EL APRIORISMO MORALIZANTE AL SERVICIO DEL «ESPÍRITU COMERCIAL»

NHP infection models use two different challenge schemes that involve either a multiple low-dose or single high-dose challenge with simian immunodeficiency virus (SIV) or a chimeric SIV/HIV virus (SHIV) (Veazey, Shattock, Klasse, & Moore, 2012). Typically the virus used to evaluate microbicides is an SIV modified to express the HIV-1 envelope (SHIV) or the HIV-1 reverse transcriptase (RT-SHIV) (Pal et al., 2012).

Using the NHP model, gel formulations of an entry inhibitor, cyanovirin-N (Tsai et al., 2003), and the reverse transcriptase inhibitors, TFV and MIV- 150 (Cranage et al., 2008; Singer et al., 2011) prevented rectal challenge of SHIV or RT-SHIV.

61 In the study by Tsai et al., macaques (Macaca fasicularis) were randomized to receive 1% cyanovirin (n = 5), 2% cyanovirin (n = 5), placebo (n = 5), or no gel (n = 4). Twenty minutes after a 2 mL rectal gel application the animals were challenged with 1,000 TCID50 of SHIV89.6P (Tsai et al., 2003). All the

animals receiving cyanovirin gel were protected from SHIV infection whereas the placebo gel or no gel animals all became infected with SHIV.

In the study by Cranage et al., a total of 20 Indian rhesus macaques were used to evaluate the protective efficacy of topical TFV (Cranage et al., 2008). Nine animals received rectal TFV 1% gel up to 2 hours prior to virus challenge, four macaques received placebo gel, and four macaques remained untreated. In addition, three macaques were given TFV gel 2 hours after virus challenge. Following intrarectal instillation of 20 median rectal infectious doses (MID50) of a non-cloned, virulent stock of

SIVmac251/32H, all animals were analysed for virus infection, by virus isolation from PBMC, quantitative proviral DNA load in PBMC, plasma viral RNA (vRNA) load by sensitive RT-PCR, and presence of SIV-specific serum antibodies by ELISA. Eight of nine macaques given TFV per rectum up to 2 hours prior to virus challenge were protected from infection (n = 6) or had modified virus outcomes (n = 2), while all untreated macaques and three of four macaques given placebo gel were infected, as were two of three animals receiving TFV gel after challenge. Moreover, analysis of lymphoid tissues post mortem failed to reveal sequestration of SIV in the protected animals. There was a strong positive association between the concentration

62 of TFV in the plasma 15 minutes after rectal application of gel and the degree of protection in the six animals challenged with virus at this time point. Moreover, colorectal explants from non-SIV challenged TFV-treated macaques were resistant to infection ex vivo, whereas no inhibition was seen in explants from the small intestine. Tissue-specific inhibition of infection was associated with the intracellular detection of TFV. Intriguingly, in the absence of seroconversion, Gag-specific gamma secreting T cells were detected in the blood of four of seven protected animals tested, with frequencies ranging from 144 spot forming cells (SFC)/106 PBMC to 261 SFC/106 PBMC (Cranage et al., 2008).

Singer et al. have evaluated the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150, in a carageenan formulation, in a rhesus macaque rectal challenge model (Singer et al., 2011). A total of four macaques received the MIV-150 gel either 30 minutes or four hours before rectal challenge with either 103 _{or 10}4 _TCID

50 of a SHIV-RT

(SIVmac239/HIV-1HXB2). A control group was treated with a placebo methyl

cellulose placebo gel. All the MIV-1 treated macaques exposed to 103 TCID50 of the SHIV-RT were protected from infection whereas only two of the

four macaques exposed to the higher dose of SHIV-RT (104 TCID50) were

protected from infection.

The most recent NHP rectal challenge study evaluated maraviroc (MVC) that had been formulated in a rectal specific hydrogel based formulation (Dobard et al., 2013). The MVC gel formulation was designed to have a neutral pH

63 and to be close to iso-osmolar. Chinese Rhesus macaques were assigned to a matching rectal placebo gel (n = 5) or MVC gel (n = 6). Twice-weekly rectal SHIVSF162p3 challenges (500 TCID50) were performed for 5 weeks. Gel (4

mL) was applied rectally 30 minutes before each challenge. MVC was measured in plasma 30 minutes after gel application using LC/MS. Infection was monitored by PCR of SHIV in plasma. Infected macaques continued to receive gel for an additional six weeks to monitor the potential impact of drug exposure on systemic viremia. All the placebo-treated macaques were infected after a median of four challenges. In contrast, four of six macaques receiving MVC gel remained protected after ten challenges, demonstrating high efficacy (84%, p=0.020; Fisher’s exact test). Low levels of MVC (median = 4 ng/ml; range: 0-19) were detected in plasma 30 minutes after rectal dosing, suggesting MVC was rapidly released from the gel and absorbed. Plasma viremia in breakthrough infections was similar to controls.

To begin to address where rectal microbicides might distribute after dosing in a more formal way, a multi-compartment pharmacokinetic study in macaques after vaginal or rectal dosing with TFV gel was done (Nuttall et al., 2012). Macaques dosed with TFV gel vaginally showed rectal drug levels only 1 log10 lower than the vaginal drug levels. Both mucosal compartments

were 4-5 log10 higher than plasma drug levels. Similar results were found

when the macaques were dosed with TFV gel rectally. These data suggest that vaginal or rectal dosing of a soluble microbicide could protect against HIV-1 regardless of the route of exposure.

