPMENT OF
TOPICAL MICROBICIDES FOR PREVENTION OF HUMAN
IMMUNODEFICIENCY VIRUS TRANSMISSION: AN UPDATE
Sheryl L. Lard-Whiteford1, Dorota Matecka2, Julian J. O’Rear2, Ita S. Yuen2*, Charles
Litterst3, and Patricia Reichelderfer4 for the International Working Group on
Microbicides#
From the 1Center for Biologics Evaluation and Research and 2Center for Drug
Evaluation and Research, Food and Drug Administration, Rockville, Maryland, 3National
Institute of Allergy and Infectious Diseases and 4National Institute of Child Health and
Human Development, National Institutes of Health, Bethesda, Maryland
# The members of the International Working Group on Microbicides are listed in the
Acknowledgements.
Disclaimer: this paper was endorsed by the International Working Group on
Microbicides, but does not necessarily reflect the policies of individual members or their
respective agencies.
ACKNOWLEDGEMENTS
I would like to acknowledge helpful discussions over the years with fellow
members of:
• International Working Group on Microbicides
• UK Medical Research Council’s Microbicide Development Programme
and with colleagues at:
•
•
•
•
•
UK Medical Research Council’s Clinical Trials Unit
London School of Hygiene & Tropical Medicine
Imperial College School of Medicine, London
St George’s Hospital Medical School, London
St Bartholomew’s Hospital Medical College, London
E-mail: [email protected]