Abstract
Aims
PTC518 is an orally administered, centrally and peripherally distributed huntingtin (HTT) pre‐mRNA splicing modifier being developed for the treatment of Huntington's disease (HD) for which there is a high unmet medical need as there are currently no approved disease‐modifying treatments. This first‐in‐human study investigated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of PTC518 in healthy volunteers.
Methods
This phase 1, single‐centre, randomized study in 77 healthy male and female volunteers evaluated the safety and tolerability and PK of PTC518 following single ascending doses and multiple ascending doses, PD as assessed by HTT mRNA and HTT protein levels after single and multiple doses, and food effects.
Results
PTC518 demonstrated a favourable safety profile. The majority of treatment‐emergent adverse events were mild and transient. PTC518 T max was reached at 6–7 h and the terminal T 1/2 was 54.0–75.3 h following a single oral dose. Exposure increased with dose though less than dose proportionally. The PTC518 concentrations in cerebrospinal fluid were approximately 2.6‐fold higher than the unbound free‐drug concentrations in plasma. A significant dose‐dependent reduction of up to approximately 60% in HTT mRNA and a significant dose‐dependent, time‐dependent and sustained reduction in HTT protein levels of up to 35% were observed after PTC518 treatment.
Conclusions
PTC518 was well tolerated, and proof of mechanism of this novel splicing modifier was demonstrated by the dose‐dependent decrease in systemic HTT mRNA and HTT protein levels. Results from this first‐in‐human study support further studies in patients with HD and demonstrate the potential for PTC518 as a breakthrough treatment for HD.
Keywords: genetic diseases, neuroscience, pharmacokinetics–pharmacodynamics, phase 1
What is already known about this subject
PTC518 is a novel, orally bioavailable, small‐molecule splicing modifier, which is being developed for the treatment of Huntington's disease.
PTC518 selectively lowers huntingtin (HTT) levels by promoting the inclusion of an inducible pseudoexon containing a premature translation termination codon in the HTT mRNA.
What this study adds
The pharmacokinetics of PTC518 in plasma were well characterized.
Proof of mechanism of PTC518‐mediated HTT pre‐mRNA splicing and HTT protein lowering were demonstrated in an exposure–response relationship.
PTC518 is well tolerated at single doses up to 135 mg and at 15 and 30 mg for 14 and 21 days.
1. INTRODUCTION
Huntington's disease (HD) is a progressive and fatal neurodegenerative disease. Patients diagnosed with HD gradually lose the ability to walk, speak, swallow or care for themselves, culminating in the need for full‐time care and, ultimately, death in late‐stage disease. 1 , 2 There are currently no disease‐modifying interventions approved for use in HD. The serious and ultimately fatal nature of the disease course, coupled with the lack of approved disease‐modifying therapies, represents a high level of unmet medical need.
This monogenic autosomal‐dominant disorder is characterized by expansion of cytosine‐adenine‐guanine (CAG) trinucleotide repeats in the huntingtin gene (HTT), which results in the production of mutant huntingtin (mHTT), a toxic gain‐of‐function protein that is ubiquitously expressed. 1 , 3 , 4 , 5 , 6 In animal models of HD, it has been shown that reducing mHTT levels alleviates motor and neuropathological abnormalities, supporting HTT lowering as a therapeutic approach. 7
PTC518 is a novel, orally bioavailable, small‐molecule splicing modifier that is being developed for the treatment of HD. This target‐selective small molecule was discovered and optimized using a drug discovery splicing platform developed to identify orally bioavailable compounds to treat diseases by decreasing gene expression. 8 This splicing platform has been successfully used to develop other target‐selective small molecules, such as risdiplam (Evrysdi™), the first ever orally bioavailable modifier of survival motor neuron 2 (SMN2) splicing for the treatment of spinal muscular atrophy (SMA). 9 , 10 , 11
PTC518 crosses the blood–brain barrier and has been designed to selectively lower HTT levels by modulating splicing of the HTT pre‐mRNA, resulting in the inclusion of a pseudoexon (from within an intron) with a premature translation termination codon. 8 This leads to the degradation of HTT mRNA and subsequent reduction in HTT protein levels, as depicted in Figure 1.
FIGURE 1.
HTT pre‐mRNA splicing and PTC518 mechanism of action. HTT, huntingtin; psiExon, pseudoexon; ss, splice site.