64 The NHP rectal microbicide challenge model provides compelling evidence to suggest that a rectal microbicide could provide significant protection from HIV acquisition in human efficacy studies. However, the model also illustrates that product efficacy may be impacted by the size of the NHP viral inoculum and the temporal association between when products are administered and when the animals are challenges with virus.

1.6 Rectal microbicide formulation considerations

Formulation of APIs for rectal use will likely be in a liquid or semi-solid dosage form to cover areas that are at most risk for HIV-1 exposure (Wang, Schnaare, Dezzutti, Anton, & Rohan, 2011). Preclinical testing of formulated APIs adds additional complexity because pH, osmolality, and viscosity of the product will impact the results. For instance, the polymers used in the formulation may enhance toxicity or efficacy due to smothering of individual cells or non-specifically binding HIV-1. Therefore, it is critical to include the vehicle control, the same formulation but without the API, in all assays to accommodate the impact the formulation may have on the testing results. As with unformulated APIs, testing algorithms have been developed (Rohan et al., 2010). Typically, the testing done with the formulations is to ensure the APIs activity has not been impaired and the formulation is safe. Mucosal tissues are used for testing the formulations, but are polarized, keeping the apical surface at the liquid/air interface (Rohan et al., 2010; Abner et al., 2005; Cummins, Jr. et al., 2007). The formulation with or without HIV-1 can be applied to the apical surface recapitulating the use by a person. Using this testing algorithm, it was recently reported the TFV 1% gel was hyperosmolar and consequently demonstrated epithelial fracture and

65 sloughing in polarized mucosal tissue (Rohan et al., 2010). The gel was reformulated to reduce the glycerin content and thus reduce the osmolality (Dezzutti et al., 2012). The reduced glycerin TFV 1% gel showed improved epithelial retention in polarized rectal and ectocervical tissue explants. These data support the clinical trial results (discussed below) that showed that gastrointestinal AEs were significantly more common when the original TFV 1% gel was used rectally compared to the reduced glycerin gel formulation (Anton et al., 2011; McGowan et al., 2012). The preclinical testing of formulations is thus important to ensure those products that move into clinical trials are safe as well as effective.

1.7 Preclinical evaluation of rectal microbicides

Preclinical testing of API, has been standardized but there are subtle variations in the specific assays used based on the preferences of the laboratory doing the testing (Buckheit, Jr. & Buckheit, 2012; Lackman-Smith et al., 2008; Lard-Whiteford, 2004). Initial tests using primary immune cells, such as peripheral blood mononuclear cells, and indicator cell lines are performed to determine mechanisms of action and potency of the API against standard laboratory and primary clinical isolates of HIV-1. Some testing is now being conducted with the newly identified primary isolates known as transmitter/founder viruses from persons who acquired HIV-1 through penile-vaginal or penile-rectal coitus (Keele et al., 2008; Dezzutti et al., 2012). The incorporation of biological fluids such as semen, cervicovaginal fluid, or their simulants is also used early in the testing to ensure the API remains potent during coitus (Neurath, Strick, & Li, 2006; Patel et al., 2007).

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1.8 Clinical evaluation of rectal microbicides

To date, the majority of rectal microbicide studies have evaluated either placebo formulations or been restricted to the Phase 1 rectal safety evaluation of antiretroviral candidates such as TFV or UC781 gels. A Phase 2 extended rectal safety study of TFV gel has just started screening and enrollment (MTN-017; http://clinicaltrials.gov/: NCT01687218). The general characteristics of Phase 1-3 microbicide development are outlined below.

1.8.1 Phase 1

Drug development including microbicide development involves a number of different stages. In the preclinical phase, new molecules are evaluated for safety and efficacy in cell lines and animal models. Compounds with an adequate safety profile are then advanced into Phase 1 safety studies where small groups of participants (10-30) are exposed to the product in very controlled circumstances for relatively short periods of time (1-2 weeks). The participants in Phase 1 RM studies are usually at very low risk of HIV acquisition and are asked to be sexually abstinent during the study.

1.8.2 Phase 2

On completion of Phase 1 studies, candidate microbicides are then evaluated in Phase 2 studies. Characteristically, Phase 2 microbicide studies are conducted in larger groups of sexually active participants (100-200) for three to six months and are designed to identify safety or acceptability issues associated with frequent use of the product. On completion of Phase 2 development a candidate microbicide then advances into an effectiveness (Phase 2B/3) study. This is the final phase of testing and seeks to determine

67 whether the product can actually reduce HIV acquisition rates in at risk populations.

1.8.3 Phase 2B/3

Phase 2B/3 evaluation of microbicides is the most arduous phase of assessment. Of necessity, the microbicide intervention has to be evaluated in populations who are already receiving a comprehensive HIV prevention package. The components of this package continue to evolve but would be expected to include diagnosis and treatment of STIs, frequent safer sex counseling, condom provision, and possibly male circumcision (UNAIDS, 2007). The net effect of these interventions is that the participants enrolled in Phase 2B/3 studies often develop a lower risk of infection than their peers not participating in the study, potentially reducing the overall HIV incidence in the study population and therefore the power to find a statistically significant result. As a consequence, Phase 2B/3 studies are usually large (2000-3000 participants), long (2-3 years), and expensive.

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Chapter 2