FormalPara Key Points

Palovarotene, a cytochrome P450 3A4 substrate, is under investigation for the treatment of fibrodysplasia ossificans progressiva (FOP), an ultra-rare genetic disorder

Despite known differences in metabolism of cytochrome P450 substrates, this trial demonstrated that palovarotene is metabolized similarly between Japanese and non-Japanese individuals

Palovarotene dose adjustments are not necessary for Japanese patients with FOP

1 Introduction

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare genetic disorder with an estimated global prevalence of up to 1.4 per million individuals, within which there are no ethnic, sex, or geographic predispositions [1,2,3]. The condition is caused by a spontaneous missense mutation in the activin receptor-like kinase-2/activin A receptor type 1 (ALK2/ACVR1) gene, which leads to activation of bone morphogenetic protein (BMP) signaling [2]. Aberrant BMP signaling results in heterotopic ossification (HO), a process that occurs through an endochondral pathway that transforms soft and connective tissues into bone [4,5,6,7]. HO, which is often preceded by painful flare-ups in which connective tissue swells, results in progressive restriction of movement, cumulative disability, and decreased quality of life [8, 9].

Until recently there were no approved disease-modifying treatments for FOP [10]. Interventions were limited to supportive care and flare-up management [11, 12]. Palovarotene, an orally bioavailable, selective retinoic acid receptor gamma (RARγ) agonist is under investigation as a treatment for FOP as part of a global clinical development program [13,14,15,16]. Palovarotene inhibits chondrogenesis through activation of the retinoid signaling pathway via RARγ, preventing HO [17]. Pooled data from phase II trials of palovarotene in FOP show a lower volume of new HO associated with flare-ups in patients receiving palovarotene compared with placebo-treated/untreated individuals [18]. Moreover, interim data from the multicenter, open-label phase III trial MOVE indicated that treatment with palovarotene results in lower mean volume of new HO per year compared with that in untreated patients [19].

In vitro studies have indicated that palovarotene is primarily metabolized by cytochrome P450 3A4 (CYP3A4) and to a minor extent by CYP2C8 and CYP2C19 [20, 21]. Ethnic differences in the expression levels of CYP450 enzymes have been observed between Japanese and Caucasian individuals [22]. It is therefore possible that palovarotene pharmacokinetics could vary between these populations. This trial was conducted to compare the pharmacokinetic, safety, and tolerability profile of palovarotene in healthy Japanese and non-Japanese participants.

2 Methods

2.1 Trial Participants

This was a double-blind, randomized, cross-over, single-dose phase I trial (NCT04829786). Participants were healthy Japanese and non-Japanese participants, matched individually (on a 1:1 basis) with respect to sex, age (±5 years), and weight (±10%). Japanese participants were required to have been born in Japan, with all biological parents and grandparents of Japanese descent. Non-Japanese individuals were required to have all biological parents and grandparents of non-Japanese descent, and to have been successfully matched with a Japanese participant at screening. Both male and female participants were eligible and needed to be aged 18–55 years and have a body mass index (BMI) of 18–30 kg/m2, a body weight of ≥ 50 kg, a resting pulse between 45 and 100 beats per minute (bpm), and a systolic and diastolic blood pressure of < 140/90 mmHg. Females of childbearing potential were required to have a negative serum pregnancy test at screening and on the day before dosing. They also needed to be willing to commit to using two consistent and acceptable methods of contraception simultaneously for at least 30 days prior to the first dose of palovarotene. Exclusion criteria included a history or current evidence of clinically significant or uncontrolled disease. The full inclusion and exclusion criteria are outlined in Supplementary Table 1.

All participants signed informed consent forms, which were reviewed and approved by the corresponding Institutional Review Board alongside any protocol updates. The trial was conducted in accordance with the Declaration of Helsinki and the International Council for Harmonisation Good Clinical Practice guidelines [23, 24].

2.2 Trial Design

A schematic of the trial design is provided in Fig. 1. Following screening, participants were randomized 1:1 on day 1 of period 1 to receive a single oral dose of palovarotene 5 mg or 10 mg, followed by the alternate dose in period 2. Between the doses, there was a 5-day washout period. Doses of palovarotene were administered 30 min after the start of a standardized breakfast (menus are provided in Supplementary Table 2).

