Safety, Tolerability and Pharmacokinetics of Benfo-Oxythiamine (B-OT) in Healthy Volunteers

• This Phase 1 study was originally designed and conducted in 2022 to assess the safety and pharmacokinetics of Benfo-Oxythiamine (B-OT) for the potential treatment of SARS-CoV-2 infection (COVID-19), as defined in the study protocol.
• B-OT targets the Transketolase (TKT)/Transketolase-like 1 (TKTL1) pathway. Inhibition of this pathway affects glucose metabolism, a mechanism relevant to both viral replication (such as SARS-CoV-2) and tumor cell proliferation (Oncology).
• Current Scientific Context (2026): While the study was conducted under a COVID-19 indication, the safety and PK data generated are now also supporting development in Oncology. Recent literature (>2022) has further validated TKT/TKTL1 as a key target in e.g., chemotherapy resistance and cell cycle control. This recent literature functions as supporting evidence for the drug target. The results of this clinical phase I study are therefore also directly relevant for future investigations in cancer indications.
• Detailed Description: Scientific Rationale, Mechanism of Action, and Preclinical Validation of Benfo-oxythiamine (B-OT)
• 1. General Introduction and Target Biology Benfo-oxythiamine (B-OT) is a novel, lipophilic S-benzoyl-O-monophosphate prodrug developed to target fundamental metabolic dependencies across multiple disease states. It is important to emphasize that all subsequent mechanisms are validated in in vitro and in vivo preclinical models, providing a robust biological basis for the potential observed clinical responses across different indications. Upon administration, B-OT crosses cellular membranes and is rapidly hydrolyzed by ecto-alkaline phosphatases and cytosolic esterases to release the thiamine antagonist oxythiamine (OT). Intracellularly, OT is metabolized by thiamine pyrophosphokinase (TPK) into its biologically active form, oxythiamine pyrophosphate (OTP). OTP functions as a potent, competitive inhibitor of thiamine diphosphate (ThDP)-dependent enzymes. Its primary therapeutic targets are Transketolase (TKT) and Transketolase-like 1 (TKTL1), which serve as critical "gatekeepers" in the non-oxidative branch of the pentose phosphate pathway (PPP). By inhibiting these enzymes, OTP disrupts the supply of Ribose-5-Phosphate (R5P) needed for nucleotide synthesis, Nicotinamide Adenine Dinucleotide Phosphate (NADPH) for redox defense, and Acetyl-CoA for lipid biosynthesis, collapsing the metabolic infrastructure required for rapid cellular proliferation and pathogen replication.
• 2. Scientific Rationale in Oncology The therapeutic rationale for targeting TKT and TKTL1 in cancer addresses cell cycle dysregulation, metabolic reprogramming, immune evasion, and therapy resistance.
• Cell Cycle Control and "Metabolic Sensing": TKTL1 acts as a direct regulator of the cell cycle. The TKTL1-TKT heterodimer generates R5P, which binds directly to TKTL1. This complex acts as a metabolic sensor, recruiting the SCF-β-TRCP ubiquitin ligase complex to the cell cycle inhibitor CDH1, triggering its proteasomal degradation and allowing the cell to transition from G1 to the S phase. Pharmacological inhibition by B-OT depletes R5P, stabilizing CDH1 and enforcing a profound G1 cell cycle arrest, which sensitizes tumor cells to apoptosis.
• Reversal of Chemoresistance and the Warburg Effect: TKT drives metabolic resistance via a physical interaction with Pyruvate Kinase M2 (PKM2), preventing its tetramerization and forcing the cell into aerobic glycolysis (the Warburg effect). This provides the ATP and lactate necessary to shield the tumor from DNA-damaging drugs. TKT inhibition dismantles this TKT-PKM2 interaction, restoring mitochondrial OXPHOS, reducing lactate, and overcoming resistance to agents like cisplatin in renal cell carcinoma. Additionally, standard genotoxic therapies (e.g., UVA irradiation, Adriamycin) induce TKTL1 and the Warburg effect as a survival mechanism against starvation and oxidative stress; inhibiting this pathway strips tumors of this acquired resistance.
