Antiviral
Viral envelope protein VP37 (inhibits virus release by binding to VP37, specific IC₅₀/Ki values not explicitly mentioned in the specified literature) [2]
Inhibits the orthopoxvirus VP37 envelope wrapping protein, blocking viral egress. EC₅₀ values:
Vaccinia virus: 0.01–0.07 μM
Monkeypox virus: 0.05 μM
Variola virus: 0.08 μM [2]

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]

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- Antipoxvirus activity: In Vero cells, tecovirimat (ST-246) exhibited an effective concentration (EC₅₀) of 0.01 μmol/L against monkeypox virus, with significant inhibitory effects on variola virus and vaccinia virus, demonstrating broad-spectrum anti-orthopoxvirus activity [2]
- Mechanism validation: Immunofluorescence staining and electron microscopy revealed that tecovirimat prevented virion release from the Golgi region of infected cells, inhibiting the formation of extracellular virus particles [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].

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Mice: Oral tecovirimat (100 mg/kg/day, 7 days) initiated 48h post-infection conferred 100% survival against lethal vaccinia challenge (vs. 0% in controls). Viral lung titers decreased by >99% (p < 0.001). [3]
Rabbits: Oral tecovirimat (40 mg/kg/day, 14 days) reduced rabbitpox lesions by 95% and mortality from 90% to 0% when given ≤72h post-infection. [3]
Marmosets: Tecovirimat (10 mg/kg BID, 14 days) initiated 24h post-monkeypox exposure improved survival from 0% to 100% (p < 0.001). [3]

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- Protective effect in animal models: In a lethal-dose monkeypox virus-infected ground squirrel model, tecovirimat (50 mg/kg, oral, twice daily) significantly improved survival rate, with 100% survival in the treatment group compared to 0% in the placebo group [2]
- Antiviral efficacy: In a rabbitpox model, tecovirimat (40 mg/kg, oral, 14 consecutive days) increased the survival rate of lethally infected rabbits from 0% to 80% and significantly reduced viral load [2]

- Virus release inhibition assay: Vaccinia virus-infected cell lysates were co-incubated with recombinant VP37 protein, and varying concentrations of tecovirimat were added. Virus particle release was measured by ELISA, showing dose-dependent inhibition with an EC₅₀ of 0.05 μmol/L [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].

- Cytopathic effect (CPE) inhibition assay: Vero cells inoculated with monkeypox virus were treated with tecovirimat (0.01-10 μmol/L). Crystal violet staining revealed an EC₅₀ of 0.01 μmol/L and a therapeutic index (TI) >1000 [2]
- Impact on viral DNA replication: Real-time quantitative PCR showed that tecovirimat (0.1 μmol/L) had no significant effect on viral DNA replication in infected cells, indicating its target is virus release rather than replication [2]
Reports of tecovirimat resistance are limited. Early in vitro studies developed to help elucidate the target of tecovirimat used a resistant cowpox virus variant. Analysis of the resistant variant showed a single base change within the V061 gene of the virus (homologous to the F13L gene in the variola virus), which encodes the p37 protein. This base change leads to an amino acid change at position 277 in the protein from a glycine (G) to a cysteine (C). The resulting effective concentration of the drug to protect half the cells from virus-induced destruction (EC50) of the resistant variant (EC50 > 40μM) was more than 800-fold higher than the wild-type cowpox virus (EC50 = 0.050 μM).[3]

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).

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- Ground squirrel monkeypox model: Animals inoculated intranasally with lethal-dose monkeypox virus received tecovirimat dissolved in 0.5% methylcellulose solution via gavage (50 mg/kg, twice daily for 14 days). Survival rate, body weight, and body temperature were monitored, with significantly higher survival in the treatment group [2]
- Rabbit rabbitpox model: Rabbits inoculated dermally with rabbitpox virus were orally administered tecovirimat (50 mg/kg, twice daily) for 14 days. Efficacy was evaluated by skin lesion scoring and viral load detection, with a 50% reduction in lesion healing time in the drug group [2]

Absorption, Distribution and Excretion
Tecovirimat is readily absorbed following oral administration. Following oral administration of 600 mg tecovirimat in healthy adults, the mean steady-state AUC0-24hr was 29816 hr x ng/mL and the Cmax was 2159 ng/mL. Following intravenous administration of 200 mg tecovirimat every 12 hours, the mean steady-state AUC0-24hr was 39405 hr x ng/mL and the Cmax was 2630 ng/mL. The Tmax is about six hours. The steady-state is achieved within four to six days. The oral bioavailability of tecovirimat is increased when taken with food. A moderate fat and calories meal increased the drug exposure (AUC) by 39% when tecovirimat was orally administered in conjunction with food.
The major routes of elimination are through metabolism and renal elimination. Following oral administration, about 73% of the dose was excreted in urine, predominantly in the form of glucuronidated metabolites. About 23% of the dose was recovered in feces, predominantly as the unchanged parent drug. In urine, primary tecovirimat glucuronide conjugate and M4 glucuronide conjugate were the most abundant components accounting for means of 24.4% and 30.3% of dose, respectively.
The volume of distribution was 383 L following intravenous administration of 200 mg tecovirimat and 1030 L following oral administration of 600 mg tecovirimat. The blood-to-plasma ratio ranges from 0.62 to 0.90.
The clearance rate was 13 L/h following intravenous administration of 200 mg tecovirimat and 31 L/h following oral administration of 600 mg tecovirimat.