Fig. 1
figure 1

Trial design. N: number of participants

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2.3 Preparation of Samples

Blood draws of 2 mL were performed pre-dose (hour 0) and at hours 1, 2, 3, 4, 6, 8, 10, 12, 16, 24, 48, and 72 post-palovarotene administration, and were used to determine palovarotene plasma concentrations. Plasma was separated and frozen until shipping for analysis. Plasma concentrations were ascertained using a validated liquid chromatography–mass spectrometry method with a lower limit of quantification of 0.005 ng/mL for palovarotene.

2.4 Bioanalytical Assay

The quantitative determination of palovarotene in human plasma samples with lithium heparin as anticoagulant was validated over a concentration range of 5 to 2000 pg/mL. Palovarotene and the internal standard, [13C6]palovarotene, were isolated from lithium heparin human plasma samples (20 μL) using a 96-well protein precipitation extraction procedure performed under yellow light. After evaporation to dryness and reconstitution, plasma extracts were analyzed by turbo ion spray liquid chromatography (reversed phase C18 column)/tandem mass spectrometry in the positive ion mode (LC-MS/MS). Linear regression with 1/x2 weighting was determined to be the best curve fit for the quantitation of palovarotene in human plasma over the calibration range.

Accuracy and precision of quality control samples were within 15% of nominal. Extraction recoveries for palovarotene from human plasma over the calibration range varied from 82.6% to 95.4%. The extraction recovery for [13C6]palovarotene was 90.4%. In addition, no carryover nor matrix effect was observed during the method validation. A 250-fold dilution factor (QC samples prepared at 250,000 pg/mL) was validated to quantify samples with concentration over the upper limit of quantification (2000 pg/mL).

Whole blood stability was demonstrated for palovarotene over 2 h in samples stored at room temperature. Benchtop stability in plasma was demonstrated over 9 h at room temperature. Frozen plasma samples (−20 °C and −70 °C) were stable for 427 days. Freeze–thaw stability was confirmed over four cycles at −20 °C and five cycles at −70 °C. No interference peaks were observed.

2.5 Trial Assessments and Endpoints

The pharmacokinetic parameters assessed in this trial were maximum plasma drug concentration (Cmax), time to reach Cmax (tmax), area under the plasma concentration–time curve (AUC) from time zero to the last timepoint (AUC(0–z)) and from time zero to infinity (AUC(0–∞)), terminal rate constant (λz), terminal elimination half-life (t1/2), apparent volume of distribution (Vd/F), and apparent total clearance from plasma (CL/F). Dose proportionality was assessed by comparing the mean body clearance (CL/F) across dose, although no formal statistical analysis was performed.

Baseline and safety measurements were conducted at screening and monitored throughout the trial. These included medical history, physical examination, vital signs (temperature, respiratory rate, systolic and diastolic blood pressure, and heart rate), 12-lead electrocardiogram (ECG) assessment, clinical laboratory parameters (including biochemistry, coagulation, lipid profile, and hematology), urine drug screens, and pregnancy testing. Prior medications were recorded at screening, and concomitant medications were assessed throughout the trial (full details provided in Supplementary Information 1).

Treatment-emergent adverse events (TEAEs), defined as those with onset after the first dose of palovarotene, were recorded throughout the trial, classified by system organ class and preferred term, and summarized using incidence rates. Severity of AEs was graded according to the following definitions: mild, events that were easily tolerated with no disruption of normal daily activity; moderate, events that caused sufficient discomfort to interfere with daily activity and/or require a simple dose medication; severe, events that incapacitated and prevented usual activity or required systemic drug therapy or other treatment. Serious AEs (SAEs) were defined as any AE occurring at any dose that resulted in any of the following outcomes: death, life-threatening situation, inpatient hospitalization or prolongation of existing hospitalization, persistent or significant disability/incapacity, congenital anomaly/birth defect, or any other medically important event. In addition, TEAEs known to be associated with retinoids (e.g., mucocutaneous events) were graded according to the Common Terminology Criteria for Adverse Events, Version 4.03.