Based on this mechanism, PTC518 targets total huntingtin (tHTT) protein expression (i.e., wild type [WT] HTT and mHTT expression). WT HTT is a large and ubiquitously expressed protein with multiple functions, including important roles in synaptic function, transcriptional regulation, and vesicular transport and is important in neurodevelopment. 7 , 12 There is a body of evidence that indicates a positive risk–benefit profile for the partial reduction (30%–50%) of either mHTT alone or both mutant and WT HTT. 7
PTC518 has been evaluated extensively in preclinical toxicology species. The absolute oral bioavailability of PTC518 is moderate to high, ranging from 49%–88% across animal species. The plasma protein binding is 80%–88% in these animal species and in humans. Upon single or multiple oral doses in mice, rats and monkeys, PTC518 exhibited high exposures in the central nervous system (CNS) tissues. PTC518 has a large volume of distribution and total plasma clearances were moderate in rodents but high in dogs and monkeys. No unique or disproportional metabolites were observed in either human liver microsomes or in human hepatocytes, and PTC518 does not appear to inhibit any major cytochrome P450 (CYP) enzymes or transporters at the expected clinical exposures.
Given its proposed mechanism of action of reducing HTT protein levels and positive preclinical safety data accumulated to date, PTC518 represents a potentially promising therapeutic for the treatment of HD.
The objectives of this phase 1, first‐in‐human study were to evaluate the pharmacokinetics (PK) of PTC518 after single ascending doses (SAD) and multiple ascending doses (MAD), food effect (FE) on PTC518 PK, CNS exposure by measuring PTC518 in cerebrospinal fluid (CSF) following multiple doses, pharmacodynamics (PD) by assessment of HTT pre‐mRNA splicing and wild‐type HTT protein lowering, and safety and tolerability of orally administered PTC518 in healthy volunteers.
2. METHODS
2.1. Study participants and ethics
Healthy male and female volunteers aged 18–65 years were enrolled into the study after successfully completing medical screening.
This study was conducted in the Netherlands in compliance with the principles of the Declaration of Helsinki and with approval from the Central Committee on Research Involving Human Subjects (CCMO, the Hague, Netherlands) according to Dutch regulations based on the mechanism of action of PTC518. The clinical study protocol and subject information and informed consent form were approved by the CCMO. Before study entry, written informed consent was obtained from each subject. A list of randomized treatment assignments was generated by the study sponsor and allocated sequentially to subjects in the order in which they enrolled; both investigators and subjects were blinded to treatment allocation.
2.2. Study design
This was a four‐part, single‐centre, SAD, MAD, CSF and FE study. An overview of the treatment groups, dose levels and sampling timepoints is presented in Appendix 1 in the Supporting Information.
The SAD and MAD parts of the study were double‐blind, randomized, and placebo‐controlled and evaluated HTT pre‐mRNA splicing, safety, PK and tolerability under fasting conditions. The MAD part of the study also evaluated HTT protein lowering in the blood. The CSF part of the study evaluated the PK of PTC518 in plasma and CSF after administration of PTC518 for 7 days and lastly, the FE part of the study enabled a preliminary assessment of the effect of food on the PK of PTC518.
2.2.1. SAD
The SAD part of the study comprised five placebo‐controlled sequential cohorts of eight subjects assessing single PTC518 doses of 5, 15, 45, 90 and 135 mg in the fasted state. Two subjects in each cohort received placebo.
The first selected dose was <1/10th of the human equivalent dose estimated from the no observed adverse effect level (NOAEL) in male rats. As PTC518 is a first‐in‐class compound with the mechanism of HTT pre‐mRNA splicing, there is no established PK/PD relationship to reliably estimate the lowest pharmacologically active dose (PAD) or minimal anticipated biological effect level (MABEL) in humans. The conventional empirical approach based on preclinical toxicology and a 10‐fold safety factor was taken to determine the starting dose in this study. The dose selection for subsequent cohorts was based upon the PK and safety from previous cohorts. PD data were not included in dose escalation decisions in the SAD part of the study due to the delay in PD response following PTC518 treatment. The incremental increases in dose were determined by the relationship of mean exposure in the cohort to that of the NOAEL. No escalations were performed beyond the highest dose level that was associated with a mean exposure exceeding half of the area under the concentration vs. time curve (AUC) at the NOAEL. Following the first cohort, the AUCs for subsequent cohorts were estimated based on a preliminary population PK model developed based on pooled blinded data of all available PK data from completed cohorts.
2.2.2. MAD
The MAD part of the study comprised three placebo‐controlled sequential cohorts assessing 15 mg PTC518 once daily (QD) for 14 days (n = 7), 30 mg PTC518 QD for 14 days (n = 8), and a loading dose (LD) of 100 mg PTC518 on Days 1–2, followed by 30 mg PTC518 QD on Days 3–21 (n = 8). Two subjects in each cohort received placebo.
