Safety, Tolerability, and Pharmacokinetics of Intravenous Doses of PF‐07304814, a Phosphate Prodrug Protease Inhibitor for the Treatment of SARS‐CoV‐2, in Healthy Adult Participants

Abstract

Studies on targeted antivirals for treatment of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the cause of the ongoing pandemic, are limited. PF‐07304814 (lufotrelvir) is the phosphate prodrug of PF‐00835231, a protease inhibitor targeting the 3C‐like protease of SARS‐CoV‐2. This phase 1 study evaluated the safety, tolerability, and pharmacokinetics (PK) of single ascending intravenous doses of lufotrelvir (continuous 24‐hour infusion of 50, 150, 500, or 700 mg) versus placebo in healthy volunteers (2 interleaving cohorts: 1, n = 8; 2, n = 7). Each dosing period was separated by a washout interval (≥5 days). Treatment‐emergent adverse events, PK, and biomarker concentrations were estimated from plasma/urine samples. Lufotrelvir was administered to 15 volunteers (mean [SD] age 39.7 [11.8] years). No serious adverse events, discontinuations, or deaths were reported. Mean maximum observed concentration of PF‐00835231 (active moiety; 97.0 ng/mL to 1288 ng/mL) were observed between median time to maximum concentration of 14 to 16 hours after the start of the lufotrelvir infusion. Near‐maximum plasma concentrations of PF‐00835231 were observed ≈6 hours after infusion start and sustained until infusion end. PF‐00835231 plasma concentrations declined rapidly after infusion end (mean terminal half‐life: 500 mg, 2.0 hours; 700 mg, 1.7 hours). Approximately 9%–11% of the dose was recovered in urine as PF‐00835231 across doses. A continuous, single‐dose, 24‐hour infusion of lufotrelvir (50–700 mg) was rapidly converted to PF‐00835231 (active moiety), with dose‐proportional PK exposures and no significant safety concerns. A daily, 24‐hour continuous infusion of 270 to 350 mg is expected to maintain PF‐00835231 concentration at steady state/above effective antiviral concentrations. Further studies exploring lufotrelvir efficacy in patients with coronavirus disease 2019 are ongoing.

Coronavirus disease 2019 (COVID‐19) is a highly contagious respiratory infection caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). COVID‐19 is responsible for the ongoing global pandemic, with high levels of socioeconomic loss and morbidity and mortality among vulnerable populations. ¹ As of July 2022, >559 million cases of infection and >6 million deaths were reported worldwide. ² Although most infections result in asymptomatic or mild disease, advanced age and the presence of comorbid conditions increase the risk of death or of developing severe disease with long‐term consequences, placing an immense burden on global health care systems. ³

The pathophysiology of severe COVID‐19 remains largely unknown. While the cause is speculated to be the result of a cytokine storm, ⁴ targeted therapies that treat the infection early are imperative and believed to have the potential to prevent worsening disease. While several vaccines are in use to reduce the spread of infection and severity of disease, ⁵ , ⁶ , ⁷ none confer sterilizing immunity. ⁸ In addition, currently available treatment options are limited, indicated in a small subset of patients, and do not cover the spectrum of disease. ⁹ The repurposed, intravenously administered RNA‐dependent polymerase inhibitor remdesivir was the earliest molecule to be approved for use in COVID‐19 treatment. ¹⁰ Emergency use authorization for remdesivir was granted by the US Food and Drug Administration (FDA) and other global regulators in May 2020 based on evidence of improvement in time to recovery in hospitalized patients with COVID‐19 involving the lower respiratory tract. ¹¹ , ¹² , ¹³ However, remdesivir use is associated with hypersensitivity reactions and an increased risk of transaminase elevations. ¹⁴ In addition, while the benefit of remdesivir in the outpatient setting has been demonstrated, there is limited and conflicting clinical evidence for mortality benefit following remdesivir use in hospitalized patients. ¹⁵ , ¹⁶

Several new molecules are currently being developed and others repurposed to satisfy this unmet treatment need, including antivirals, cytokine‐neutralizing agents, and monoclonal antibodies targeting viral surface proteins. Some of these molecules have already received emergency use authorization from global regulators, but most are indicated in very specific subsets of patients: those who are hospitalized with increasing oxygen needs or in nonhospitalized patients at high risk of severe disease. ¹⁰ However, the effectiveness of drugs currently used in the hospital setting is limited. In addition, monoclonal antibodies may be rendered ineffective by emerging viral variants. ¹⁷

The main coronavirus protease, the 3C‐like protease (3CL^pro or M^pro), is essential for replication and has low cross reactivity with human proteases owing to its substrate preference, making it a suitable drug target. ¹⁸ In addition, it is highly conserved and acts early during the coronavirus life cycle, releasing 11 of the 13 proteins required for viral replication after uncapping. ¹⁹ , ²⁰

