Ulcerative colitis is a chronic inflammatory bowel disease with a remitting and relapsing course, characterized by bloody diarrhea, abdominal cramps, and fatigue.1, 2 The pathogenesis is thought to result from inappropriate immune response to gastrointestinal antigens and environmental triggers in genetically susceptible individuals.3 The highest prevalence is reported in Europe (505 per 100 000 persons) and North America (249 per 100 000 persons).4 Ulcerative colitis has a significant negative impact on patient quality of life and presents a high economic burden on health systems.

Inflammatory bowel disease has been managed with corticosteroids, 5-aminosalicylates, and immunosuppressants, and more recently with the use of biologics targeted against specific mediators of inflammation. Therapeutic options for the long-term maintenance of remission in ulcerative colitis are limited.5 5-Aminosalicylates such as sulfasalazine, olsalazine, balsalazide, and various forms of mesalamine are effective only in mild to moderate disease, whereas patients with severe disease may be started on biologics.6 Several monoclonal antibodies against tumor necrosis factor (TNF)-α (eg, infliximab, adalimumab, golimumab, and certolizumab) are now available.7-10 Agents targeted against other cytokines involved in the inflammatory response such as ustekinumab against interleukin (IL)-12/IL-23, and tofacitinib, a pan–Janus kinase (JAK) inhibitor, are now part of the therapeutic options available for inflammatory bowel disease, and several IL-23 and sphingosine-1-phosphate receptor modulators are also currently under clinical investigation.11-13

In spite of the wide array of therapeutic options, there are still limitations in the treatment of inflammatory bowel diseases, and the agents available are not without risk. TNF-α inhibitors are ineffective in approximately one-fifth to one-third of the patients, and 10% to 15% of treated patients who show an initial benefit may lose response every year.6, 14-16 Cutaneous reactions are also the most common adverse reactions with anti-TNF treatments.17 This includes injection site reactions, cutaneous infections, immune-mediated complications such as psoriasis and lupus-like syndrome, and, rarely, skin cancers. Tofacitinib can increase the risk of infection and may increase the risk of thrombosis or thromboembolic events. There is increasing recognition that mitigation of the local inflammatory response may hold promise. Orally administered budesonide and 5-aminosalicylates are effective locally, and various other locally acting agents, including AMT-101, an oral biologic fusion protein of interleukin 10,18 TD-1473, a JAK inhibitor,19 GB004, a hydroxylase inhibitor,20 and BT-11, a lanthionine synthetase inhibitor,21 have shown promise or are undergoing clinical investigation for inflammatory bowel disease. Local delivery through oral administration may allow higher doses of drug to be delivered to the target site without increasing systemic side effects.

The α4β7 integrin, present on the cell surface of circulating memory T and B lymphocytes, is primarily involved in the recruitment of leukocytes to the gastrointestinal mucosa and associated lymphoid tissues. The major ligand for α4β7, mucosal addressin cell adhesion molecule, is selectively expressed on the endothelium of the gastrointestinal vasculature and is present in increased concentrations in inflamed tissues.

Vedolizumab is an intravenously administered humanized immunoglobulin G monoclonal antibody directed against α4β7 that has been approved for the treatment of moderate to severe ulcerative colitis and Crohn disease in adult patients who are not responding to ≥1 conventional treatments, such as steroids, immunosuppressive agents, or TNF inhibitors.22-24 Due to the inconvenience and potential systemic risks of injectable treatments, an oral, gastrointestinal-restricted therapeutic that selectively targets the α4β7 integrin may provide a significant benefit to patients with ulcerative colitis. Results from a small clinical study with PTG-100, a first-generation α4β7 integrin antagonist, have demonstrated dose-dependent mucosal healing suggesting that an oral, gut-restricted, α4β7 integrin antagonist may be effective in ulcerative colitis.25 PN-943 is an orally stable peptide and a structural analog of PTG-100 that binds specifically to the α4β7 integrin on leukocytes with a higher in vitro potency than PTG-100.26 Preclinical studies have shown that PN-943 has minimal systemic absorption (<1%) in animals and is more effective than PTG-100 as measured by greater levels of target engagement and effects on T-cell trafficking. In a trinitrobenzenesulfonic acid–induced colitis rat model, PN-943 dosing caused a significantly lower mean colon histopathology score compared to rats treated with vehicle or PTG-100.26 Through the blockade of leukocyte trafficking in the gut and local lymphocyte activation,27 PN-943 may inhibit colon inflammation, reducing the signs and symptoms of ulcerative colitis. The present study investigated the safety, tolerability, pharmacokinetics, and pharmacodynamics of oral PN-943 in healthy male subjects.