Dose levels in the MAD part of the study were selected based on available SAD data, including safety, PK and HTT mRNA. A preliminary PK/PD model was developed to analyse the exposure–response relationship. The initial dose and escalation scheme in the MAD part of the study was designed to achieve 30%–50% reduction from baseline in HTT protein levels at steady state, as projected from the PK/PD model.
2.2.3. CSF
The CSF part of the study comprised a single cohort assessing an LD of 90 mg PTC518 on Days 1–2, followed by 30 mg PTC518 QD on Days 3–7 (n = 3). All three subjects in this cohort received PTC518. The dose level was determined based upon a review of the safety, tolerability and PK data of the SAD and MAD parts of the study. Samples were taken for determination of PTC518 concentrations in CSF and plasma. No samples were collected for HTT mRNA and HTT protein assessments.
2.2.4. FE
The FE part of the study comprised two separate single‐dose open‐label cohorts assessing single PTC518 doses of 15 (n = 6) and 45 mg (n = 5) following a standardized high‐fat, high‐calorie meal. All 11 subjects received PTC518 at the designated dose. These cohorts enabled a preliminary assessment of the effect of food on the PK of PTC518 by comparison of PK in the 15 and 45 mg fed state cohorts with PK in the fasted state from the equivalent SAD dose cohorts.
2.3. Safety assessment
The overall safety profile of PTC518 in the study was characterized by type, frequency, severity, timing and relationship to study treatment of any adverse events, vital signs, laboratory abnormalities, physical examination abnormalities, neurological examination abnormalities, Columbia‐Suicide Severity Rating Scale scores or electrocardiogram (ECG) abnormalities.
2.4. Pharmacokinetics assessment
Blood samples for the determination of PTC518 concentrations in plasma were collected at specified timepoints via venipuncture or via intravenous (IV) catheter placed in a vein in the arm and CSF samples were serially collected by means of an indwelling CSF catheter (Appendix 1 in the Supporting Information).
Methods for quantitation of PTC518 in human plasma and CSF were validated in the range of 0.1–50 ng mL−1 using liquid chromatography–tandem mass spectrometry with a lower limit of quantitation (LLOQ) of 0.1 ng mL−1 (Appendix 2 in the Supporting Information).
2.5. Pharmacodynamics assessment
Blood samples for the determination of total HTT mRNA levels in the SAD and MAD parts of the study and tHTT protein levels in the MAD part of the study were collected at specified timepoints via venipuncture or via IV catheter placed in a vein in the arm (Appendix 1 in the Supporting Information).
A reverse transcription‐quantitative polymerase chain reaction (qPCR) assay was qualified to measure HTT (target gene) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH, reference gene) mRNA levels in blood samples (Appendix 2 in the Supporting Information). Control RNA was diluted from 50 ng μL−1 to 1.5625 ng μL−1 on every plate. The lowest dilution was considered the LLOQ of the assay.
Total HTT protein levels in blood samples were measured by electrochemiluminescence on the Meso Scale Discovery (MSD) platform according to a qualified method in the range of 1.10–2000 pM with an LLOQ of 1.10 pM (Appendix 2 in the Supporting Information).
2.6. Statistical analysis
Safety was reported descriptively, summarizing treatment‐emergent adverse events (TEAEs) by system organ class and preferred term. The measured individual plasma concentrations of PTC518 were used to estimate the PK parameters by the non‐compartmental analysis (NCA) method using Phoenix WinNonlin (version 8.1; Certara, Princeton, NJ, USA). Summary statistics (mean, median, standard deviation [SD], minimum, maximum) and changes from baseline were provided for HTT mRNA and HTT protein levels. All safety and statistical programming was conducted with SAS 9.4 (SAS Institute Inc., Cary, NC, USA). Food effects were assessed from the geometric least squares mean ratio (GMR) and 90% confidence interval (CI) calculated from natural log‐transformed primary PK parameters in each state (fed or fasted). A post hoc two‐sample t‐test was conducted to compare percentage of baseline HTT protein between cohorts.
2.7. Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY, and are permanently archived in the Concise Guide to PHARMACOLOGY 2023/24.
3. RESULTS
3.1. Subject demographics
The study was conducted in 77 healthy male and female volunteers with an age range of 18–63 years. Demographics by study part are summarized in Appendix 3 in the Supporting Information.
3.1.1. SAD
A total of 40 subjects (14 male, 26 female) were enrolled, of whom 37 completed the study. Thirty subjects (10 male, 20 female) received PTC518 and were 18–63 years old (mean age: 34.5 years).