PF‐00835231, a small‐molecule protease inhibitor (identified in 2003), binds to the essential 3CL protease of SARS‐CoV‐1 (responsible for the SARS outbreak). ²¹ Evidence from in vitro binding and thermal shift assays indicate that it also binds to the catalytic pocket of the analogous polyprotease of SARS‐CoV‐2, which is identical in genetic sequence to that of the SARS‐CoV‐1 protease. ²² , ²³ In in silico molecular docking experiments comparing the effectiveness of 6 different antivirals, PF‐00835231 was found to be the most effective against the wild‐type 3CL protease, with high but variable effectiveness against modeled mutants as well. ²⁴ In human airway models and cell lines enriched with angiotensin‐converting enzyme 2 expression and infected with SARS‐CoV‐2, the potency of PF‐00835231 to inhibit viral replication was found to be equivalent to or higher than remdesivir. ¹⁶ Data from nonclinical studies also indicate that PF‐00835231 may act synergistically in combination with remdesivir. This combination may be clinically relevant since both molecules target different components of the viral replication pathway. PF‐00835231 acts first to inhibit 3CL^pro, and remdesivir subsequently targets the RNA polymerase pathway. ²³ PF‐07304814 (lufotrelvir) is a phosphate ester prodrug of PF‐00835231 (phosphate prodrug formulation) with increased solubility, suitable for intravenous (IV) administration and converted rapidly after administration to the active form PF‐00835231 (active moiety) by alkaline phosphatase. Crystal structures of both lufotrelvir and PF‐00835231 are previously described, including binding and inhibition kinetics with 3CL^pro23. Prior assessments of the pharmacokinetic (PK) profile of PF‐00835231 in human liver microsomes have indicated that it is mainly metabolized by cytochrome P450 3A4. In preclinical studies of intravenous PF‐00835231 in rats, dogs, and monkeys, moderate plasma clearance and a low volume of distribution was observed. ²³

In this study, we report the safety, tolerability, and PK of lufotrelvir administered via continuous IV infusion as a single ascending dose in healthy adult participants.

Methods

Study Participants

Healthy adults aged 18 to 60 years capable of providing informed consent, including women of childbearing potential on contraceptive medication, were eligible. Health status was assessed using medical history, physical examination, standard 12‐lead electrocardiogram, laboratory tests, cardiac monitoring, and body mass index (between 17.5 and 30.5 kg/m² was permissible). Adults with clinically significant hematological, renal, endocrine, pulmonary, gastrointestinal, cardiovascular, hepatic, psychiatric, neurological, and allergic disease were excluded. Potential participants presenting with a history of HIV, hepatitis B, or hepatitis C infection; or venous thromboembolic event occurrence; or substance or alcohol abuse were also excluded. In addition, those adults with prescription/nonprescription/herbal supplement use within 7 days (or 5 half‐lives) before the first scheduled dose of study intervention were excluded.

Study Design

This phase 1 randomized, double‐blind, sponsor‐open, placebo‐controlled study (NCT04627532) was designed to assess the safety, tolerability, and PK of single ascending doses of lufotrelvir administered as a 24‐hour, constant‐rate, continuous infusion in healthy participants. The study was conducted at New Haven Clinical Research Unit (New Haven, Connect) between October 23, 2020, and December 17, 2020. The protocol and informed consent forms were approved by the institutional review board (IntegReview, USA), and the study was conducted according to the principles of the Declaration of Helsinki, International Conference on Harmonisation–Good Clinical Practice, and all applicable local regulations. Enrollment of 16 participants in 2 interleaving cohorts was planned, with a 2‐period crossover in each cohort. An optional third period could be added to cohort 1 to further explore the dose range based on emerging safety, tolerability, and PK assessments. For each period, approximately 6 participants received a single dose of lufotrelvir administered as a 24‐hour continuous IV infusion, and ≈2 participants received placebo. Between each dose administration to a participant, a washout interval of at least 5 days between dosing was planned (Figure 1). After the last dose in the final period and completion of day 5 (96 hours) activities, all participants were to be released from the clinical research unit. Two follow‐up telephone calls were scheduled, the first between day 8 and day 10 and the second between day 30 and day 37. From screening to last follow‐up, the overall study duration was 11 weeks (including the optional third period in cohort 1).

Figure 1.

Study design and randomization scheme are shown. Four doses (50, 150, 500, and 700 mg) of the phosphate prodrug lufotrelvir (PF‐07304814) administered as a 24‐hour infusion were assessed in 4 different treatment periods among 15 participants. Each cohort experienced 2 treatment periods, separated by a washout period of ≥5 days. Two telephone follow‐ups were planned. *All study participants received the intervention as planned and completed the study. AP, active pharmaceutical (lufotrelvir; PF‐07304814); Pbo, placebo; R, randomization.

The main end point of the study was the assessment of safety and tolerability following single ascending 24‐hour IV infusions of lufotrelvir in healthy adult participants. Other end points included the assessment of the plasma PK of lufotrelvir and PF‐00835231 and urine PK of PF‐00835231. The inhibitory potential of PF‐00835231 on organic anion transporting polypeptide 1B (OATP1B) and multidrug and toxic compound extrusion (MATE) transporters using endogenous biomarkers coproporphyrin I (CP‐I) and N‐methylnicotinamide (NMN), respectively, were also assessed.

Dose Selection

The approach for dose selection for this study included consideration of all relevant information obtained in nonclinical pharmacology and toxicity studies with lufotrelvir or PF‐00835231 (active moiety). Dose escalation was planned in incremental increases of one‐half log or less (up to ≈3.3‐fold) based on exposure predictions. The proposed doses of lufotrelvir were 50, 150, 500, and 1000 mg for a 24‐hour continuous infusion based on predicted human PK from in vivo animal PK studies. At the highest planned dose of 1000 mg, at least 1.1‐fold safety margins for both PF‐00835231 and lufotrelvir were predicted relative to the PK stopping limits.