Methods Study Design

Two studies were conducted at a single clinical center (Nucleus Network, Melbourne, Australia). Study 1 was a 3-part first-in-human study in healthy male volunteers to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of a liquid solution formulation of PN-943. Part 1 was a randomized, placebo-controlled, double-blind study of single ascending doses of PN-943 in 40 male subjects divided into 4 equal cohorts. Dose escalation proceeded from 100 mg to 300 mg, 1000 mg, and 1400 mg. Subjects in the 300-mg dose cohort received treatment in the fasted state on 1 occasion and following a high-fat meal on a second occasion in a crossover fashion. The high-fat meal consisted of 2 eggs fried in butter, 2 strips of bacon, 2 slices of toast with butter, 4 ounces of hash brown potatoes, and 240 mL of whole milk. During part 1, subjects refrained from food and drink except water for 10 hours before and for 4 hours after dosing with the exception of subjects in the 300-mg dose cohort during the fed treatment. Part 2 was a randomized, placebo-controlled, double-blind multiple-ascending-dose study in 50 male subjects divided equally into 5 cohorts. Subjects received once-daily dosing of PN-943 or placebo for 14 days. Doses evaluated in part 2 included 100 mg, 300 mg, and 1000 mg. During part 2, two cohorts of subjects (100 mg and 300 mg) received food approximately 30 minutes before each dose and another 2 cohorts of subjects (300 mg and 1000 mg) refrained from food for 10 hours before and for 1 hour after dosing. An additional cohort of 9 subjects in part 2 received 300 mg of PN-943 in a crossover fashion to evaluate the effect of meal timing on the pharmacokinetics and pharmacodynamics of PN-943. Subjects in this cohort received a meal 30, 60, or 90 minutes after PN-943 dosing. Part 3 was an open-label, randomized, crossover multiple-dose comparison of 900-mg once-daily and 450-mg twice-daily dosing of PN-943 as a liquid solution for 5 days. Subjects in part 3 refrained from food for 10 hours before and for 1 hour after dosing of PN-943.

The second study was a 5-day multiple-dose pharmacokinetic and pharmacodynamic study comparing the liquid formulation and a tablet formulation administered as 450 mg PN-943 twice daily in healthy male and female subjects. Subjects held food for 10 hours before and for 1 hour after the morning dose and for 1 hour before and after the evening dose of each day.

Both studies were conducted at a single clinical center, and the study protocols, subject information, and informed consent form were reviewed and approved by independent human research ethics committees (Alfred Health Human Research Ethics Committee, Melbourne VIC 3004, Australia for study 1; and Bellberry Human Research Ethics Committee, Eastwood South Australia 5063, Australia for study 2). The studies were conducted in accordance with the Declaration of Helsinki on biomedical research involving human subjects and International Conference on Harmonization Good Clinical Practice guidelines, and all study procedures were conducted by scientifically and medically qualified personnel. Written informed consent explaining the nature, purpose, and potential risks and benefits of the study was provided by subjects before they participated in any study-related activities.

Study Subjects

Both studies used similar procedures for screening and enrollment. Subjects were screened within 21 days of enrollment. Eligible subjects were aged 18 to 55 years inclusive with a body mass index between 18 and 30 kg/m2, who were in good general health, with no significant medical history or clinically significant abnormalities on physical examination. The first-in-human study (study 1) enrolled only male subjects, while the study evaluating the tablet formulation (study 2) enrolled men and women who agreed to use highly effective methods of contraception based on the Clinical Trials Facilitation and Coordination Group for the duration of the study and for 90 days after the last dose.