• Nucleotide Stress and DNA Repair Impairment: Rapidly dividing cells require a continuous supply of R5P from the PPP to synthesize nucleotides for repairing double-strand DNA breaks. OT treatment induces severe "nucleotide stress," impairing homologous recombination and non-homologous end joining, evidenced by the accumulation of the DNA damage marker γ-H2AX. Consequently, B-OT serves as a potent radiosensitizer and chemosensitizer in highly aggressive tumors.
• Lipid Starvation and Epigenetic Disruption: TKTL1 utilizes a unique one-substrate phosphoketolase reaction to cleave xylulose-5-phosphate into glyceraldehyde-3-phosphate and Acetyl-CoA without CO2 loss. This provides cytosolic Acetyl-CoA essential for de novo lipid synthesis and histone acetylation, driving neurogenesis and rapid proliferation. As intracellular Acetyl-CoA is the absolute bottleneck for histone acetylation and growth gene activation, inhibiting TKTL1 starves the cancer cell of this crucial epigenetic and structural substrate.
• Growth Factor Independence: Ectopic TKTL1 expression renders tumors resistant to serum withdrawal, bypassing apoptotic triggers caused by growth factor deprivation. TKT/TKTL1 upregulation allows cancers to maintain redox homeostasis (NADPH) and survive independently of hormonal signaling, driving resistance to endocrine therapies.
• Metastasis and Tumor Microenvironment Acidification: High TKTL1/TKT expression accelerates lactic acid production, creating an acidic tumor microenvironment. This acidosis promotes extracellular matrix degradation via the dysregulation of matrix metalloproteinases (MMPs), driving tumor invasion. OT treatment downregulates AKT-mediated PFKFB3 signaling, suppresses MMP-2 and MMP-9, and restores Tissue Inhibitors of Metalloproteinases (TIMP-1 and TIMP-2), significantly inhibiting metastatic dissemination in vitro and in vivo.
• Immunosuppression and Macrophage Polarization: Tumor-derived lactic acid paralyzes the local immune response, inhibiting TNF secretion, blunting natural killer (NK) and T cell immunosurveillance, and inducing PD-L1 expression on cancer cells. Reversing this acidosis systematically reactivates NK cells to express IFN-gamma and control tumors. Furthermore, PPP activity in macrophages fuels the UDP-glucose-STAT1-IRG1-itaconate cascade, polarizing them into an immunosuppressive M2-like phenotype. B-OT through its metabolite OT downregulates this axis, reprogramming macrophages toward a pro-inflammatory M1-like state, boosting IL-6 while decreasing IL-10, and dramatically enhancing antibody-dependent cellular phagocytosis (ADCP).
• The Lactylation Axis in Cancer: Lactate drives the epigenetic "lactylation" of histone and non-histone proteins. Lactylation of the DNA repair protein MRE11 enhances homologous recombination, promoting chemoresistance. By halting TKT-driven lactate production, B-OT cuts off the substrate required for lactylation, dismantling these epigenetic defenses.
• 3. Scientific Rationale in Virology Viruses act as obligate intracellular parasites, completely dependent on the host's metabolic machinery. B-OT acts as a host-directed therapy, targeting the specific metabolic reprogramming induced by viral infections.
• Disruption of Viral Supply Lines: Viral infection redirects host glucose into the PPP via TKT and TKTL1 to meet extreme anabolic demands. This supplies ATP for assembly, Ribose-5-Phosphate for viral RNA/DNA genome synthesis, and Acetyl-CoA/NADPH for the lipid biosynthesis needed to form viral replication complexes and envelopes. For example, dengue viruses specifically require this Warburg-like glycolytic flux for optimal replication. By inhibiting TKT/TKTL1, B-OT triggers a targeted metabolic trap, causing nucleotide and lipid starvation.
