Antiviral

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. [2]

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[2].

ST-246 exhibited potent antiviral activity against a broad spectrum of orthopoxviruses in CPE assays while showing little activity against unrelated RNA and DNA containing viruses. The EC50 values for inhibition of viral replication ranged from 0.01 μM for vaccinia virus to 0.07 μM for ectromelia virus to greater than 40 μM for unrelated viruses. Notably, cowpox appears to be less susceptible to ST-246 when compared on the same cell lines (5 to 50-fold). The mechanism of reduced susceptibility to ST-246 is unknown but may reflect a different mode of virus spread that is less dependent upon formation of extracellular virus. ST-246 was active against a CDV-resistant (CDVr) cowpox virus (EC50 = 0.05 μM), suggesting that the mechanism by which ST-246 inhibits virus replication is distinct from that of CDV. Furthermore, ST-246 inhibited clinical isolates from both of the major clades of monkeypox and variola viruses in cell culture. ST-246 inhibited orthopoxvirus replication in a variety of cell types including human embryonic lung fibroblasts, primary human keratinocytes, and organotypic endothelial raft cultures [2].

Efficacy of ST-246 in Small Animal Models of Orthopoxvirus Disease[2]
Models of orthopoxvirus disease were developed in mice, including BALB/c, NMRI, ANC/R and Nu/nu, rabbits, prairie dogs, and ground squirrels. These models provided opportunities to evaluate the antiviral activity of ST-246 against multiple species of orthopoxviruses, including vaccinia virus strains IHD-J, Lister, and WR, ectromelia virus, strain Moscow, cowpox virus, rabbitpox virus, and monkeypox virus. Infections were established by a variety of routes including intranasal, intravenous, intradermal, subcutaneous and aerosol delivery of virus. In all cases, ST-246 protected animals from severe disease and death. ST-246 treatment has been demonstrated to inhibit poxvirus dissemination virus shedding and systemic disease in mice. These models were used to optimize dosing strategies for antiviral efficacy and studies were conducted to evaluate the effect of varying the dose level, dose duration, and time of treatment post-infection on disease outcome (Table 1). From these studies, we have determined that once per day oral dosing in mice at 100 mg/kg, for a period of greater than seven days appears to be optimal for providing protective efficacy. Treatment can be initiated as late as 72 hours post-infection for full protection. In one experiment in prairie dogs infected with monkeypox virus, treatment initiated 10 days post-infection resulted in 100% protection from death. This result is striking in that the mean time to death in this experimental system is 11 days.
Mice that survive lethal infection due to ST-246-treatment are resistant to subsequent challenge with lethal doses of vaccinia virus due to acquisition of protective immunity during the initial infection. ST-246 has also been shown to protect in mice from lethal infection that are deficient in either CD4+ or CD8+ T cells, but not both, regardless of the presence or absence of B-cell deficiency. ST-246 treatment in combination with smallpox vaccination does not appear to diminish the immune response raising the possibility that ST-246 could be co-administered with the smallpox vaccine to reduce vaccine-related side-effects and protect individuals from infection prior to acquisition of protective immunity. Taken together these results support further development of ST-246 for treatment of pathogenic orthopoxvirus infections.

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Non-human Primate Models of Orthopoxvirus Infection[2]
Infection of non-human primates (NHP) via intravenous injection (IV) of monkeypox virus has been used to evaluate efficacy of ST-246. ST-246 administered at three days post-infection (dpi) at four different doses, from 100 mg/kg down to 3 mg/kg, once a day for 14 days, protected NHP 100% from a lethal infection with monkeypox virus (MPX) and reduced the viral load and lesion formation. In NHP, a ST-246 dose of 10 mg/kg/day for 14 days resulted in blood exposure comparable to levels attained in humans administered 400 mg in the fed state. A randomized double blind, placebo controlled study was conducted to evaluate the efficacy of ST-246 in cynomolgous macaques inoculated with a lethal dose of monkeypox virus via intravenous injection. Treatment was initiated at three and four days post-infection and ST-246 delivered at 10 mg/kg or placebo was administered by oral gavage once per day for 14 consecutive days. The results show that ST-246 administered at three or four days post infection protected animals from lethal infection and reduced lesion formation and viral DNA levels in the blood. In this model, five of the 16 NHPs showed lesion onset on Day 3 while the remaining 11 animals in the study all had lesions by Day 4 post-inoculation.