Subjects were excluded if they had a history of clinically significant endocrine, gastrointestinal, cardiovascular, hematologic, hepatic, immunologic, renal, respiratory, or genitourinary abnormalities or diseases, or had clinically significant laboratory abnormalities, including impaired renal function (serum creatinine >106 μmol/L or estimated creatinine clearance <80 mL/min) or alanine aminotransferase or aspartate aminotransferase values >1.2 times the upper limit of normal.

Procedures Study 1

The single- and multiple-ascending-dose phase of the study consisted of sequential dose escalations in 10 subject per dose cohort. Participants were randomized to receive PN-943 or matching placebo as a 60-mL oral solution in a ratio of 8:2. Blood samples for pharmacokinetics were collected before dosing and for 48 hours after dosing following single doses. In the multiple-ascending-dose phase, blood samples were obtained on days 1 to 3 and 14 to 16; on day 8, samples were obtained before dosing and at 4 and 12 hours after dosing. On day 10 of the multiple-ascending-dose phase, subjects were required to collect all urine for the 0- to 6-, 6- to 12-, 12- to 18-, and 18- to 24-hour intervals after dosing, and on day 11, subjects were required to collect all fecal samples for 24 hours. The decision to proceed to the next dose level was made by the investigator and the safety monitoring committee based on acceptable safety and tolerability of the lower dose.

Study 2

This study was a randomized, open-label, 2-treatment, 2-period, multiple-dose study to determine the safety, tolerability, pharmacokinetics, and pharmacodynamics of an immediate-release (IR) tablet and a liquid solution of PN-943. The study allowed comparison of a solid dose formulation to the liquid formulation that had been investigated in the first-in-human study. Subjects received 450 mg of PN-943 twice daily for 5 days as one 300-mg and one 150-mg dosage strength IR tablet administered every 12 hours and 450 mg of PN-943 twice daily for 5 days as a liquid solution administered every 12 hours in a randomized fashion.

Stability studies were conducted for the dosing solutions over the anticipated concentration range (1-25 mg/mL) and demonstrated that dosing solutions were stable for up to 3 months when stored at 2 to 8°C. Dose solutions were formulated in 50 mM of phosphate buffer, pH 7.4, and were prepared weekly by a qualified pharmacist.

Dose Selection Rationale

The starting dose in the first-in-human single- and multiple-dose study was based on consideration of the no-observed-effect level from 28-day toxicology studies in rats and cynomolgus monkeys, and the receptor occupancy noted in cynomolgus monkeys. The no-observed-effect level determined in rats and monkeys translated to a human equivalent dose of approximately 145 mg using standard allometric scaling and a 10-fold safety margin. A starting dose of 100 mg was selected, with initial stepwise escalations of approximately 3-fold.

The dose selected for study 2, comparing a tablet and the oral solution formulation, was based on the pharmacokinetic and pharmacodynamic profile from part 3 of the first-in-human study and the anticipated dose planned in an efficacy study in patients with moderate to severe ulcerative colitis.

Sample Analysis Method for PN-943 in Humans

Concentrations of PN-943 in plasma, urine, and fecal samples from study 1 and plasma and fecal samples from study 2 were assayed using a validated high-performance liquid chromatography–tandem mass spectrometry method.

