First-in-human trial evaluating safety and pharmacokinetics of AT-752, a novel nucleotide prodrug with pan-serotype activity against dengue virus

ABSTRACT AT-752 is a novel guanosine nucleotide prodrug inhibitor of the dengue virus (DENV) polymerase with sub-micromolar, pan-serotype antiviral activity. This phase 1, double-blind, placebo-controlled, first-in-human study evaluated the safety, tolerability, and pharmacokinetics of ascending single and multiple oral doses of AT-752 in healthy subjects. AT-752 was well tolerated when administered as a single dose up to 1,500 mg or when administered as multiple doses up to 750 mg three times daily (TID). No serious adverse events occurred, and the majority of treatment-emergent adverse events were mild in severity and resolved by the end of the study. In those receiving single ascending doses of AT-752, no pharmacokinetic sensitivity was observed in Asian subjects, and no food effect was observed. Plasma exposure of the guanosine nucleoside metabolite AT-273, the surrogate of the active triphosphate metabolite of the drug, increased with increasing dose levels of AT-752 and exhibited a long half-life of approximately 15–25 h. Administration of AT-752 750 mg TID led to a rapid increase in plasma levels of AT-273 exceeding the target in vitro 90% effective concentration (EC90) of 0.64 µM in inhibiting DENV replication, and maintained this level over the treatment period. The favorable safety and pharmacokinetic results support the evaluation of AT-752 as an antiviral for the treatment of dengue in future clinical studies. Registered at ClinicalTrials.gov (NCT04722627).

and adults in Europe, Indonesia, Thailand, and Brazil (7-9) .Critically, however, both vaccines offer imbalanced protection across serotypes, and data indicate low efficacy in younger children (7, 9, 10).Consequently, there are several vaccines and antivirals in development, including repurposed drugs (5-7).To date, clinical trials of repurposed drugs with antiviral activity such as balapiravir, chloroquine, lovastatin, and celgosivir have not demonstrated significant efficacy in reducing viremia and/or improving clinical outcomes (11)(12)(13)(14).Thus, there remains an unmet need for an effective direct-acting antiviral for the treatment of dengue, especially considering that a higher viral burden has been linked to severe dengue disease (6,15).
AT-752 was developed by Atea Pharmaceuticals, Inc and is an orally available novel guanosine nucleotide prodrug inhibitor of the DENV polymerase with sub-micromo lar, pan-serotype antiviral activity (16).AT-752 is the hemisulfate salt of AT-281, a dual guanosine nucleotide prodrug which undergoes multistep metabolic activation to the active 5′ triphosphate metabolite AT-9010, which selectively inhibits the viral RNA-dependent RNA polymerase (16,17) (Fig. 1).Dephosphorylation of AT-9010 results in the formation of the guanosine nucleoside metabolite AT-273, which is regarded as a surrogate plasma marker for intracellular concentrations of AT-9010 (18).
AT-752 is a potent inhibitor of DENV2 and DENV3, as well as other flaviviruses, in vitro, and demonstrated in vivo activity against DENV2 in a mouse model of disease (16).The starting dose of 250 mg (4.2 mg/kg for a human body weight of 60 kg) AT-752 (expressed as the free base AT-281) in this first-in-human study was determined based on initial pre-clinical toxicology studies of AT-752 in rats and monkeys.The no-observed-adverseeffect level after 14 days of dosing of AT-752 in rats and monkeys was 1,000 mg/kg/day.Based on the body surface area conversion factor of 0.162 in rats and 0.324 in monkeys (19), the human equivalent doses calculated were 162 and 324 mg/kg/day, respectively, in the two animal species, providing 39-and 77-fold of margin of safety for the human starting dose.
This first-in-human phase 1 study was designed to evaluate the safety, tolerability, and pharmacokinetics (PK) of oral single ascending doses (SAD) and multiple ascending doses (MAD) of AT-752 (free base AT-281 and metabolites AT-551, AT-229, and AT-273) in healthy male or female subjects.Additionally, the effect of food on the PK of single oral doses of AT-752, and PK sensitivity of AT-752 in Asian ethnicity were assessed.

Study enrollment and completion
A total of 65 subjects were enrolled into one of six SAD or three MAD cohorts (see Materials and Methods).Among the SAD cohorts, 41 subjects were enrolled, and 40 subjects (97.6%) completed the trial.One subject (2.4%) randomized to receive AT-752 discontinued due to withdrawal of consent after receiving AT-752 500 mg (cohort B) and was replaced.All 41 subjects were included in the safety population, and 31 subjects (75.6%) who received at least one dose of AT-752 were included in the PK population.Among the MAD cohorts, 24 subjects were enrolled, and all completed the trial.All 24 subjects were included in the safety population, and 18 subjects (75.0%) who received at least one dose of AT-752 were included in the PK population.