• Reversal of Viral Immune Evasion via Lactylation: Viruses hijack glycolysis to cause an infection-induced lactate surge. This lactate is utilized to lactylate the host immune sensor IFI16 at lysine 90, preventing it from recruiting DNA-PK and suppressing the induction of antiviral cytokines like IFN-beta. Lactylation of histones (e.g., H3K18la) also upregulates HSPA6, a negative regulator that blocks TRAF3/IKKε to shut down the interferon response. Furthermore, hyperlactatemia drives macrophages into an M2 state, impairing viral clearance, and is clinically correlated with severe dengue mortality. By lowering lactate production, B-OT unmasks the virus and sustains the host's innate antiviral interferon response.
• Vascular Integrity: In dengue, viral-induced MMP overproduction degrades the endothelial glycocalyx, causing plasma leakage. As previously noted, B-OT through it metabolite OT suppresses MMPs and restores TIMPs, offering a protective effect against vascular permeability.
• Validation in Viral Models: Preclinical data confirms the antiviral capacity of B-OT and OT. Pharmacological thiamine deficiency with OT increased resistance to the Lansing strain of poliomyelitis in mice. OT reversibly inhibited the growth of the psittacosis virus in tissue culture and protected cells from cytopathic effects induced by myxoviruses, including influenza and mumps. B-OT specifically inhibited SARS-CoV-2 replication in human cells in a concentration-dependent manner, showing synergy with glycolysis inhibitors.
• 4. Scientific Rationale in Mycology Thiamine antivitamins demonstrate selective antifungal activity. OT exerts potent, selective fungistatic activity against Malassezia pachydermatis and other Malassezia species (e.g., M. restricta, M. globosa), likely due to their unique cell wall structure and specific thiamine-dependent enzyme expression profiles. OT also reduces the proliferation of yeast such as Saccharomyces cerevisiae. Importantly, OT acts synergistically with standard antifungals like ketoconazole, significantly lowering the effective concentration needed to clear fungal strains, presenting a viable avenue for combination adjuvant therapies. Conversely, Candida albicans and standard dermatophytes/molds showed resistance to OT.
• 5. Scientific Rationale in Bacteriology OT functions as a potent antimetabolite against multidrug-resistant (MDR) bacterial strains, specifically Pseudomonas aeruginosa. OT inhibits ThDP-dependent enzymes like pyruvate dehydrogenase, causing severe metabolic disruption. It exhibits a low minimal inhibitory concentration (MIC50 ≈ 1.4 µM) in minimal media, retaining activity against efflux-pump mutants. OT sensitizes P. aeruginosa to standard antibiotics (e.g., tetracyclines) and non-antibiotics (e.g., 5-fluorouracil), demonstrating dramatic synergy and load reduction in murine ocular infection models. Its ability to dismantle bacterial central carbon metabolism provides a strong rationale for combination of antibacterial strategies.
• 6. Scientific Rationale in Parasitology Vitamin B1 biosynthesis and utilization are critical for parasite survival. OT targets thiamine pyrophosphokinase (TPK) in Leishmania donovani, competitively inhibiting the conversion of thiamine to its active cofactor form. This significantly impairs energy production and oxidative stress defense, demonstrating potent antiparasitic activity against both intracellular amastigotes and promastigotes without macrophage cytotoxicity. Similarly, Plasmodium falciparum (malaria) relies on scavenged thiamine; parasites overexpressing TPK become 1,700-fold more sensitive to OT, confirming lethal intracellular bioactivation. In vivo murine models confirmed that OT significantly reduces parasitemia and prolongs survival.
• 7. Safety and Selectivity Profile The broad therapeutic applicability of B-OT relies on a unique kinetic selectivity. The human TKT enzyme binds its physiological cofactor, thiamine diphosphate (ThDP), with exceptionally high, quasi-irreversible affinity via specific residues (e.g., Gln189) that lock the cofactor into the active site. Consequently, the active B-OT metabolite OTP cannot displace ThDP from pre-existing, stable holo-enzymes found in healthy tissues. Instead, OTP preferentially targets and inhibits newly synthesized apo-enzymes. Because rapidly proliferating cancer cells and virus-infected cells have extremely high rates of de novo protein turnover compared to resting cells, they are selectively starved and inhibited. This mechanism safeguards healthy cells, providing a therapeutic window.