PN-943 and its stable isotope-labeled internal standard were isolated from plasma using a protein precipitation procedure using acetonitrile : methanol : formic acid solution (74.9% : 25.0% : 0.1%). PN-943 and its stable-labeled internal standard were isolated from urine stabilized with 10% Triton X-100 (100 μL/10 mL urine) using a dilution procedure with aqueous solution of 75% acetonitrile. Feces samples were homogenized with ethanol and phosphate buffer solution (50% : 50%). PN-943 and its stable isotope-labeled internal standard was extracted from feces homogenate using a solid-phase extraction procedure with 5% ammonia solution in ultra-pure water and eluted with methanol/trifluoroacetic acid mixture (99% : 1%). Analysis of PN-943 in all matrices was conducted using a Zorbax 300 StableBond C18 column (2.1 × 150 mm) with 5-μm particle size (Agilent Technologies, Santa Clara, California) and a C18 guard cartridge (4.0 × 21 mm; Phenomenex, Torrance, California). Mobile phase A comprised 5% acetonitrile, 94.9% water, and 0.1% formic acid; and mobile phase B comprised 94.9% acetonitrile, 5% water, and 0.1% formic acid. The samples were analyzed using a liquid chromatography coupled with tandem mass spectrometry using an API4000 detector (AB SCIEX, Framingham, Massachusetts) using a turbospray ion source with positive multiple reaction monitoring mode with m/z for Q1 mass set to 918.0.

Calibration curves for PN-943 in human plasma were linear from 0.200 to 100 ng/mL using 200 μL of plasma with correlation coefficients ≥0.9889. Calibration curves for PN-943 in urine were linear from 20.0 to 10,000 ng/mL using 50 μL of urine with correlation coefficients ≥0.9881. Calibration curves for PN-943 in fecal homogenate were linear from 0.100 to 50.0 μg/mL using 50 μL of fecal homogenate with correlation coefficients ≥0.9926. The interday (between-day) precision (% coefficient of variation) ranged from 7.2% to 13.2% for plasma, 4.1% to 18.0% for urine, and 4.3% to 8.2% for fecal homogenate. Intraday (within-day) precision (% coefficient of variation) ranged from 1.3% to 15.8% for plasma, 1.7% to 13.5% for urine, and 2.2% to 8.7% for fecal homogenate. The interday accuracy (% bias) ranged from –4.7% to 0.7% for plasma, –6.8% to 7.2% for urine, and –5.6% to 4.0% for fecal homogenate. Intraday accuracy ranged from –19.5% to 4.9% for plasma, –19.5% to 20.0% for urine, and –7.7% to 6.0% for fecal homogenate.

Study End Points

The objective of this first-in-human study was the safety and tolerability assessments following single and multiple dosing with PN-943. In addition, the study characterized the single- and multiple-dose pharmacokinetics and pharmacodynamics of PN-943, and evaluated the effect of a high-fat meal on PN-943 pharmacokinetics, and compared twice-daily and once-daily dosing. Safety assessments, adverse events, and laboratory assessments are summarized descriptively for the placebo and each PN-943 dose.

The end points for the second study comparing the oral solution and the tablet formulation were pharmacokinetics and pharmacodynamics.

Pharmacokinetic Analyses

Pharmacokinetic parameters were estimated by noncompartmental methods (Phoenix WinNonlin; Certara, Princeton, New Jersey). Peak plasma concentration (Cmax) and time to peak plasma concentration were observed values. The elimination rate was estimated from the slope of the least squares regression on the terminal log-linear phase. Area under the plasma concentration–time curve from time 0 to the last quantifiable concentration (AUCt) was estimated by a linear trapezoidal method and was extrapolated to infinity (AUCinf) by dividing the last quantifiable concentration by the elimination rate. For calculation of plasma concentration summary statistics, values below the limit of quantification were set to 0. Steady-state fluctuation in the plasma concentration was calculated as . Accumulation was estimated as the ratio of the parameter (Cmax and AUCinf) following the last dose of the multiple-dose regimen to the value on day 1 (AUCinf Day 14 or Day 5/AUCinf Day 1 and Cmax Day 14 or Day 5/Cmax Day 1). Bioavailability of the IR tablets was estimated relative to the liquid solution using the ratio of the AUCinf for the 2 treatments.