2.6 Statistical Analysis

Pharmacokinetic parameters and endpoints were derived from plasma concentrations using Phoenix WinNonlin Version 6.4 (Certara USA, Inc., Princeton, NJ) and a noncompartmental model with oral drug input. All analyses were performed using the statistical software SAS Version 9.2 (SAS Institute, Cary, NC) or higher. Pharmacokinetic parameters were summarized using descriptive statistics [number of non-missing observations (n), mean/median, standard deviation, and minimum/maximum]. The natural log-transformed parameters were summarized, and estimates of the geometric mean difference between dose and Japanese and non-Japanese groups were calculated, along with the 90% confidence intervals (CI). The geometric mean differences and the confidence limits were transformed back to the original scale in order to give estimates of the individual ratios by dose and Japanese and non-Japanese groups with 90% CIs. The CIs for the ratio of the geometric means for Cmax, AUC(0–z), and AUC(0–∞) were compared with the constant value of 1. Further information on statistical analyses can be found in Supplementary Information 2.

3 Results

3.1 Trial Participants

Of the 77 individuals screened, 18 were enrolled, all of whom completed the trial. One participant did not meet eligibility criteria as their body weight was not within 10% of their matched Japanese counterpart but was enrolled in the trial following sponsor review and approval. Within the group of 18 enrolled and randomized participants, there were 8 pairs of matched non-Japanese and Japanese participants (6 female and 2 male), and 2 unmatched Japanese participants. The Japanese and non-Japanese groups were well matched with respect to mean age (39.1 and 40.6 years), sex (70.0% and 75.0% female), and BMI (21.6 and 21.5 kg/m2). Medical history was also similar between the groups. Demographic and baseline characteristics are summarized in Table 1.

Table 1 Baseline demographics and participant characteristics
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3.2 Palovarotene Pharmacokinetic Evaluations

The mean plasma concentration–time profiles were similar between Japanese and non-Japanese groups at both the 5 mg and 10 mg dose levels (Fig. 2). Mean plasma concentration levels for both cohorts and dose levels declined from a peak 4 h post-dose until 16 h post-dose (Fig. 2).

Fig. 2
figure 2

Concentration–time profile curves for palovarotene. Error bars represent standard deviation; N: number of participants

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Mean pharmacokinetic parameters were similar between Japanese and non-Japanese groups, at both the 5 mg and 10 mg dose levels (Table 2). Cmax and AUC values appeared dose-proportional between doses in both participant groups. Mean CL/F values ranged from 22.7 to 24.2 L/h across the 5 mg and 10 mg dose levels and across Japanese and non-Japanese participants, confirming dose proportionality and similar pharmacokinetics between Japanese and non-Japanese groups (Table 2). Mean t½ ranged from 9.7 to 13 h across both groups and doses (Table 2).

Table 2 Pharmacokinetic parameters by palovarotene dose and Japanese/non-Japanese group
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The geometric means for Cmax and AUC were slightly lower for the Japanese versus the non-Japanese participants at both dose levels. The coefficients of variation were larger for both AUC(0–t) and AUC(0–∞) parameters for the non-Japanese participants, indicating more variability relative to the mean. However, the coefficients of variation were similar for Cmax (Table 3).

Table 3 Pharmacokinetic results by palovarotene dose and Japanese/non-Japanese group
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The geometric mean ratio for AUC(0–∞) between the Japanese and non-Japanese groups was 0.892 (90% CI 0.667, 1.192) at the 5 mg dose level and 0.950 (90% CI 0.711, 1.270) at the 10 mg dose level. The geometric mean ratio for Cmax between the Japanese and non-Japanese groups was 0.962 (90% CI 0.723, 1.280) at the 5 mg dose level and 0.956 (90% CI 0.718, 1.272) at the 10 mg dose level.