Study Assessments

Safety Analyses

Safety was assessed based on adverse event (AE) monitoring (by system‐organ class) and results from routine laboratory safety tests, including complete blood counts; blood chemistry panel; kidney, liver, and lipid profiles; and assessment of blood coagulation parameters. Serious and nonserious AE monitoring was scheduled at screening, time of admission to the clinical research facility, and continued through the completion of the study/study termination. Safety laboratory assessments were conducted at screening; 1 day before dosing; at 6, 24, 48, and 96 hours after the start of infusion of each period; or at study termination. Dose escalation could be stopped if it was determined that the limits of safety and/or tolerability were reached. In addition, due to the increased risk of development of thrombotic events (eg, deep vein thrombosis [DVT]) in the intended patient population (hospitalized patients with COVID‐19), any evidence of DVT (or any other thrombotic event) confirmed by ultrasound, computed tomography angiogram or ventilation–perfusion scan, and relevant laboratory results, dosing would be paused, and, regardless of causality, would resume only with agreement from the sponsor's independent protocol review committee and the regulatory authority.

Pharmacokinetic Evaluation

Concentrations of lufotrelvir and PF‐00835231 in plasma and urine (PF‐00835231 only; PPD Inc., Middleton, Wisconsin) samples were analyzed using validated liquid chromatography–tandem mass spectrometry (LC‐MS/MS) methods at Pfizer, Inc. (Groton, Connecticut). Sample processing for lufotrelvir and PF‐00835231 was performed by protein precipitation using isotopically labeled internal standards (PF‐07304814‐d4 and PF‐00835231‐d4). Lufotrelvir and PF‐00835231 were separated using an Acquity UPLC BEH Phenyl column (2.1 × 50 mm, 1.7 μm; Waters Corp., Milford, Massachusetts); gradient elution using 2% formic acid in water (mobile phase A) and in acetonitrile (mobile phase B) at a flow rate of 0.600 mL/min was performed. An API 5500 triple quadrupole mass spectrometer (Sciex, Framingham, Massachusetts) equipped with a turbo ion spray source was employed for detection in positive ionization mode. Quantification was performed based on multiple reaction monitoring (MRM) of the transitions of m/z 553.2/267.0 for lufotrelvir, 473.3/187.0 for PF‐00835231, 557.4/267.0 for PF‐07304814˗d4, and 477.4/187.0 for PF‐00835231˗d4. A linear calibration curve, with a 1/concentration² weighting factor with a dynamic quantitation range of 40–10000 ng/mL for lufotrelvir and PF‐00835231 was used in plasma analysis. Interday precision (expressed as coefficient of variation) of the quality control samples in human plasma ranged from 3.0% to 3.5% for lufotrelvir and 3.6% to 4.7% for PF‐00835231. The corresponding interday accuracies (expressed as percent relative error) of the quality control samples in plasma ranged from 1.7% to 3.7% for lufotrelvir and 2.5% to 3.4% for PF‐00835231. For analysis of PF‐00835231 in urine samples, a linear calibration curve with a dynamic quantitation range of 5 to 1000 ng/mL was used, while interday precision ranged from 2.5% to 9.4% and the interday accuracy ranged from –1.3% to 2.7%.

Plasma PK parameters for both lufotrelvir and PF‐00835231 were calculated from plasma concentration–time data, using a standard noncompartmental analysis method, and included area under the plasma concentration–time curve (AUC) from time 0 to the last quantifiable concentration (AUC_last), AUC from time 0 extrapolated to infinite time (AUC_inf), dose normalized AUC_last (AUC_last [dn]), dose normalized AUC_inf (AUC_inf [dn]), maximum observed concentration (C_max), time to maximum concentration (t_max), terminal half‐life (t_1/2), concentration at 24 hours, concentration at steady state (C_ss), and dose normalized C_ss (C_ss [dn]). Urine PK parameters were assessed for PF‐00835231, including the amount of unchanged drug excreted in urine and percentage of unchanged drug excreted in urine.

Biomarker Analyses

The inhibitory potential of lufotrelvir and PF‐00835231 on MATE (MATE1/2) and OATP1B (OATP1B1 and ‐1B3) transporters was assessed using the endogenous biomarkers NMN (MATE substrate; plasma and urine levels measured) and CP‐I (OATP1B substrate; plasma levels measured), respectively. ²⁵ , ²⁶ Plasma levels of both biomarkers were assessed before dose; at 0.5, 3, 6, 9, 12, 16, 24, 26, 28, 30, 32, and 48 hours after infusion start during each dosing cycle; and at early study termination, if applicable. Urine levels of NMN were assessed before dosing, 0 to 12, 12 to 24, and 24 to 36 hours after infusion start during each dosing cycle. NMN concentrations were also determined from plasma and urine samples from the placebo group. For NMN analysis, data from participants receiving placebo were analyzed and used as a baseline control. For CP‐I, the predose concentration value for each participant was used as a baseline control. To obtain 24‐hour baseline profiles, CP‐I concentrations were assumed to be static and the predose concentration was substituted for all CP‐I concentrations for the corresponding PK profile over 24 hours, and AUC from time 0 to 24 hours (AUC₂₄) postdose: AUC₂₄ predose ratios were calculated (ad hoc) using geometric and arithmetic means.

Plasma samples for CP‐I and NMN concentrations and urine samples for NMN concentrations were analyzed at PPD, Inc. using validated, sensitive, and specific LC‐MS/MS methods.