Pharmacodynamic Assays for α4β7 Receptor Occupancy and Receptor Expression

Translational biomarkers such as receptor occupancy have been validated as pharmacodynamic markers through use in preclinical studies and in clinical trials with vedolizumab binding against the α4β7 receptor.28, 29 In this study, a flow cytometry–based assay was designed to quantify the amount of α4β7 integrin on the cell surface that is occupied by PN-943 or the amount of α4β7 expression on the cell surface of circulating lymphocytes in response to engagement by PN-943. Similar approaches for measuring receptor occupancy and receptor expression using flow cytometry have been used previously.30-33 Briefly, in this assay, each heparinized whole blood sample is first treated with saturating amount of an unlabeled competing peptide serving as the “blocked” control for 100% receptor occupancy, or no peptide, serving as the “unblocked” sample to measure the level of blocking by orally administered PN-943. After incubation, the blood is stained with a subsaturating concentration of Alexa647-labeled peptide, followed by staining with the cell surface marker panel (CD45, CD3, CD4, CD45RA, CD19, immunoglobulin D, and the anti-α4β7 antibody vedolizumab). After staining is completed, the samples are treated with a red blood cell lysis and fixation buffer, washed and acquired on a flow cytometer. To quantify receptor occupancy on α4β7 expressing memory CD4 T cells, the median fluorescence intensity (MFI) of Alexa647-labeled peptide within the vedolizumab + memory CD4+ T cells was used. Receptor occupancy was calculated according to the following formula: [Percent RO] = (1 – ([Unblocked] – [Blocked]) / ([Baseline Unblocked] – [Baseline Blocked])) × 100.

Expression of α4β7 is defined by MFI of vedolizumab within the memory CD4+ T cells from the unblocked samples. Receptor expression was calculated as percent change of MFI from baseline for the vedolizumab stain.

Pharmacokinetic/Pharmacodynamic Analysis

The in vivo PN-943 plasma concentration-receptor occupancy relationship was characterized using a sigmoid Emax (Hill) model, using Prism version 9.0.0 (GraphPad Software, San Diego, California).

Statistical Analyses

No formal sample size estimations were performed. Eight subjects received oral PN-943, and 2 subjects received placebo in each dose cohort in the single- and multiple-ascending-dose study. Ten subjects were enrolled in the second study comparing the immediate-release tablet formulation to the oral solution. The enrollment in each study was considered adequate to assess the tolerability and safety and to allow characterization of the pharmacokinetics and pharmacodynamics of PN-943.

Results Subject Characteristics and Disposition

A total of 97 healthy male subjects were enrolled in study 1 with 40 subjects enrolled in the single-dose phase and 57 subjects in the multiple-dose phase. An overview of the disposition of subjects is presented in Figure S1. All subjects completed their dosing with PN-943 or placebo as planned. Two subjects withdrew consent for personal reasons unrelated to safety; 1 subject did not want to remain in the clinical unit, and a second subject was uncomfortable with the venous cannula. Subject characteristics are summarized in Table S1. The average age was 28.7 years in the single-dose phase and 30.9 years in the multiple-dose phase.

Ten subjects were enrolled in study 2, and 9 subjects completed both treatments. One subject discontinued the study on day 1 following the oral solution treatment due to an adverse event of acute tonsillitis that was considered unrelated to the study drug. Subject characteristics for study 2 are presented in Table S1.

Safety and Tolerability

An overview of the treatment-emergent adverse events (TEAEs) following single doses of PN-943 (study 1) is presented in Table S2. A total of 23 TEAEs were reported by 14 subjects during the single-ascending-dose phase. Of the 14 subjects who experienced TEAEs, 12 received PN-943 (21 events) and 2 received placebo (2 events). Of the 21 TEAEs following PN-943, 7 were considered related to treatment; both TEAEs following placebo were considered treatment related. Incidence of TEAEs did not demonstrate a systematic dose relationship. All TEAEs were mild or moderate except for a severe headache in a subject treated with 100 mg of PN-943 that was not considered related to treatment. All subjects recovered from the adverse events (AEs) and no subjects were withdrawn due to AEs. Nervous system disorders were the most frequently reported TEAEs. AEs reported in ≥2 subjects included nausea, upper respiratory tract infections, headache, presyncope, and somnolence. No clinically relevant changes were observed in respiratory rate or vital signs, clinical laboratory parameters (hematology, coagulation, serum chemistry, or urinalysis), or in the interpretation of electrocardiograms or QTc interval.