Patient demographics and baseline characteristics
Demographics and baseline characteristics in the SAD cohorts were generally similar across cohorts and treatments (Table 1).The proportion of male (51.2%) and female (48.8%) subjects in the SAD cohorts was similar, whereas the majority were male (62.5%) in the MAD cohorts.Overall, subjects were predominately white, except for one SAD cohort where most participants were Asian.

Safety and tolerability of AT-752
Overall, AT-752 was well tolerated in both the SAD and MAD cohorts, with no serious adverse events (SAEs) or discontinuations due to adverse events (AEs).Non-serious treatment-emergent adverse events (TEAEs) were mild or moderate in severity and resolved by the end of the study.The most frequently reported TEAEs are summarized in Table 2. Sporadic cases of gastrointestinal-related events, including mild-to-moderate vomiting, occurred mostly at higher doses.No treatment-related or dose-related trends in clinical laboratory values, vital sign measurements, or 12-lead electrocardiogram (ECG) parameters were observed.

SAD cohorts
Following single oral administration of AT-752 under fasting conditions, plasma concentrations of the parent prodrug AT-281 increased rapidly and were transient.AT-281 was rapidly eliminated with a short plasma half-life (t 1/2 ) of approximately 0.5 h regardless of dose, resulting in mostly unquantifiable levels 4-6 h post dose.The intermediate L-alanyl metabolite AT-551 also exhibited a transient exposure with a mean t 1/2 of approximately 2-3 h across dose groups.The N 6 -methyl nucleoside metabolite, AT-229, subsequently peaked and exhibited a slower elimination phase (Fig. 2).Plasma concentrations of AT-273 (surrogate for the intracellular active triphosphate AT-9010) appeared more gradually than AT-281 and other metabolites, and exhibited a long t 1/2 of approximately 15-25 h across doses, reflecting sustained intracellular exposure of the active metabolite AT-9010.
Plasma PK parameters for the SAD cohorts are summarized in Table 3.Following single oral administration of AT-752 at 250-1,500 mg, peak concentration (C max ) and extent of exposure (AUC) were slightly higher numerically (with, however, largely overlapping ranges) for the Asian cohort compared with the mostly white cohort at the same dose level (AT-752 1,000 mg) for AT-281, AT-551, and AT-229.For AT-273, the guanosine circulating metabolite representing the intracellular active triphosphate metabolite AT-9010, C max , and AUC was similar for the Asian cohort and the mostly white cohort at the same dose level.Time to peak concentration (T max ) was also similar for the Asian cohort and the mostly white cohort for all metabolites.The comparable PK profiles observed between the Asian and mostly Caucasian subject cohorts suggest an absence of PK sensitivity in Asian ethnicity.Plasma exposure of AT-281 and its metab olites increased over the studied dose range of 250-1,500 mg: AT-229 increased in a dose-proportional manner, and AT-281 and AT-551 increased in a greater than dose-pro portional manner, whereas AT-273 was slightly less than dose-proportional (Fig. 3).A high-fat/high-calorie meal delayed and decreased peak levels of AT-281, AT-551, and AT-229 but had limited-to-no impact on their total exposure, and slightly increased the plasma exposure of AT-273 in comparison to fasted conditions.This demonstrates that AT-752 can therefore be taken with or without food.The extent of urine elimination was low for AT-281 (up to 2.4%) and AT-551 (up to 0.7%), and moderate for AT-229 (10.7-17.9%)and AT-273 (6.7-8.8%).Total urine recovery ranged from approximately 20% to 30% of administered doses across cohorts, and renal clearance (CL R ) of AT-229 and AT-273 exceeded estimated glomerular filtration rate (eGFR).

MAD cohorts
Following repeat dosing of AT-752 1,000 mg once daily (QD), 750 mg twice daily (BID), and 750 mg three times daily (TID), plasma AT-281 (the unchanged protide) did not meaningfully accumulate due to its rapid elimination (Fig. 4).Plasma exposure of AT-273 increased by approximately 25%, 60%, and 80% with QD, BID, and TID, respectively, due to its long plasma half-life, reflecting rapid accumulation and sustained intracellu lar exposure of the active metabolite AT-9010.Steady-state plasma PK parameters are summarized in Table 4. Based on the trough AT-273 concentrations, steady state was essentially achieved by day 3 in all MAD cohorts (Fig. 5).As depicted in Fig. 4, among the MAD cohorts, only AT-752 750 mg TID led to a rapid increase in plasma AT-273 levels, with mean trough levels within the first day exceeding the 90% effective concentration (EC 90 ) of the drug in inhibiting DENV replication in vitro (0.64 µM or 200 ng/mL of equivalent AT-273) and maintained these levels over the treatment period.