Plasma samples were analyzed for CP‐I concentrations using a validated, sensitive, and specific ultra‐performance LC‐MS/MS (UPLC‐MS/MS) method. A 200‐μL plasma sample was fortified with 50 μL of 4.00 ng/mL coporoporphyrin‐I‐ ¹⁵N ₄ internal standard solution and extracted by solid supported liquid extraction with ethyl acetate. The supernatant was evaporated to dryness under nitrogen and reconstituted using 125 μL of water/acetonitrile/formic acid (750:250:1, v/v/v). The extracted sample was injected to a UPLC column (Acquity BEH C18, 2.1×100 mm, 1.7 μm; Waters Corp.) with a gradient mobile phase containing water/formic acid (1000:1, v/v) and acetonitrile/formic acid (1000:1, v/v). Detection was performed using a API 6500+ (Sciex) in the positive ion mode. The MRM ion transition was m/z 328→238 for CP‐I and m/z 330→240 for the internal standard. The calibration standard responses were linear over the range 100 to 5000 pg/mL using a weighted (1/concentration²) linear least squares regression. The between‐day assay accuracy, expressed as percent relative error for quality control (QC) concentrations, ranged from –1.41% to 3.07%. Assay precision, expressed as the between‐day percent coefficient of variation (%CV) of the mean estimated concentrations of QC samples, was ≤3.9%.

Plasma samples were analyzed for NMN concentrations using a validated, sensitive, and specific UPLC‐MS/MS method. A 50‐μL plasma sample was fortified with 50 μL of 250 ng/mL N1‐methylnicotinamide‐d₃ internal standard solution in acetonitrile and extracted by protein precipitation with acetonitrile. Portion of the extracted sample was diluted with 450 μL of acetonitrile/water/formic acid (900:100:1, v/v/v). The extracted sample was injected to a UPLC column (Acquity BEH HILIC, 2.1×50 mm, 1.7 μm; Waters Corp.) with a gradient mobile phase containing water/acetonitrile/ammonium formate 1M/formic acid (900:100:10:1, v/v/v/v) and acetonitrile/water/ammonium formate 1M/formic acid (900:100:10:1, v/v/v/v). Detection was performed using a Xevo TQ‐S (Waters Corp.) in the positive ion mode. The MRM ion transition was m/z 137→94 for NMN and m/z 140→97 for the internal standard. The calibration standard responses were linear over the range 1.00 to 1000 ng/mL using a weighted (1/concentration²) linear least squares regression. The between‐day assay accuracy, expressed as percent relative error for QC concentrations, ranged from –6.88% to 4.94%. Assay precision, expressed as the between‐day %CV of the mean estimated concentrations of QC samples, was ≤10.9%.

Urine samples were analyzed for NMN concentrations using a validated, sensitive, and specific UPLC‐MS/MS method. A 25‐μL urine sample was fortified with 50 μL of 5000 ng/mL N1‐methylnicotinamide‐d₃ internal standard solution in acetonitrile and extracted by protein precipitation with acetonitrile. Portion of the extracted sample was diluted with 450 μL of acetonitrile/water/formic acid (900:100:1, v/v/v). The extracted sample was injected to an UPLC column (Acquity BEH HILIC, 2.1×50 mm, 1.7 μm) with a gradient mobile phase containing water/acetonitrile/ammonium formate 1M/formic acid (900:100:10:1, v/v/v/v) and acetonitrile/water/ammonium formate 1M/formic acid (900:100:10:1, v/v/v/v). Detection was performed using a Xevo TQ‐S in the positive ion mode. The MRM ion transition was m/z 137→94 for NMN and m/z 140→97 for the internal standard. The calibration standard responses were linear over the range 100 to 100,000 ng/mL using a weighted (1/concentration²) linear least squares regression. The between‐day assay accuracy, expressed as percent relative error for QC concentrations, ranged from –4.67% to 2.01%. Assay precision, expressed as the between‐day %CV of the mean estimated concentrations of QC samples, was ≤3.14%.

Results

Baseline Characteristics and Disposition

A total of 15 participants were enrolled, 8 in cohort 1 and 7 in cohort 2. Each participant was randomized to either placebo or a single IV dose of lufotrelvir during any given treatment period. During the conduct of the study, the highest selected dose of 1000 mg was adjusted to 700 mg based on emerging PK results from lower doses to remain within predefined PK stopping limits. Among the 2 cohorts and 4 treatment periods, 8 doses of placebo, 6 doses each of 50 mg of lufotrelvir and 500 mg of lufotrelvir, and 5 doses each of 150 mg of lufotrelvir and 700 mg of lufotrelvir were administered (Figure 1). All participants completed the study, and no discontinuations were reported throughout the duration of the study.

Baseline characteristics were balanced between cohorts. The overall mean (standard deviation [SD]) age was 39.7 (11.8) years, and most participants were African American men with a mean (SD) body mass index of 24.1 (2.9) (Table 1).

Table 1.

Key Demographics and Baseline Characteristics (Safety Analysis Set)

Parameter	Cohort 1, N = 8	Cohort 2, N = 7	Total, N = 15
Age, y, mean (SD) 18–44, n (%) 45–60, n (%)	41.1 (9.6) 6 (75.0) 2 (25.0)	38.0 (14.5) 4 (57.1) 3 (42.9)	39.7 (11.8) 10 (66.7) 5 (33.3)
Sex, n (%) Male Female	7 (87.5) 1 (12.5)	5 (71.4) 2 (28.6)	12 (80.0) 3 (20.0)
Race, n (%) White Black/African American Asian	1 (12.5) 6 (75.0) 1 (12.5)	2 (28.6) 5 (71.4) 0	3 (20.0) 11 (73.3) 1 (6.7)
BMI, mean (SD)	23.6 (3.0)	24.7 (3.0)	24.1 (2.9)

BMI, body mass index; SD, standard deviation.