3.1.2. MAD
A total of 23 subjects (11 male; 12 female) were enrolled and completed the study. Seventeen subjects (8 male, 9 female) received PTC518 and were 18–63 years old (mean age: 42.2 years).
3.1.3. CSF
A total of 3 subjects (2 male, 1 female) were enrolled and completed the study. All subjects received PTC518 and were 57–63 years old (mean age: 60.3 years).
3.1.4. FE
A total of 11 subjects (4 male, 7 female) were enrolled and completed the study. All subjects received PTC518 and were 27–63 years old (mean age: 42.8 years).
3.2. PTC518 safety and tolerability
The majority of TEAEs reported in the study were mild and had resolved by the end of the study. Headache was the most commonly occurring individual TEAE, experienced by 6 (37.5%) subjects who received placebo and 15 (24.6%) subjects who received PTC518, followed by dry skin in 7 subjects (placebo: 2 [12.5%] subjects; PTC518: 5 [8.2%] subjects), fatigue in 7 subjects (placebo: 3 [18.8%] subjects; PTC518: 4 [6.6%] subjects), and catheter‐site‐related reaction in 6 subjects (placebo: 1 [6.3%] subject; PTC518: 5 [8.2%] subjects). There were no deaths or TEAEs leading to discontinuation of PTC518. One treatment‐emergent serious adverse event (TESAE) of psychogenic tremors that was considered moderate and unrelated to the study drug was reported in the MAD part of the study; of note, the TESAE occurred 7 days after the last dose of study drug. The TESAE had resolved at the conclusion of the study. No treatment‐ or dose‐related effects on clinical laboratory parameters, vital signs or ECG results were observed in the study, and there was no effect of food on the safety of subjects.
3.3. PTC518 pharmacokinetics
3.3.1. Single ascending doses
PTC518 showed delayed absorption after oral administration, where time to maximum observed plasma concentration (T max) was 4–12 h (Table 1 and Figure 2), with a median of 6 h, and PTC518 exhibited a long apparent elimination half‐life (T 1/2) of 54.0–75.3 h across cohorts. The apparent oral clearance (CL/F) of PTC518 ranged from 81.5–135 L h−1 across cohorts.
TABLE 1.
PK parameters of PTC518 in plasma following a single oral dose of 5, 15, 45, 90 or 135 mg PTC518 under fasted conditions in healthy subjects.
| Parameters | PTC518 | PTC518 | PTC518 | PTC518 | PTC518 |
|---|---|---|---|---|---|
| 5 mg | 15 mg | 45 mg | 90 mg | 135 mg | |
| Mean (SD) a | Mean (SD) a | Mean (SD) a | Mean (SD) a | Mean (SD) a | |
| (n = 6) | (n = 6) | (n = 6) | (n = 6) | (n = 6) | |
| C max (ng mL−1) | 1.43 (0.404) | 3.75 (0.791) | 8.50 (1.34) | 16.4 (3.56) | 17.2 (3.00) |
| T max (h) | 6.00 (4.02, 8.00) | 6.00 (6.00, 8.00) | 6.00 (4.00, 8.00) | 6.00 (6.00, 6.02) | 6.00 (6.00, 12.00) |
| AUC0–24 (h ng mL−1) | 22.1 (6.02) | 52.4 (6.97) | 141 (22.6) | 259 (53.5) | 281 (35.1) |
| AUC0‐inf (h ng mL−1) | 71.1 (28.9) | 178 (12.7) | 524 (142) | 863 (393) | 1020 (193) |
| T 1/2 (h) | 54.0 (16.2) | 58.7 (13.7) | 62.4 (2.83) | 75.3 (9.30) | 62.0 (8.75) |
| CL/F (L h−1) | 81.5 (35.7) | 84.5 (6.33) | 90.1 (23.5) | 116 (53.0) | 135 (25.5) |
Abbreviations: AUC0–24, area under the concentration–time curve from time 0 to 24 h; AUC0‐inf, area under the concentration–time curve from time 0 to infinity; AUC0‐t , area under the concentration–time curve from time 0 to time t, where t is the time of the last measured (or measurable) concentration; CL/F, apparent oral clearance; C max, maximum observed plasma concentration; PK, pharmacokinetic; SD, standard deviation; T 1/2, apparent elimination half‐life; T max, time to maximum observed plasma concentration.
Data presented are arithmetic mean (SD) for all parameters except for T max, which are median (minimum, maximum).
FIGURE 2.
