- 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) paradoxically 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.