Safety and Tolerability

All 15 participants from both cohorts were included in the safety analysis set. No discontinuations were reported, and none of the treatment‐emergent AEs (TEAEs) were adjudicated as related to treatment by the investigators, indicating tolerability. Safety was assessed using a combination of AE analyses (Table 2) and abnormalities detected in standard laboratory tests. Across all treatment groups, no clinically significant changes were noted in high sensitivity C‐reactive protein or parameters evaluating the coagulation system (activated partial thromboplastin time, prothrombin time, fibrinogen, and D‐dimer). All other laboratory abnormalities were transient and not considered clinically significant, all reported AEs were mild, and no serious AEs were reported. The total number of AEs reported were similar in participants receiving placebo and any dose of lufotrelvir, with the lowest number of AEs (n = 1) reported in the 50‐ and 700‐mg treatment groups. The most common AEs classified by system‐organ class were skin/subcutaneous tissue disorders (reported by a total of 4 participants administered any dose of lufotrelvir), with contact dermatitis being the most common. No deaths were reported in any group.

Table 2.

Summary of TEAEs (Safety Analysis Set)

Parameter	Placebo, N = 8	50 mg, N = 6	150 mg, N = 5	500 mg, N = 6	700 mg, N = 5
Number of AEs	5	1	6	7	1
Participants with AEs, n (%) by SOC GI disorders General disorders/administration site conditions Injury/poisoning/procedural complications Nervous system disorders Psychiatric disorders Reproductive system/breast disorders Skin/subcutaneous tissue disorders	4 (50.0) 0 2 (25.0) 0 0 1 (12.5) 1 (12.5) 1 (12.5)	1 (16.7) 0 0 1 (16.7) 0 0 0 0	3 (60.0) 1 (20.0) 1 (20.0) 0 0 0 0 2 (40.0)	3 (50.0) 1 (16.7) 0 0 1 (16.7) 0 0 2 (33.3)	1 (20.0) 0 0 0 0 0 0 1 (20.0)

AE, adverse event; GI, gastrointestinal; SOC, system organ class; TEAE, treatment‐emergent adverse event.

Pharmacokinetics

Phosphate Prodrug Lufotrelvir

The prodrug (lufotrelvir) PK analysis set comprised data from all participants across the doses studied, except for 1 participant in the 50‐mg treatment period whose plasma concentrations were all below the limit of detection. Plasma PK parameters are summarized in Table 3. Following a 24‐hour IV infusion of lufotrelvir, mean C_max was observed between a median t_max of 3 to 16 hours after infusion start across the dose range studied, with declining concentrations observed before the end of the continuous infusion period (Figure 2). C_max appeared to increase in a dose‐proportional manner across all doses, and interparticipant variability (indicated by %CV) was between 13% and 24% across the dose range studied. Values for t_½ could not be determined since all plasma samples collected after the 16 hours following the start of infusion were below the limit of detection.

Table 3.

Summary of PK Parameters (Phosphate Prodrug Lufotrelvir) (PK Analysis Set)

Parameter (Units) a	50 mg, N = 5 b	150 mg, N = 5	500 mg, N = 6	700 mg, N = 5
AUClast, ng • h/mL	283.3 (127); 412.6 ± 401.8	1962.0 (36); 2062.0 ± 737.6	7391.0 (18); 7488.0 ± 1300.8	13310.0 (23); 13580.0 ± 3161.8
AUClast [dn], ng • h/mL/mg	5.7 (127); 8.3 ± 8.0	13.1 (36); 13.8 ± 4.9	14.8 (18); 15.0 ± 2.6	19.0 (22); 19.4 ± 4.5
Cmax, ng/mL	48.0 (13); 48.4 ± 6.5	137.2 (24); 140.3 ± 31.6	459.3 (16); 464.2 ± 73.4	875.2 (24); 895.8 ± 220.0
tmax, h	16.0 (3.0–16.0)	9.0 (3.0–16.0)	12.5 (3.0–16.0)	3.02 (0.5–16.0)
C24, ng/mL	NA; 9.0 ± 20.2	NA; 15.8 ± 35.3	86.6 (27); 89.3 ± 24.9	219.8 (82); 270.6 ± 191.2
Css, ng/mL	19.4 (92); 24.3 ± 16.9	118.9 (27); 122.2 ± 31.7	370.0 (19); 375.3 ± 68.8	641.5 (22); 654.2 ± 153.1
Css [dn], ng/mL/mg	0.39 (92); 0.5 ± 0.3	0.8 (27); 0.8 ± 0.2	0.74 (19); 0.8 ± 0.1	0.9 (22); 0.9 ± 0.2

AUC, area under the plasma concentration–time curve; AUC_last, area under the plasma concentration–time curve from time 0 to time of last quantifiable concentration; AUC_last [dn], dose normalized AUC_last; C₂₄, concentration at 24 hours; C_max, maximum observed concentration; C_ss, concentration at steady state; N, number of participants in the treatment group contributing to summary statistic; NA, not presented (below detection limit); PK: pharmacokinetics; t_max: time to maximum concentration.

^a Geometric mean (geometric % coefficient of variation) and arithmetic mean ± standard deviation for all parameters except median (range) for t_max.

^b Data from 1 participant with all concentrations below the limit of detection was not included in the summary statistics.

Figure 2.

Mean plasma lufotrelvir (PF‐07304814) concentration–time profiles following single ascending intravenous infusions at 50, 150, 500, and 700‐mg doses are shown in linear and semilog (inset) representation.

Systemic exposure based on mean AUC_last values appeared to increase in a dose‐proportional manner between the 150‐ and 700‐mg doses, and mean C_ss appeared to increase in a dose‐proportional manner across the dose range studied. The high interparticipant variability in AUC_last (indicated by %CV) at the 50‐mg dose (127%) may be due to values being near the lower limit of quantification, while %CV for the 150‐ to 700‐mg dose ranged between 18% and 36% (Table 3).