• 8. Study Design and Cohort Progression This First-in-Human (FIH) study was conducted in a single centre and was divided into two sequential parts.
• Part 1: Single Ascending Dose (SAD): In this part, subjects received a single oral dose of B-OT. Cohorts: Up to 5 cohorts were planned with escalating doses (starting at 0.5 mg). The first two cohorts included 3 subjects each; subsequent cohorts included 6 subjects each. Sentinel Dosing: To maximize safety, each cohort began with a single sentinel subject. The remaining subjects in the cohort were dosed only after a safety observation period of at least 48 hours for the sentinel subject. Confinement: Subjects were admitted to the clinical unit on Day -1 and discharged on Day 4 (72 hours post-dose).
• Part 2: Multiple Ascending Dose (MAD): In this part, subjects received oral B-OT once daily for 7 consecutive days. Cohorts: 4 cohorts were planned with escalating doses (starting at 1 mg). Each cohort included 6 subjects. Sentinel Dosing: Similar to the SAD part, each cohort began with a sentinel subject. The remaining subjects were dosed after a safety observation period of at least 72 hours for the sentinel subject. Confinement: Subjects were admitted on Day -1 and confined through Day 8 (24 hours after the final dose) for intensive monitoring.
• Dose Escalation and Safety Review: Dose escalation between cohorts was not automatic. A Safety Review Committee (SRC) consisting of the Principal Investigator and Sponsor representatives reviewed cumulative safety, tolerability, and pharmacokinetic data from the current cohort before approving escalation to the next dose level. Stopping rules were defined for both individual subjects (e.g., occurrence of drug-related Serious Adverse Events) and for whole cohorts (e.g., if clinically relevant signs of similar nature occur in 2 or more subjects in a group).
• Pharmacokinetic and Pharmacodynamic Assessments: Serial blood samples were collected throughout the confinement periods and at follow-up visits to characterize the pharmacokinetic profile of B-OT and its metabolite oxythiamine. In the MAD part, steady-state parameters were evaluated. As an exploratory pharmacodynamic assessment, transketolase activity was measured in erythrocytes (SAD part) or white blood cells (MAD part) to determine the biological effect of the drug on its target enzyme.

Inclusion criteria

• Signed and dated informed consent form.
• Subjects capable to understand the purposes and risks of the study.
• Male volunteers willing to comply with contraception requirements.
• Aged 18-60 years, inclusive.
• Healthy participants, as determined by screening assessments and Principal Investigator's judgment (absence of active/chronic disease).
• Body Mass Index (BMI) of 18-30 kg/m² inclusive.
• Male participants with female partners of childbearing potential must agree to be abstinent or use a male condom plus partner use of a contraceptive method.

Exclusion criteria

• Clinically relevant history of respiratory, gastrointestinal, renal, hepatic, hematological, lymphatic, neurological, cardiovascular, psychiatric, musculoskeletal, genitourinary, immunological, dermatological, endocrine, or connective tissue disorders.
• Fridericia's correction factor for QT (QTcF) > 450 ms or history of QT interval prolongation.
• Acute gastrointestinal symptoms at screening/admission.
• Any abnormal laboratory value of Grade 2 or higher considered clinically significant.
• Values ≥ 10% above upper limit of normal for ALT, AST, Alkaline Phosphatase, Creatinine, or Urea.
• Clinically relevant surgical history.
• History of relevant drug hypersensitivity, alcoholism, or drug abuse.
• Significant infection or known inflammatory process.
• Use of prescription/non-prescription medicines within 2 weeks of admission.
• Receipt of investigational drug within 30 days prior to screening.
• Use of tobacco/nicotine products within 3 months of screening.
• Positive alcohol or drug screen.
• Blood donation within 3 months prior to screening.
• Vaccinated with a Covid-19 vaccine within 2 weeks prior to screening.