Non-clinical evaluation of microbicides: scientific approaches Alan Stone ( London, UK) Regional meeting on regulatory issues in microbicide research 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.

Cell-based in vitro assays Standard lab strains of HIV. R5 and X4 strains (use respectively CCR5 and CXCR4 co- receptors on surface of human lymphocytes). Cell-free and cell-associated virus. 1 or 2 dilutions of compound. Screening a large number of compounds to identify antiviral activity

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

In vitro data from laboratory of Professor D J Jeffries (London, UK) PRO 2000

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. Effects on other microorganisms and sperm

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. Drug resistance

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.

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. IWGM non-clinical recommendations J Acquir Immune Defic Syndr 2004, 36:

ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS I would like to acknowledge helpful discussions over the years with fellow members of: International Working Group on Microbicides International Working Group on Microbicides UK Medical Research Council’s Microbicide Development Programme UK Medical Research Council’s Microbicide Development Programme and with colleagues at: UK Medical Research Council’s Clinical Trials Unit UK Medical Research Council’s Clinical Trials Unit London School of Hygiene & Tropical Medicine London School of Hygiene & Tropical Medicine Imperial College School of Medicine, London Imperial College School of Medicine, London St George’s Hospital Medical School, London St George’s Hospital Medical School, London St Bartholomew’s Hospital Medical College, London St Bartholomew’s Hospital Medical College, London