Active Moiety PF‐00835231

The PK analysis set for the active moiety PF‐00835231 included data from all participants across all doses studied. Plasma and urine PK parameters are summarized in Table 4. Following 24‐hour IV infusions of lufotrelvir, PF‐00835231 mean C_max was observed between a median t_max of 14 to 16 hours after infusion start across the dose range studied. Near maximum plasma concentrations of PF‐00835231 generally occurred at ≈6 hours and were sustained to the end of the infusion period (Figure 3), with a rapid decrease following the end of the infusion. T_½ values of 2.0 hours and 1.7 hours were observed for the 500‐ and 700‐mg doses and could not be determined for the 50‐ and 150‐mg doses (Table 4).

Table 4.

Summary of PK Parameters (Active Moiety PF‐00835231) (PK Analysis Set)

Parameter (Units) a	50 mg, N = 6	150 mg, N = 5	500 mg, N = 6	700 mg, N = 5
Plasma
AUCinf, ng• h/mL	NR; NR	NR; NR	23,650 (32); 24,700 ± 8237.7	28,680 (23); 29,300 ± 6963.5
AUCinf [dn], ng • h/mL/mg	NC; NC	NC; NC	47.3 (32); 49.4 ± 16.5	40.9 (23); 41.8 ± 9.9
AUClast, ng • h/mL	1915 (32); 2005 ± 740.1	5289 (18); 5360 ± 1026.9	23,530 (32); 24,570 ± 8187.2	28,520 (23); 29,140 ± 6960.1
AUClast [dn], ng • h/mL/mg	38.2 (32); 40.1 ± 14.8	35.3 (18); 35.7 ± 6.8	47.0 (32); 49.1 ± 16.4	40.8 (23); 41.7 ± 10.0
Cmax, ng/mL	97.0 (26); 100.0 ± 29.3	251.0 (17); 253.8± 44.8	1093.0 (29); 1133.0 ± 342.3	1288.0 (21); 1312.0 ± 286.1
t1/2 ± SD, h	NC	NC	2.0 ± 0.2	1.7 ± 0.1
tmax, h	16.0 (9.0–23.8)	16.0 (9.0–16.0)	14.0 (9.0–16.0)	16.0 (11.8–23.8)
C24, ng/mL	77.9 (39); 82.9 ± 34.5	179.9 (34); 188.8 ± 73.3	790.6 (41); 845.7 ± 357	1049.0 (29); 1084.0 ± 328.8
Css, ng/mL	88.3 (30); 91.8 ± 30.7	223.3 (19); 226.8 ± 47.7	983.2 (33); 1028.0 ± 347.4	1180.0 (24); 1208.0 ± 302.6
Css [dn], ng/mL/mg	1.8 (30); 1.8 ± 0.6	1.5 (19); 1.5 ± 0.3	2.0 (33); 2.1 ± 0.7	1.7 (24); 1.7 ± 0.4
Urine
Ae, mg	4.8 (17); 4.8 ± 0.8	11.9 (22); 12.1 ± 2.5	43.5 (17); 44.0 ± 7.4	55.9 (15); 56.4 ± 8.0
Ae, %	10.9 (17); 11.1 ± 1.9	9.2 (22); 9.3 ± 1.9	10.1 (17); 10.2 ± 1.7	9.3 (15); 9.4 ± 1.3

Ae, amount of unchanged drug excreted in urine; AUC, area under the plasma concentration–time curve; AUC_inf, area under the plasma concentration–time curve from time 0 extrapolated to infinite time; AUC_last, area under the plasma concentration–time curve from time 0 to time of last quantifiable concentration; AUC_last [dn], dose normalized AUC_last; C₂₄, concentration at 24 hours; C_max, maximum observed concentration; C_ss, concentration at steady state; dn, dose normalized; N, number of participants in the treatment group contributing to summary statistic; NC, not calculated; NR, not reported; PK, pharmacokinetics; SD, standard deviation; t_1/2, terminal half‐life; t_max, time to maximum concentration.

^a Geometric mean (geometric % coefficient of variation) and arithmetic mean ± standard deviation for all parameters except median (range) for T_max.

Figure 3.

Mean plasma PF‐00835231 concentration–time profiles following single ascending lufotrelvir (PF‐07304814) intravenous infusions at doses of 50, 150, 500, and 700 mg are shown in linear and semilog (inset) representation.

Systemic exposure based on mean AUC_last and mean C_max values appeared to increase in a dose‐proportional manner across all doses administered. Interparticipant variability (based on %CV) for AUC_last was between 18% and 32% and for C_max was between 17% and 29% across the dose range studied. Approximately 9% to 11% of the administered dose was recovered in urine as the active moiety PF‐00835231 across the 50‐ to 700‐mg dose range (Table 4).

NMN PK Parameters

Mean plasma NMN exposure (AUC from time 0 to 12 hours [AUC₁₂] and C_max) was similar in participants receiving placebo compared with those receiving lufotrelvir. Mean renal clearance of NMN also appeared to be similar between participants receiving placebo and those receiving lufotrelvir (Table 5). Similarly, the interparticipant variability (geometric %CV) for renal clearance, AUC₁₂, and C_max of NMN were comparable between participants receiving placebo and those receiving lufotrelvir.

Table 5.