3.3 Safety Evaluations

The overall adverse event (AE) profile of palovarotene is summarized in Table 4. There were no deaths, serious AEs (SAEs), or AEs leading to discontinuation of treatment. Six participants (four Japanese and two non-Japanese) reported a total of eight AEs, most of which were mild or moderate in severity (five mild, two moderate, one severe). The most frequently occurring AE was headache, which was reported by one Japanese participant receiving palovarotene 10 mg (who also experienced nausea) and one non-Japanese participant receiving palovarotene 5 mg (who also experienced cellulitis).

Table 4 Reported adverse events
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Four participants experienced five AEs during the trial that were assessed by the Investigator as possibly or probably related to the study treatment. These AEs included nausea, arthralgia, headache, and migraine in three Japanese participants, and an elevated lipase value of 155 U/L in one non-Japanese, male participant (higher than the upper limit of the normal range of 7–60 U/L). This AE was assessed by the Investigator as severe (grade 3) but was resolved without sequelae, and the participant completed the trial.

The majority of serum chemistry parameters were within the normal reference range at all timepoints in both the Japanese and non-Japanese groups. No differences from baseline in mean coagulation, lipid profile, hematology, or urinalysis changes were observed between the Japanese and non-Japanese groups. Overall, vital signs and mean ECG parameters remained within normal limits during the trial with no clinically relevant differences between the groups. All observed abnormal ECG findings were considered by the Investigator not to be clinically significant.

4 Discussion

In vitro studies have indicated that palovarotene is primarily metabolized by CYP3A4 and to a minor extent by CYP2C8 and CYP2C19, and so inter-ethnic differences in CYP-mediated metabolism could possibly affect palovarotene pharmacokinetics [20,21,22]. This phase I trial characterized the pharmacokinetic profile of palovarotene in healthy Japanese and non-Asian individuals.

When investigating the absorption and elimination of palovarotene, mean palovarotene plasma concentrations reached a peak at the same time (4 h post-dose) for both the Japanese and non-Japanese groups, at both dose levels investigated. Coupled with the finding that mean plasma concentration–time profile curves were alike between Japanese and non-Japanese individuals, these results indicate that palovarotene is absorbed and eliminated in a similar manner for the two groups. Additionally, the pharmacokinetic parameters of palovarotene were similar between Japanese and non-Japanese groups at both dose levels based on geometric mean ratios and CIs for Cmax, AUC(0–∞), and AUC(0–z). The CIs for the ratio of the geometric means for these parameters were compared with the constant value of 1. Given the expected variability in the responses, and that this trial was not statistically powered as an equivalence trial, comparison of the CIs with the conventional 80–125% reference boundaries was not considered appropriate. The 90% CIs for all pharmacokinetic parameters for both Japanese and non-Japanese cohorts at both dose levels crossed unity, meaning equivalence could not be ruled out for these pharmacokinetic parameters. Cmax and AUC values appeared to be dose-proportional between doses in Japanese and non-Japanese groups. Additionally, mean CL/F values ranged from 22.7 to 24.2 L/h across doses and Japanese and non-Japanese groups, confirming dose proportionality and similar pharmacokinetics between the groups. These results indicate that dose adjustment is not necessary for palovarotene for the treatment of FOP.

No new safety concerns were reported for the two single palovarotene dose regimens used in the trial. Three Japanese individuals experienced AEs considered by the Investigator as possibly or probably related to palovarotene. Despite this, no AEs led to discontinuation, and there were no deaths or SAEs, confirming that both palovarotene doses were well tolerated. Nevertheless, only a small group of 18 healthy participants were enrolled in this trial, at a single site in the USA, meaning that the generalizability of these results to patients with FOP should be carefully considered [25].

Furthermore, the general applicability of these pharmacokinetic results must be considered given that single dose regimens were used in this trial, whereas multiple dose regimens are intended for treatment of FOP. However, results from a palovarotene drug–drug interaction trial in which multiple dose regimens were used indicate no accumulation of palovarotene [26].

5 Conclusion

The results of this trial indicate that the pharmacokinetic profile of palovarotene is similar in Japanese and non-Japanese populations, suggesting that palovarotene dose adjustments are not required for Japanese patients receiving palovarotene as a treatment for FOP. In combination with ongoing efficacy and safety studies, these findings will therefore support palovarotene clinical trials in Japanese patients with FOP.