Development of ST-246® for Treatment of Poxvirus Infections

Abstract

ST-246 (Tecovirimat) is a small synthetic antiviral compound being developed to treat pathogenic orthopoxvirus infections of humans. The compound was discovered as part of a high throughput screen designed to identify inhibitors of vaccinia virus-induced cytopathic effects. The antiviral activity is specific for orthopoxviruses and the compound does not inhibit the replication of other RNA- and DNA-containing viruses or inhibit cell proliferation at concentrations of compound that are antiviral. ST-246 targets vaccinia virus p37, a viral protein required for envelopment and secretion of extracellular forms of virus. The compound is orally bioavailable and protects multiple animal species from lethal orthopoxvirus challenge. Preclinical safety pharmacology studies in mice and non-human primates indicate that ST-246 is readily absorbed by the oral route and well tolerated with the no observable adverse effect level (NOAEL) in mice measured at 2000 mg/kg and the no observable effect level (NOEL) in non-human primates measured at 300 mg/kg. Drug substance and drug product processes have been developed and commercial scale batches have been produced using Good Manufacturing Processes (GMP). Human phase I clinical trials have shown that ST-246 is safe and well tolerated in healthy human volunteers. Based on the results of the clinical evaluation, once a day dosing should provide plasma drug exposure in the range predicted to be antiviral based on data from efficacy studies in animal models of orthopoxvirus disease. These data support the use of ST-246 as a therapeutic to treat pathogenic orthopoxvirus infections of humans.

Keywords: ST-246; Smallpox; Tecovirimat; antiviral drug; egress inhibitor; orthopoxvirus; p37.

Figure 1.

Structure activity relationships and chemical information for ST-246. A summary of antiviral activity of chemical analogs of ST-246 (adapted from Bailey et al. [18]). EWG—Electron withdrawing groups; EDG—Electron donating groups. R refers to modifications of the phenyl ring.

Figure 2.

ST-246 inhibits production of extracellular virus and systemic virus spread in vitro and in vivo. (A) Diagram showing the four infectious forms of vaccinia virus and viral genes required for each step in the morphogenesis process. Vaccinia virus genes involved in each step in the pathway are shown (adapted from Smith et al. [23]). (B) ST-246 inhibits extracellular virus (CEV and EEV) formation. BSC-40 cell monolayers were infected with vaccinia virus at 5 pfu/cell in the presence and absence of 10 μM ST-246. The cell monolayers were radiolabeled with ³⁵-S methionine and vaccinia virus particles, either cell-associated (upper graph) or released into the culture medium (lower graph) were fractionated by equilibrium centrifugation on cesium chloride gradients. The radiolabeled material in each fraction was quantified by liquid scintillation. The assignment of each type of virus particle was based upon their reported density (adapted from Chen et al.) [48] (C) ST-246 inhibits plaque formation. BSC-40 cell monolayers (1 × 10⁶ cells/well) were infected with 10-fold serial dilutions of vaccinia virus in the presence and absence of 5 μM ST-246. At 3 days post-infection, the cultures were fixed in 5% glutaraldehyde and stained with crystal violet to visualize plaques. (D) ST-246 protects mice from systemic disease. Mice were inoculated with a lethal dose of vaccinia virus (WR) via the intranasal route. ST-246 was administered at 100 mg/kg as a liquid suspension by oral gavage once per day for 14 days. Shown are mice treated with ST-246 or placebo at day 8 post-infection.

Figure 3.

Dose normalized exposure of ST-246 in BALB/c mice from Day 1 of multiple studies. Dose normalized area-under-the curve values are plotted versus dose for mouse toxicology studies. The results suggest that absorption limits increased exposure as the dose is increased. AUC_inf = Area under concentration time curve for plasma drug levels after oral dosing from time 0 extrapolated to infinite time.

Figure 4.

Correlation between maximum monkeypox viral DNA levels in the blood versus maximum lesion number in non-human primates infected with monkeypox virus. Viral DNA levels were determined by quantitative PCR assay. Maximum viral DNA levels and lesion number were determined over a period of 21 days starting at the time of infection (Day 0). All doses were administered by oral gavage. Dose levels ranged from 0.3 mg/kg/day to 300 mg/kg/day for 14 days. Treatment was initiated on Day 1 (E3), Day 5 (E6), Days 3 or 4 (21G) or lesion onset on Day 3 or 4 (26G). Studies 21G and 26G were randomized, double blind, placebo-controlled studies conducted in compliance with GLP guidelines. Pearson’s correlation coefficient was calculated to show the degree of correlation.

Figure 5.

Efficacy of ST-246 in a non-human primate model of monkeypox virus infection. Cynomolgus macaques were infected with 5 × 10⁷ pfu of monkeypox virus via intravenous inoculation. At 3 days post-inoculation, ST-246 was administered by oral gavage once per day for 14 consecutive days. Viral DNA in the blood was determined by quantitative PCR. Viral DNA and lesion counts were measured every day for 21 days (adapted from Jordan et al.) [63]. (A) The maximum viral DNA levels in the blood and lesion number for individual animals over the course of the experiment were plotted per treatment group. The average value (bar) for each treatment group is shown. (B) Daily lesion numbers and viral DNA levels in the blood were shown for vehicle-treated animals (blue circles) and ST-246-treated animals at 3 mg /k g (red squares) with the average values plotted as a single black line. Cross symbols indicate day of death. The dashed horizontal line shows the limit of quantification of viral DNA.

Figure 6.

A comparison of ST-246 exposure (area under the concentration time curve, AUC) in monkeys and humans. Monkey doses were converted to human equivalent doses by multiplying first by 0.32 in accordance with FDA Guidance for Industry (entitled Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, published July 2005) and then multiplying by the average weight of human (78.6 kg) from the clinical study. The maximum, minimum and mean AUC for monkeys is shown in the green triangles, brown squares, and blue diamonds, respectively. The range in AUC values in humans is indicated by the symbols. The red dashed line indicates the dose of ST-246 that protects non-human primates from lethal infection.