Descriptive Summary of NMN PK Parameters (PK Analysis Set)

Parameter (Units) a	Placebo, N = 8	50 mg, N = 6	150 mg, N = 5	500 mg, N = 6	700 mg, N = 5
Plasma
AUC12, ng • h/mL	642.1 (34); 673.8 ± 230.4	737.6 (38); 778.2 ± 272.9	677.1 (24); 692.6 ± 166.3	715.5 (33); 746.5 ± 232.7	557.6 (32); 579.6 ± 175.6
Cmax, ng/mL	23.3 (37); 24.8 ± 10.3	29.2 (56); 32.5 ± 15.5	26.1 (19); 26.5 ± 4.5	26.4 (27); 27.2 ± 7.0	19.7 (43); 21.0 ± 8.6
tmax, h	23.8 (0.0–48.0)	13.4 (0.0–48.0)	3.0 (0.0–48.0)	23.8 (0.5–28.0)	23.8 (16.0–48.0)
Urine
Ae12, mg	2.9 (63); 3.3 ± 1.9	4.1 (49); 4.5 ± 1.8	3.5 (20); 3.5 ± 0.7	3.1 (70); 3.6 ± 1.8	2.4 (33); 2.5 ± 0.9
CLr, L/h	19.0 (20); 19.3 ± 3.5	22.5 (30); 23.3 ± 6.8	20.1 (23); 20.5 ± 4.4	19.0 (27); 19.5 ± 4.9	19.8 (21); 20.2 ± 4.4

Ae₁₂, amount of endogenous biomarker excreted in urine from 0 to 12 hours; AUC₁₂, area under the plasma concentration–time curve from time 0 to 12 hours; CL_r:, renal clearance; C_max, maximum observed concentration; NMN, N‐methylnicotinamide; PK, pharmacokinetics; t_max, time to maximum concentration. N, number of participants in the treatment group contributing to summary statistic.

^a Geometric mean (geometric % coefficient of variation) and arithmetic mean ± standard deviation for all parameters except median (range) for t_max..

Coproporphyrin I PK Parameters

Plasma CP‐I PK parameters are summarized descriptively in Table 6. Simulated endogenous exposure of CP‐I (AUC₂₄ predose) was similar to what was observed following lufotrelvir administration (AUC₂₄ postdose). The ratio of AUC₂₄ postdose to AUC₂₄ predose values was up to 1.2 across the dose range assessed, indicating nearly equivalent CP‐I exposures before and after dosing.

Table 6.

Descriptive Summary of CP‐I PK Parameters (PK Analysis Set)

Parameter (Units) a	50 mg, N = 6	150 mg, N = 5	500 mg, N = 6	700 mg, N = 5
Cmax, ng/mL	0.4 (7); 0.4 ± 0.0	0.3 (13); 0.3 ± 0.1	0.4 (10); 0.3 ± 0.0	0.3 (27); 0.3 ± 0.1
tmax, h	26.0 (16.0–28.0)	16.0 (11.8–28.0)	16.0 (9.0–30.0)	9.0 (3.0–16.0)
AUC24 predose, ng • h/mL	6.7 (32); 6.9 ± 1.8	7.1 (8); 7.2 ± 0.6	6.5 (13); 6.5 ± 0.8	5.7 (24); 5.8 ± 1.2
AUC24 postdose, ng • h/mL	8.3 (5); 8.3 ± 0.4	7.3 (10); 7.4 ± 0.8	7.4 (8); 7.4 ± 0.6	6.5 (27); 6.6 ± 1.6
AUC24 postdose: AUC24 predoseb	1.2 1.2	1.0 1.0	1.1 1.1	1.1 1.2

AUC₂₄, area under the plasma concentration–time curve from time 0 to 24 hours; AUC₂₄ predose, simulated area under the plasma concentration–time curve from time 0 to 24 hours using the CP‐I predose concentration value of each participant for all time points; C_max, maximum observed concentration; CP‐I, coproporphyrin I; N, number of participants in the treatment group contributing to summary statistic; PK, pharmacokinetics; t_max, time to maximum concentration.

^a Geometric mean (geometric % coefficient of variation) and arithmetic mean ± standard deviation for all parameters except median (range) for t_max.

^b Ratio calculated based on geometric and arithmetic means.

Discussion

Despite the development and approval of several effective vaccines, SARS‐CoV‐2 continues to cause severe disease and significant socioeconomic loss worldwide. The development of effective, targeted antivirals that can be administered during various stages of disease is necessary to tackle infections occurring due to lack of access to vaccination, vaccine hesitancy, waning immunity following vaccination, and rapidly emerging viral mutants of concern resulting in breakthrough infections. ⁸ Results from previous nonclinical studies supported the clinical development of PF‐00835231 as a potential SARS‐CoV‐2 treatment. ¹⁶ , ²³ Here, we report the results of the first phase 1, randomized, double‐blind, placebo‐controlled, single‐ascending‐dose study to evaluate the safety, tolerability, and PK of lufotrelvir (the phosphate prodrug of the active moiety PF‐00835231) as a 24‐hour IV infusion in healthy adult participants. This study demonstrates that the administration of lufotrelvir as a continuous 24‐hour IV infusion across a dose range of 50 to 700 mg was safe, with no tolerability issues identified. There were no deaths, serious AEs, or instances of discontinuation reported. All AEs were mild. No TEAEs were considered treatment related, and all laboratory abnormalities were transient and not considered clinically significant. Following the single‐dose administration of lufotrelvir, exposures increased in a dose‐proportional manner across the dose range (50–700 mg) evaluated. A rapid conversion from the phosphate prodrug lufotrelvir to the active moiety PF‐00835231 was demonstrated with detection of plasma concentrations of PF‐00835231 immediately after the start of the infusion. Sustained conversion to PF‐00835231 was also observed until the end of the infusion. In addition, high C_max (between 97.0 ng/mL and 1288 ng/mL) and C_ss (between 88.26 ng/mL and 1180 ng/mL) of PF‐00835231 were achieved. Similar to other antiviral molecules, ²⁷ , ²⁸ , ²⁹ PF‐00835231 is expected to exhibit therapeutic anti−SARS‐CoV‐2 activity in patients when the free plasma exposure is maintained at or above the in vitro antiviral 90% maximal effective concentration value of 0.5 μM (or a total plasma concentration of 526 ng/mL). The observed PK profiles indicated that this can be achieved at doses higher than 270 mg to 350 mg, assuming linear kinetics and similar PK in patients. The constant rate of IV infusion of lufotrelvir is expected to result in a sustained stable plasma exposure, unlike the peak‐to‐trough variation usually observed following an oral administration. The decreased variability of plasma PK through continuous infusion is also expected to translate into more consistent viricidal activity, indicating a potential for reduction in the emergence of drug‐resistant viral mutants. For anti‐infective therapies, combination therapy is a common approach. In addition to the complementary mechanism of action of antiviral activity, preliminary assessments based on in vitro studies indicated a low potential of lufotrelvir or PF‐00835231 as a perpetrator for drug‐drug interaction related to inhibition of various cytochrome P450 enzymes. Therefore, lufotrelvir may be considered a potential candidate in combination therapy.