An overview of the TEAEs following multiple doses of PN-943 is shown in Table S3. Thirty subjects in the group receiving multiple doses of PN-943 reported a total of 68 AEs. All but 2 occurrences were mild in severity. One report of upper respiratory tract infection was characterized as moderate, and 1 report of influenza that occurred after release from the clinical unit was categorized as severe and considered a serious AE. Four subjects receiving placebo reported a total of 6 mild TEAEs, primarily gastrointestinal disorders. TEAEs reported in ≥2 subjects in the multiple-ascending-dose phase included abdominal discomfort, flatulence, upper respiratory tract infection, back pain, dizziness, and headache. Nervous system disorders, particularly headache, were the most commonly reported TEAEs. No clinically relevant changes were observed in respiratory rate, vital signs, or clinical laboratory parameters or on the electrocardiograms.

Of the 10 subjects enrolled in study 2 comparing dosing with IR tablets to an oral solution, 9 subjects completed both treatments. One subject experienced an AE of moderate tonsillitis unrelated to the treatment that led to discontinuation from the study. The incidence of TEAEs was similar across both treatments. The most common AE was headache, with all other AEs being reported in only 1 subject.

Pharmacokinetics

The mean plasma concentration-time profiles following single doses of PN-943 are presented in Figure 1A. The single-dose pharmacokinetics of PN-943 are summarized in Table 1. Median time to peak plasma concentration was 2 to 4 hours. Cmax increased from 2.11 ng/mL to 23.5 ng/mL, and AUCinf increased from 16.5 ng • h/mL to 260 ng • h/mL as PN-943 doses increased from 100 mg to 1400 mg. There was a dose-proportional increase in AUCinf and a slightly less than dose-proportional increase in Cmax over the dose range of 100 mg to 1400 mg PN-943. The mean elimination half-life at the lower doses (100 and 300 mg) was 3.1 to 4.0 hours and at higher doses (1000 and 1400 mg) was 5.3 to 5.7 hours.

Single dose pharmacokinetics (A) and pharmacodynamics of PN-943 based on receptor occupancy (B) and receptor expression (C).

Table 1. Single-Dose Pharmacokinetics of PN-943 Following Oral Dosing (Mean ± SD) 100 mg (N = 8) 300 mg Fasted (N = 8) 300 mg High Fat Meal (N = 8) 1000 mg (N = 8) 1400 mg (N = 8) Cmax, ng/mL 2.11 ± 1.15 6.55 ± 3.38 1.58 ± 0.71 15.3 ± 4.11 23.5 ± 19.0 tmax, ha 2.0 (1.0, 4.0) 3.0 (1.0, 8.0) 4.0 (2.0, 4.0) 4.0 (0.5, 4.0) 4.0 (1.0, 12.0) AUCt, ng • h/mL 12.9 ± 7.27 44.3 ± 21.5 11.5 ± 4.62 138 ± 33.7 257 ± 173 AUCinf, ng • h/mL 16.5 ± 8.70b 57.6 ± 23.6b –c 151 ± 31.7d 260 ± 173 t1/2, h 3.05 ± 0.71b 4.02 ± 1.37b –c 5.26 ± 0.91d 5.74 ± 1.35 AUCt, area under the plasma concentration–time curve to the last observed concentration; AUCinf, area under the plasma concentration–time curve extrapolated to infinity; Cmax, maximum observed plasma concentration; SD, standard deviation; tmax, time to maximum concentration; t1/2, half-life. aMedian (min, max) bN = 4. cNot reported due to insufficient data. dN = 7.