DISCUSSION
AT-752, when metabolized to the active triphosphate AT-9010, has been demonstrated to target the NS5 RNA-dependent RNA polymerase (RdRp) of DENV1-4 (17).NS5, the largest and most conserved non-structural protein encoded by flaviviruses, is a promising target for antivirals, particularly nucleoside/nucleotide analogs in the treatment of dengue ( 5).This first-in-human phase 1 study of AT-752 evaluated the safety, tolerability, and PK of ascending single and multiple doses in healthy subjects.AT-752 demonstrated a favorable safety profile and was well tolerated in healthy subjects when administered as a single oral dose up to 1,500 mg or when administered as multiple oral doses up to 750 mg TID.No SAEs were observed, and no subjects experienced AEs that lead to study drug discontinuation.Most TEAEs were mild in severity, and all TEAEs resolved by the end of the study.There were no clinically significant physical examination findings, vital signs, or ECGs observed.
In the SAD cohorts, plasma exposure of AT-281 and its metabolites was approxi mately dose-proportional over the studied dose range of 250-1,500 mg AT-752.Fed conditions led to a 48% reduction in C max compared with fasted conditions, whereas AUC values were similar.The extent of urinary elimination of AT-281 and its metabolites was low-to-moderate.Renal clearance of the nucleoside metabolites AT-229 and AT-273 exceeded eGFR, suggesting involvement of active secretion in their renal elimination.Furthermore, the similar PK profiles observed between the Asian and mostly white cohorts suggest an absence of PK sensitivity in Asian participants; therefore, dose adjustment of AT-752 in this population does not appear necessary.In the MAD cohorts, C max of AT-281 was attained approximately 0.5 h post dose (median estimates) for all dose levels.Plasma exposure of AT-273 increased with increasing dose levels due to its long plasma half-life, reflecting rapid accumulation and sustained intracellular exposure of the active metabolite AT-9010.AT-752 750 mg TID led to a rapid increase in plasma AT-273 levels, exceeding the target EC 90 of 0.64 µM, and maintained this level over the treatment period.
While both male and female subjects were well represented in most cohorts and overall in the study, cohort F inadvertently enrolled only males.However, there did not seem to be any gender-related trends in AEs or PK based on preliminary analyses.Additional analyses, preferably via population PK, will be needed to ultimately assess intrinsic factors (including gender) on the safety and PK of AT-752 using cumulative data from multiple trials in the future.
In summary, the results of this study demonstrate that AT-752 was well tolerated when administered as a single dose up to 1,500 mg or when administered as multiple doses up to 750 mg TID.In the SAD cohorts, AT-752 exhibited no PK sensitivity in South/ Southeast/East Asian participants, and no food effect was observed, demonstrating that dose adjustment is not necessary for Asian ethnicity and that AT-752 can be taken with or without food.In the MAD cohorts, AT-752 TID exceeded the target antiviral level for inhibition of DENV replication.The favorable safety and PK results reported here demonstrate AT-752 as an attractive antiviral for the treatment of dengue, and support dose selection of AT-752 in future clinical studies.

MATERIALS AND METHODS
The study protocol was approved by the Human Research Ethics Committee before the trial began, and written informed consent was obtained from each subject before entering the study.This study was conducted according to the principles of the International Council for Harmonisation Harmonised Tripartite Guideline E6(R2): Good Clinical Practice, and the ethical principles from the Declaration of Helsinki.

Study design
This was a phase 1, first-in-human, randomized, double-blind, placebo-controlled study consisting of two sequential parts: SAD and MAD.The SAD cohorts included a foodeffect cohort, and both SAD and MAD cohorts evaluated the safety, tolerability, and PK of AT-752 ( ClinicalTrials .govregistration no.NCT04722627).All subjects were screened up to 28 days prior to the first study drug administration on day 1, which included clinical history, physical examination, 12-lead ECG, vital signs, and laboratory tests of blood and urine.

SAD cohorts
Subjects in the SAD cohorts were assigned to one of five sequential dose cohorts (eight subjects per cohort) on day 1 prior to the first dose.Subjects were randomized 3:1 within each cohort to receive AT-752 or a matching placebo (Table 5).Sentinel dosing was employed for two subjects in each SAD cohort in a ratio of 1:1 (one active, one placebo), and were dosed at least 48 h before the remainder of the cohort was dosed.All treatments were administered orally under fasting conditions, except for the food effect cohort.As part of the food effect evaluation, subjects in cohort B (n = 7) remained confined to the clinic for an additional study drug administration on day 7 under fed conditions.Subjects fasted overnight for at least 8 h and received a high-fat and high-calorie breakfast approximately 30 minutes (± 5 minutes) before study drug administration.Subjects consumed the meal within 30 minutes or less and consumed at least 75% of the meal.The high-fat (approximately 50% of total calorific content of the meal) and high-calorie (approximately 800-1,000 calories) breakfast followed Food and Drug Administration guidance recommendations and provided approximately 150, 250, and 500-600 calories from protein, carbohydrate, and fat, respectively.