We also assessed the potential of lufotrelvir and PF‐00835231 to inhibit renal and hepatic transporters using endogenous biomarkers. In agreement with nonclinical in vitro studies, preliminary data suggest a low risk of hepatic (OATP1B‐mediated) and renal (MATE‐mediated) transporter inhibition. ²³ Plasma exposure and renal clearance of NMN were similar among participants who received lufotrelvir and those who received placebo. Plasma CP‐I exposure was similar before and after dosing, indicating minimal changes due to lufotrelvir administration.

Several molecules are currently being administered for the treatment of SARS‐CoV‐2, many of which have unfavorable adverse reaction profiles. ³⁰ , ³¹ The presence of preexisting comorbidities may exacerbate these effects and have the potential to render available treatment options unusable. The evaluation of treatment options for patients with COVID‐19 presenting with kidney injury and disease is urgent since many are at high risk of mortality, and existing treatment options, such as remdesivir, are contraindicated. Current consensus statements indicate that remdesivir use is not recommended in those with end‐stage renal disease and impaired kidney function even though they are at higher risk of contracting severe COVID‐19. ¹⁴ Previous PK studies on remdesivir have demonstrated that renal excretion accounts for 74% of elimination from the human body. ¹⁴ From our results, for lufotrelvir, only 9% to 11% of the active moiety (metabolized product PF‐00835231; Table 4) was detected in the urine of healthy participants, indicating that renal excretion does not constitute the main pathway for elimination. Therefore, lufotrelvir may represent a viable treatment option for patients with COVID‐19 with existing kidney disease or other comorbidities, like uncontrolled diabetes and hypertension, that result in severely impaired kidney function. In addition, an increase in deep vein thrombosis and other circulatory problems has also been reported in patients treated with remdesivir. ³¹ Initial data from our study indicate that up to 700 mg of lufotrelvir does not cause any abnormalities in laboratory coagulation parameters when administered as a single 24‐hour infusion in healthy participants.

Although several candidates are being evaluated for their ability to inhibit the 3CL^pro, the availability of in vivo PK data in humans is limited, and most studies focus on in vitro or in silico data. ³² Previous in vitro experiments were conducted using 2 SARS‐CoV‐2 clades, the original basal clade (clade A) and the spike protein D614G clade (clade B). From these data in angiotensin‐converting enzyme 2 expressing SARS CoV‐2 infected cells, the half maximal effective concentration reported for a 24‐hour administration of PF‐00835231 was 0.22 μM at 24 hours for clade A–infected cells and 0.18 μM for clade B–infected cells, ²³ indicating that the concentrations achieved across the dose range in the current PK evaluation are likely to be within the therapeutic range. From in vitro data, the half maximal effective concentration reported for remdesivir was higher than for PF‐00835231 (0.44 μM and 0.28 μM, respectively, for each clade). In addition, in vitro data from human airway models also suggest that remdesivir and PF‐00835231 can have a synergistic inhibitory effect on SARS‐CoV‐2 if used together early during infection. ¹⁶ Previous in silico binding data also demonstrated that PF‐00835231 had the best fit while binding to the active pocket of the wild‐type 3CL^pro enzyme when compared with 5 other inhibitors currently in development (bedaquiline, boceprevir, efonidipine, manidipine, and lercanidipine). ²⁴

Conclusions

In summary, single doses of the phosphate prodrug lufotrelvir, from 50 mg to 700 mg administered as a 24‐hour continuous infusion, were demonstrated to be safe, with no tolerability issues identified. In addition, a rapid and sustained conversion to the active moiety PF‐00835231, as well as a dose‐proportional increase in exposures of PF‐00835231, were observed across the studied dose ranges. PK characteristics suggested that a daily dose of 270 to 350 mg, administered over 24 hours as a continuous infusion, is expected to maintain the C_ss of PF‐00835231 at or above the antiviral 90% maximal effective concentration. Based on these results, further studies in patients with COVID‐19 are warranted. Currently, a phase 1b study evaluating the safety and PK of single and multiple ascending doses of lufotrelvir in hospitalized patients with COVID‐19 is complete. ³³

Conflicts of Interest

All authors are employees of Pfizer, Inc. and may hold stock/stock options in Pfizer.

Funding

This study was sponsored by Pfizer Inc.

Data Availability Statement

The data sets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.