The mean plasma concentration-time profiles following multiple doses of PN-943 are presented in Figure 2A. The pharmacokinetics of PN-943 following multiple doses is summarized in Table 2. There was an approximate dose-proportional increase in Cmax and in AUCinf for the 100- and 300-mg dose groups in the fed condition on day 14 and between the 300-mg and 1000-mg dose groups in the fasted condition on day 14. Median time to peak plasma concentration ranged from 2 to 4 hours. The mean elimination half-life was 5.2 to 7.7 hours. Consistent with the half-life, a comparison of the Cmax and the AUCt values for individual subjects on day 1 and on day 14 at 300 mg and 1000 mg suggested minimal (≤30%) accumulation with once-daily dosing. Comparison of the AUCinf values on day 1 and AUCt values on day 14 indicated the absence of time-dependent changes in the pharmacokinetics of PN-943.

Multiple-dose pharmacokinetics (A) and pharmacodynamics of PN-943 based on receptor occupancy (B) and receptor expression (C).

Table 2. Multiple-Dose Pharmacokinetics of PN-943 Following Oral Dosing (Mean ± SD) 100 mg Fed (N = 8) 300 mg Fed (N = 8) 300 mg Fasted (N = 8) 1000 mg Fasted (N = 8) Day 1 Day 14 Day 1 Day 14 Day 1 Day 14 Day 1 Day 14 Cmax, ng/mL 0.745 ± 0.409 0.898 ± 0.454 2.32 ± 1.34 2.80 ± 1.66 7.23 ± 4.29 4.72 ± 1.85 13.8 ± 3.86 17.1 ± 6.05 tmax, ha 4.0 (2.0, 8.0) 4.0 (2.0, 4.0) 4.0 (1.0, 4.0) 4.0 (4.0, 8.0) 2.0 (0.5, 2.0) 2.0 (1.0, 4.0) 2.0 (0.25, 4.0) 2.0 (1.0, 4.0) AUCt, ng • h/mL 4.58 ± 3.12 5.87 ± 3.38 16.4 ± 10.8 21.0 ± 9.99 42.9 ± 17.9 39.5 ± 11.5 141 ± 43.6 184 ± 81.8 AUCinf, ng • h/mL 9.81b 8.33b 39.7b – 46.3 ± 19.2 43.9 ± 13.0d 157 ± 55.7 207 ± 125 t1/2, h 3.85b 7.40b 7.96b –c 5.16 ± 1.92 7.31 ± 3.86d 6.60 ± 1.99 7.70 ± 3.69 AUCt, area under the plasma concentration–time curve to the last observed concentration; AUCinf, area under the plasma concentration–time curve extrapolated to infinity; Cmax, maximum observed plasma concentration; SD, standard deviation; tmax, time to maximum concentration; t1/2, half-life. aMedian (min, max). bN = 1. cNot reported due to insufficient data. dN = 7.

During the multiple-ascending-dose phase, 24-hour collection of urine and feces was undertaken in the 300-mg and 1000-mg dose groups. Only a small fraction of PN-943 was recovered intact in urine over 24 hours, with recoveries of 0.028%, 0.056%, and 0.056% in the 300-mg fasted, 300-mg fed, and 1000-mg dose groups, respectively. There was a dose-related increase in the 24-hour fecal recovery of PN-94 with 0.73%, 1.78%, and 16.8% PN-943 recovered intact in the 300-mg fasted, 300-mg fed, and 1000-mg dose groups, respectively.

Effect of Food

The effect of a high-fat meal on the pharmacokinetics of PN-943 was evaluated in a crossover fashion at 300 mg during the single-ascending-dose portion of study 1. Administration of PN-943 within 30 minutes of consuming a high-fat meal reduced the peak concentration and exposure compared to the fasted state (Table 1). Mean PN-943 peak plasma concentrations were 6.55 ng/mL in the fasted state and 1.58 ng/mL in the fed state. Median time to peak concentration was delayed by 1 hour following a high-fat meal.

