Tivoxavir marboxil, an Influenza Inhibitor Targeting Cap-dependent Endonuclease: Results from a Phase I Trial Demonstrating Safety, Tolerability and Pharmacokinetics and Demonstrating Protection Against H5N1 Bird Flu in a Rodent Challenge Model Ekaterina Dokukina1 , Viktoriya Moskovich1 , Boris Rogovoy1 , Ruben Karapetian2 , Alexander Ivaschenko2 , Robert R. Redfield3 , Iain D. Dukes3 , Nikolay Savchuk3 , Werner Cautreels3 , and C. David Pauza3 1 Viriom, Inc. Rockville, MD, 2 Chem Div, San Diego, CA, 3 Traws Pharma, Inc., Rockville, MD Tivoxavir marboxil (TXM) is a prodrug inhibitor of the Influenza virus cap-dependent endonuclease (CEN), which is encoded by the viral RNA segment 3 (Acidic polymerase). Due to its critical role in virus replication and very high level of sequence conservation across virus strains, CEN inhibition has become an important strategy for treatment and prevention of seasonal or pandemic influenza. In laboratory testing, the TXM metabolite TXA demonstrated an effective concentration 50% (EC50) in the picomolar range for a variety of Influenza A isolates and was up to 10-times more potent than Baloxavir (BXA), for inhibiting the BXA-resistant I38T variant of H1N1 or H3N2 A/Influenza. TXA also demonstrated picomolar EC50 against multiple highly pathogenic avian influenza H5N1 strains circulating in nature during 2022-2023. Oral dosing with TXM protected 100% of mice from a lethal, intranasal challenge with H5N1 (A/Texas/37/2024), a virus isolated from a dairy worker who was infected through contact with dairy cattle. The TXM Phase I clinical study evaluated 3 dose levels, single dose, in healthy volunteers. Pharmacokinetic studies of the highest dose level showed plasma drug levels at or above the predicted therapeutic window for ~25 days after a single dose. The laboratory and clinical findings for TXM support further development of this compound for treatment of seasonal or pandemic influenza and the pharmacokinetic data support additional studies on TXM as a potential treatment or prophylactic agent for H5N1 bird flu. The viral replication complex binds and cleaves host mRNA leaving the 5’ cap. Sequences in the 3’ end of viral genomic RNA (-strand) are complementary to the remaining fragment of host mRNA and serve to prime +strand RNA synthesis. Cap-dependent endonuclease inhibitors (CENI) prevent cleavage of host mRNA, reduce viral + strand synthesis and block genomic RNA replication. Adapted from Dou, et al., 2018. Front. Immunol. 9:1581. Adapted from: Mostafa, et al. (2025) Replication kinetics, pathogenicity and virus-induced cellular responses of cattle-origin influenza A(H5N1) isolates from Texas, United States, Emerging Microbes and Infections, 14:1 2447614, DOI: 10.1080/22221751.2024.2447614 Tivoxavir marboxil inhibits the cap-independent endonuclease of influenza and suppresses replication of baloxavir-resistant viruses. Tivoxavir marboxil was well-tolerated up to a single dose of 480 mg in a Phase I clinical trial with healthy adult participants Tivoxavir (dose equivalent to 240 mg in humans) protected 100% of mice from lethal infection with the human isolate A/Texas/37/2024 (H5N1) The cap-dependent endonuclease coding region in PA is highly conserved among H5N1 viruses from multiple species 5 Strain Designation Tivoxavir (TRX101) H5N1 viruses EC50 (nM) A/chicken/Idaho/22-011347-004/2022 0.40 A/Canada_goose/Wyoming/22-011671-001/2022 0.52 A/Red_Shoulder_Hawk/Minnesota/22-012000-004/2022 0.43 A/dolphin/Florida/22-025319-002/2022 0.34 A/black_vulture/Florida/22-012333-001/2022 - c30 0.41 Reference A(H3N2) Viruses A/Louisiana/50/2017 (Wt) 1.21 A/Louisiana/49/2017 (PA-138M) 10.28 Subtype/Lineage A(H1N1)pdm2009 A(H3N2) A/Illinois/37/2018 I38L 6.23 ± 1.05 (1) 13.71 ± 1.33 (7.70) A/Illinois/08/2018 I38T 22.26 ± 4.45 (11.97) 73.9 ± 14.31 (41.52) 36.33 ± 9.86 (19.53) 86.69 ± 19.18 (48.14) A/Illinois/08/2018 I38S 8.46 ± 3.62 (10.85) 11.94 ± 3.47 (12.44) A/Louisiana/50/2017 I38M 8.24 ± 1.73 (24.24 0 33.61 ± 7.61 (80.14) A/Yokohama/133/2018 I38T Source: Adapted from Chesnokov, A., et al. 2024. Antimicrob. Agents Chemother. 68: e0172723 Source: United States Centers for Disease Control and Prevention, Atlanta, Georgia 26.78 ± 4.62 (41.2) 81.41 ± 16.25 (92.51) A/Bangladesh/3007/2017 I38T Virus PA Mutation TXA BXA Mean EC50 +SD, nM (fold-change) Tivoxavir marboxil Tivoxavir exhibits sub-nanomolar potency against A/H5N1 circulating in nature during 2022-2023 Bovine to human transmission resulted in 9 amino acid changes across 5 influenza genome segments, that are associated with increased replication in mammalian cells Day 3 Lung Virus Titers (TCID 50) Animal 31 32 33 34 35 46 47 48 49 50 Virus titer - TCID 50/gram of tissue BLLQ-below lower limit of quantitation (2.2x10³ TCID 50/gram) Group Virus Titer 7x108 Vehicle TXM Day 1 dose of 50 mg/kg 1.3x107 1.3x108 8.2x108 1.2x107 BLLQ BLLQ BLLQ BLLQ BLLQ Cohort 1 (80 mg fasted) Sentinels Active (1); Placebo (1) Main Group Active (5); Placebo (1) Cohort 2 (120 mg fasted) Sentinels Active (1); Placebo (1) Main Group Active (5); Placebo (1) Cohort 3 (240 mg fasted) Sentinels Active (1); Placebo (1) Main Group Active (5); Placebo (1) Cohort 4 (480 mg fasted) Sentinels Active (1); Placebo (1) Main Group Active (5); Placebo (1) Virus challenge stock: H5N1 (A/Texas/37/2024) Stock titer: 1.47x109 TCID 50/mL Intranasal challenge: 7.34x100 TCID 50/dose Oral treatment - TXM given 0 and 12 hours after virus inoculation (total 50 mg/kg) 15 animals per group - 5 animals per group sacrificed on D3 for determination of virus titer in lungs Phase I Clinical Trial Schema for Oral Tivoxavir Marboxil in Healthy Adults: Safety, Tolerability and Pharmacokinetics 20 days Pharmacokinetics of Tivoxavir in Healthy . PB2 PB1 PA PA-X NA NS1 E E L K K I R N S R Q 40 E K v I K M G E L LEGEND: Amino acids in red are substitutions found in the human isolate A/Texas/37/2024 (H5N1) Amino acids in blue are amino acids found in the corresponding bovine isolate: A/bovine/Texas/24-029328-02/2024 (H5N1) 3 4 2 1 6 71 142 219 497 142 392 362 627 631