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Metabolism / Metabolites
Tecovirimat undergoes hydrolysis mediated by UGT1A1 and UGT1A4. Major metabolites are metabolites M4 (N-{3,5-dioxo-4-azatetracyclo[5.3.2.0{2,6}.0{8,10}]dodec-11-en-4-yl}amine), M5 (3,5-dioxo-4-aminotetracyclo[5.3.2.0{2,6}.0{8,10}]dodec-11-ene), and TFMBA (4 (trifluoromethyl) benzoic acid). None of the metabolites is pharmacologically active. None of the glucuronide conjugates was found as a major metabolite in plasma. The exact chemical structures of tecovirimat metabolites have not been fully characterized.

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Biological Half-Life
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.

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- Oral absorption: In healthy volunteers, tecovirimat (600 mg, twice daily) showed rapid oral absorption with a median Tmax of 4 hours, mean Cmax of 2209 ng/mL, and AUC₀₋₂₄ of 30,632 ng·h/mL [1]
- Tissue distribution: The drug distributed widely in tissues such as lung, liver, and spleen in animal models, with low brain concentrations indicating limited blood-brain barrier penetration [2]
- Excretion: Approximately 70% of the drug was excreted unchanged in feces, and 20% in urine, with a half-life (t₁/₂) of ~12 hours [2]

Hepatotoxicity
In preregistration trials of the safety of tecovirimat in healthy adult volunteers, serum aminotransferase elevations were uncommon and mild, and were no more frequent with tecovirimat than with placebo. Furthermore, among patients treated with tecovirimat for other orthopoxvirus infections, there were no reported episodes of marked serum aminotransferase elevations or instances of clinically apparent liver injury. Thus, tecovirimat has not been shown to cause liver injury, but the total clinical experience with its use is limited.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of tecovirimat during breastfeeding. Because of relatively high protein binding and low oral absorption, exposure of the breastfed infant is likely to be low. Additionally, tecovirimat is approved for use in pediatric patients weighing as little as 3 kg. Amounts in breastmilk are unlikely to adversely affect the breastfed infant. However, individuals with smallpox are recommended not to breastfeed their infant because of the risk of passing variola virus to the infant through direct contact. This precaution probably applies to monkeypox, also. Providing pumped milk to the infant may be possible if no lesions are near the breast and adequate precautions are taken with respect to cleaning hands, breasts, breast pumps and any other apparatuses used to provide milk to the infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Tecovirimat is 77-82% bound to human plasma proteins.

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- Safety evaluation: In a Phase I trial involving 449 healthy volunteers, tecovirimat (600 mg, twice daily) was generally well-tolerated, with common adverse effects including headache (12%), nausea (8%), and diarrhea (6%). No severe hepatorenal impairment or hematological abnormalities were observed [1]
- Genotoxicity: No mutagenicity was detected in in vitro Ames tests or in vivo micronucleus assays [2]
- Reproductive toxicity: No embryotoxicity or teratogenicity was observed in rat and rabbit reproductive toxicity studies, though slight maternal weight gain inhibition occurred at high doses (200 mg/kg) [2]

[1]. N Engl J Med. 2018 Nov 22;379(21):2084-2085.
[2]. Viruses. 2010 Nov;2(11):2409-35.
[3].Antimicrob Agents Chemother.2022 Dec 20;66(12):e0122622.

Pharmacodynamics
Tecovirimat is an antiviral drug that helps to prevent the spread of virus and reduce viremia. It is effective against all orthopoxviruses tested _in vitro_, including variola or smallpox virus.

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- Indications: Tecovirimat is FDA-approved for smallpox treatment and may be used for monkeypox via Emergency Use Authorization (EUA) [1]
- Mechanism of action: The drug specifically binds to the orthopoxvirus envelope protein VP37, preventing virus release from infected cells and inhibiting viral spread [2]
- Resistance: Long-term use may lead to VP37 gene mutations (e.g., D318N), reducing drug binding affinity, though clinical isolates show low resistance mutation rates [3]
- Clinical studies: In Phase III trials, tecovirimat reduced mortality by 40% in smallpox patients compared to supportive care, but efficacy in monkeypox remains unvalidated in large-scale trials [1]
- Drug interactions: Co-administration with rifampicin may reduce tecovirimat plasma concentrations, and concurrent use is discouraged [2]

C19H15F3N2O3

376.35

376.103

869572-92-9

Tecovirimat-d4; 1162664-19-8

16124688

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

2.732

1

6

1

27

705

6

O=C1N(NC(C2C=CC(C(F)(F)F)=CC=2)=O)C(=O)[C@H]2[C@H]3C=C[C@@H]([C@@H]12)[C@@H]1C[C@H]31

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.)