The effect of the interval between PN-943 dosing and consumption of a meal was examined in study 1. Subjects received a meal 30, 60, or 90 minutes after a single dose of 300-mg PN-943. The median time-to-peak PN-943 plasma concentrations was 1, 2, and 4 hours for the 30-, 60-, and 90-minute treatment groups. There was a small increase in the Cmax and AUCt values when food was delayed 60 or 90 minutes compared to 30 minutes following PN-943, with minor differences noted between the 60- and 90-minute delay. Based on the more favorable Cmax and AUCt values noted for the 60-minute delay in food compared to the 30-minute delay, dosing for additional cohorts in the multiple ascending dose incorporated a 1-hour fasting interval before and after dosing of PN-943.

Table 2 presents a comparison of the pharmacokinetics of 300 mg of PN-943 following a meal compared to refraining from a meal within 1 hour of dosing PN-943 as part of the multiple-ascending-dose phase of study 1. The median time-to-peak concentration was 4 hours when PN-943 was administered following a meal, whereas it was 2 hours when food was withheld for 1 hour following PN-943. Peak plasma concentrations were lower when PN-943 was administered shortly after a meal compared to when food was administered 1 hour following dosing (day 1 Cmax were 7.23 ng/mL and 2.32 ng/mL for the fasted and fed 1 hour after dosing, respectively).

Pharmacokinetics of Once-Daily and Twice-Daily Dosing

The effect of dosing regimen was evaluated following 900 mg once daily for 5 days and 450 mg twice daily for 5 days in a randomized crossover fashion in part 3 of study 1. A summary of the pharmacokinetic comparison of once-daily and twice-daily dosing is presented in Table S4. Peak concentrations were noted at a median of 2 hours for both dosing regimens on day 1 and on day 5. The steady-state peak concentrations were 14.2 ng/mL for once-daily dosing and 9.96 ng/mL for twice-daily dosing. Dose-adjusted AUC over the dosing interval was comparable for the 2 treatment regimens. Consistent with the half-life of PN-943, there was minimal accumulation with once-daily dosing, and the accumulation was approximately 1.6- to 1.7-fold with twice-daily dosing. Twice-daily dosing of 450 mg of PN-943 as a liquid solution resulted in sustained plasma concentrations as reflected by the lower peak-to-trough fluctuation (143% vs 245%) and higher trough concentrations (3.25 ng/mL vs 1.78 ng/mL) compared to 900 mg once daily (Table S4).

Pharmacokinetics of a Liquid Solution and an IR Tablet Formulation of PN-943

The steady-state pharmacokinetics of an IR tablet of PN-943 administered as 450 mg twice daily for 5 days compared to the liquid solution used in the first-in-human study is summarized in Table 3. Figure 3A presents the mean steady-state plasma concentration–time profile for the 2 formulations. Both formulations had a similar median time to peak concentration (2 hours), while the peak concentration was  ≈20% lower for the IR tablet compared to the liquid solution. The IR tablet formulation had a bioavailability of ≈85% relative to the liquid solution. Twice-daily dosing of the tablet formulation resulted in an accumulation of ≈2.2-fold based on Cmax and 1.6-fold based on AUC. Steady-state trough concentrations of PN-943 were comparable for the IR tablet and the liquid solution (1.86 ng/mL and 1.98 ng/mL, respectively).

Table 3. Steady-State Pharmacokinetics of PN-943 Following Oral Dosing of 450 mg Twice-Daily as a Liquid Solution and as an Immediate-Release Tablet (Mean ± SD) Liquid Solution 450 mg Twice Daily (N = 9) Immediate Release Tablet 450 mg Twice Daily (N = 9) Cmax, ng/mL 9.36 ± 4.81 7.67 ± 2.97 tmax, ha 2.0 (1.0, 8.0) 2.0 (2.0, 4.0) AUC0-24, ng • h/mL 106 ± 34.7 86.3 ± 30.9 AUC0-12, ng • h/mL 65.6 ± 38.6 53.8 ± 20.9 AUC12-24, ng • h/mL 39.3 ± 15.2 32.4 ± 11.4 Bioavailability,b % – 85.3 ± 36.2 Accumulationc Cmax 1.25 ± 0.46 2.19 ± 1.0 Accumulationc AUC