MAD cohorts
Subjects in the MAD cohorts were assigned to a repeat-dose cohort on day 1 prior to the first dose.Subjects were randomized 3:1 to receive AT-752 or a matching placebo (Table 6).Sentinel dosing was employed for two subjects in each MAD cohort in a ratio of 1:1 (one active, one placebo), and were dosed at least 48 h before the remainder of the cohort was dosed.All treatments were administered orally under fasting conditions.Dose escalation to cohort G occurred based on review of available safety data (through day 12) and PK data (through day 8) of cohort F. Similarly, dose escalation to cohort H occurred based on review of available safety data (through day 10) and PK data (through day 6) of cohort G.

Study subjects
Healthy subjects between the ages of 18 and 65 years who met the inclusion and exclusion criteria were considered eligible for this study.All subjects were required to weigh at least 50 kg and have a body mass index of between 18 and 29 kg/m 2 , inclusive.Subjects in cohort D (Asian cohort) must have been of first to third generation South/ Southeast/East Asian descent.Exclusion criteria included clinically relevant abnormal

Statistical analysis
Plasma concentration-time data were analyzed by non-compartmental approach using Phoenix WinNonlin Version 8.3 (Certara USA Inc., Princeton, New Jersey) or SAS Version 9.4 (SAS Institute Inc., Cary, North Carolina), as appropriate.For the SAD cohorts, dose proportionality was assessed on day 1 for AT-281, AT-551, AT-229, and AT-273 in the plasma using the power regression model for the AUCs and C max PK parameters (20).
To assess the impact of a high-fat meal on exposure to AT-281, AT-551, AT-229, and AT-273, the log-transformed values of AUC inf and C max were analyzed in cohort B for AT-281, AT-551, AT-229, and AT-273 using a linear mixed effect model with fed/fasted status as a fixed effect and subject as a random effect.Sensitivity analysis for analyte AT-752 PK was performed by visual inspection of mean concentration vs time profiles and assessment of PK parameters in summary tables.

Laboratory and safety assessment
Safety assessments included monitoring of AEs, clinical laboratory tests (hematology, serum chemistry, urinalysis, coagulation tests, and cardiac biomarkers), vital sign measurements, 12-lead ECGs, and physical examination.All AEs were coded using Medical Dictionary for Regulatory Activities Version 24.1.

FIG 3
FIG 3 Dose proportionality plots in SAD cohorts.AUC inf , area under the curve extrapolated to infinity.

FIG 4
FIG 4 Mean (+ SD) plasma concentration-time profiles of AT-281 and metabolites AT-551, AT-229, and AT-273 following oral administration of multiple ascending doses of AT-752.Dashed line, first dose on day 1; solid line, last dose at steady state.

FIG 5
FIG 5 Mean (+ SD) plasma trough concentration-time profiles of AT-273 with following oral administration of multiple ascending doses of AT-752.EC 90 , 90% effective concentration.

TABLE 1
Baseline characteristics of study subjects a

TABLE 2
Summary of most frequent TEAEs reported by >2 subjects

TABLE 3
Summary of plasma PK parameters of AT-281 and metabolites AT-551, AT-229, and AT-273 following oral administration of single ascending doses of AT-752 a,b a C max , AUC inf , and t 1/2 are represented as mean ± SD.T max is represented as median (minimum-maximum).CL R is represented as mean ± SD. b AUC inf , area under the curve extrapolated to infinity; CI, confidence interval; CL R , renal clearance; C max , maximum plasma concentration; SD, standard deviation; t 1/2 , half-life; T max , time to reach maximum concentration.c Food effect is represented as geometric mean ratio (90% CI).

TABLE 4
Summary of steady-state plasma PK parameters of AT-281 and metabolites AT-551, AT-229, and AT-273 following oral administration of multiple ascending doses of AT-752 a,b C max and AUC tau represented as mean ± SD.T max is represented as median (minimum-maximum).b AUC tau , area under plasma concentration-time curve over the dosing interval. a

TABLE 5
vaccine against COVID-19 in the 14 days before the first dose.Subjects could not have used a prescription medicine, over-the-counter medicine, or herbal or dietary supplements during the 7 days before the first dose.

TABLE 6
MAD cohorts