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Pharmacokinetics[2]
The nonclinical pharmacokinetic profile of ST-246 was evaluated in several in vivo studies in BALB/c mice, Spraque-Dawley rats, New Zealand White rabbits and Cynomolgus monkeys. Although the solubility of ST-246 is low it is highly permeable (Biopharmaceutics Classification System (BCS) Class II) and has high levels of oral bioavailability, which increases when the compound is co administered with food. The initial evaluation of bioavailability in mice showed that approximately 40% of the compound was bioavailable when the area under the concentration time curve (AUC) value of a 1 mg/kg intravenous infusion was compared to an oral dose of 30 mg/kg of ST-246. Higher doses had lower apparent bioavailability. This was most likely due to decreased absorption that was observed as the dose was increased. In rats, the bioavailability was 90% and 33%, respectively, for males and females after oral administration of 30 mg/kg ST-246. The lower concentrations of ST-246 exposure observed in female rats was consistent with first pass metabolism while multiple dose administration resulted in much lower exposure in both male and female rats, suggesting induction of metabolism. Over the course of extensive repeat dose studies in mice, however, there was no consistent evidence of induced metabolism, suggesting that this phenomenon was rat specific. The predominant cause of nonlinearity in the pharmacokinetics of ST-246 observed in mice was the apparent decreased absorption with increasing dose. The decreased absorption was observed in both the observed maximum plasma concentrations as well as the exposure (as determined by AUC values). Thus, as the doses were increased, exposures also increased but not dose proportionally (Figure 3).

[1]. N Engl J Med.2018 Nov 22;379(21):2084-2085; [2]. Viruses.2010 Nov;2(11):2409-35.

Tecovirimat is an antiviral prescription medicine approved by the U.S. Food and Drug Administration (FDA) for the treatment of smallpox in adults and children. (Smallpox is a disease caused by the variola virus, a member of the orthopoxvirus group of viruses. Since 1980, smallpox has been considered eradicated worldwide.)

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Although tecovirimat is only approved for smallpox treatment, it may also be used to treat other types of orthopoxvirus infections, including mpox. Tecovirimat can be used for mpox treatment through an expanded access program (also known as compassionate use) or through clinical trials.

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The World Health Organization declared smallpox, a contagious and sometimes fatal infectious disease, eradicated in 1980. However, there have been longstanding concerns that smallpox may be used as a bioweapon. Tecovirimat is an antiviral drug that was identified via a high-throughput screen in 2002. It is effective against all orthopoxviruses, including vaccinia, cowpox, ectromelia, rabbitpox, monkeypox, and Variola (smallpox) virus. Tecovirimat was approved by the FDA in July 2018 as the first drug ever approved to treat smallpox. Tecovirimat was later approved by Health Canada in December 2021, followed by the approval from the European Commission in January 2022. Other than smallpox, tecovirimat is also indicated to treat complications due to replication of the vaccinia virus following vaccination against smallpox, and to treat monkeypox and cowpox in adults and children. Tecovirimat is available as both oral and intravenous formulations.

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Tecovirimat is an Orthopoxvirus VP37 Envelope Wrapping Protein Inhibitor. The mechanism of action of tecovirimat is as a Cytochrome P450 3A Inducer, and Cytochrome P450 2C8 Inhibitor, and Cytochrome P450 2C19 Inhibitor, and Breast Cancer Resistance Protein Inhibitor.

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Tecovirimat is an orally available antiviral agent with activity against smallpox and other orthoviruses that is approved for use against smallpox virus (variola) infection in humans and has been used on a compassionate use basis disseminated vaccinia and monkeypox (now renamed “mpox”) infection. Tecovirimat therapy has not been linked to serum aminotransferase elevations or to instances of clinically apparent liver injury.

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Tecovirimat is an orally available small molecule with activity against orthopoxviruses. Tecovirimat is an inhibitor of viral p37 and blocks the ability of virus particles to be released from infected cells.

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The elimination half-life (CV%) was 21 (45%) hours following intravenous administration of 200 mg tecovirimat and 19 (29%) hours following oral administration of 600 mg tecovirimat.

C19H15F3N2O3

376.35

376.103

869572-92-9

Tecovirimat-d4

16124688

Typically exists as white to off-white solids at room temperature

2.73

66.48

O=C(NN(C(C1C2C(C3)C3C(C=C2)C41)=O)C4=O)C5=CC=C(C(F)(F)F)C=C

CSKDFZIMJXRJGH-VWLPUNTISA-N

InChI=1S/C19H15F3N2O3/c20-19(21,22)9-3-1-8(2-4-9)16(25)23-24-17(26)14-10-5-6-11(13-7-12(10)13)15(14)18(24)27/h1-6,10-15H,7H2,(H,23,25)/t10-,11+,12+,13-,14-,15+

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2.5 mg/mL (6.64 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.64 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)