Mean plasma PTC518 concentration–time profiles following a single oral dose of 5, 15, 45, 90 and 135 mg PTC518 under fasted conditions in healthy subjects (semi‐log scale).
Using a power model, PTC518 area under the plasma concentration curve from time 0 to last (AUC0‐last) increased dose proportionally in the clinically relevant dose range of 5–45 mg following a single dose of PTC518 (slope β = 0.91, 90% CI: 0.79, 1.04). PTC518 maximum observed plasma concentration (C max) increased less than dose proportionally (slope β = 0.82, 90% CI: 0.71, 0.94).
Although sample sizes were small, no apparent sex difference was observed across all doses except for the 5‐mg dose cohort in which C max and AUC were almost double in females (n = 5) compared to males (n = 1).
3.3.2. Multiple ascending doses
PTC518 showed delayed absorption after multiple oral doses, where T max was 4–12 h, with a median of 6–7 h at Days 1, 14 and 21 (Table 2). The T 1/2 was 336–481 h after the last dose on Days 14 and 21. The average concentration on Day 14 after 15 and 30 mg daily dosing was 5.1 and 9.7 ng mL−1, respectively, and on Day 21 was 11.3 ng mL−1. The accumulation ratio (AR) was <2 based on C max and 2.2–2.5 based on AUC when Day 14 was compared with Day 1. PTC518 CL/F was 115–130 L h−1 across cohorts. The observed terminal T 1/2 was 336–481 h and the effective half‐life (T 1/2eff) was 33.3 h, based on the dosing interval (τ) and PTC518 accumulation over time following multiple dose administration and the AR of PTC518 (area under the concentration–time curve within the dosing interval [AUC0‐tau] at Day 14/AUC0‐tau at Day 1) using the equation: T 1/2eff = τ * ln2/ln[AR/(AR − 1)]. With a T 1/2eff of 33 h, steady state is expected to be attained around Day 7.
TABLE 2.
PK parameters of PTC518 in plasma following multiple daily oral doses of 15 or 30 mg PTC518 under fasted conditions in healthy subjects.
| Parameters | PTC518 15 mg (14‐day dosing) | PTC518 30 mg (14‐day dosing) | PTC518 30 mg (21‐day dosing) (100 mg LD x 2 days and 30 mg QD for 19 days) | |||
|---|---|---|---|---|---|---|
| Mean (SD) a | Mean (SD) a | Mean (SD) a | ||||
| (n = 5) | (n = 6) | (n = 6) | ||||
| Day 1 | Day 14 | Day 1 | Day 14 | Day 1 | Day 21 | |
| C max (ng mL−1) | 3.52 (1.32) | 6.29 (1.23) | 7.59 (2.12) | 11.7 (1.44) | 16.2 (4.18) | 14.2 (3.88) |
| T max (h) | 6.00 (4.05, 8.02) | 6.02 (6.00, 6.07) | 6.00 (6.00, 12.02) | 6.00 (6.00, 8.02) | 7.00 (6.00, 12.00) | 6.00 (0.5, 8.02) |
| C min (ng mL−1) | NA | 3.90 (0.565) | NA | 7.78 (1.10) | NA | 9.04 (2.26) |
| C avg (ng mL−1) | NA | 5.13 (0.686) | NA | 9.68 (0.978) | NA | 11.3 (2.57) |
| AUC0‐tau (h ng mL−1) | 51.2 (16.7) | 123 (16.5) | 112 (24.9) | 232 (23.5) | 269 (58.9) | 272 (61.6) |
| T 1/2 (h) | NA | 336 (33.4) | NA | 481 (228) | NA | 380 (131) |
| CL/F (L h−1) | NA | 124 (19.4) | NA | 130 (13.8) | NA | 115 (25.2) |
| ARcmax (ratio) | NA | 1.89 (0.370) | NA | 1.68 (0.658) | NA | NA |
| ARauc (ratio) | NA | 2.54 (0.537) | NA | 2.20 (0.745) | NA | NA |
ARauc, accumulation ratio based on AUC0‐tau; ARcmax, accumulation ratio based on C max; AUC0‐tau, area under the concentration–time curve within the dosing interval, calculated by linear up/log down trapezoidal method; C avg, average concentration over a dosing interval; CL/F, apparent oral clearance; C max, maximum observed plasma concentration; C min, minimum concentration over a dosing interval; LD, loading dose; NA, not applicable; PK, pharmacokinetic; QD, once daily; SD, standard deviation; T 1/2, apparent elimination half‐life; T max, time to maximum observed plasma concentration.
Data presented are arithmetic mean (SD) for all parameters except for T max, which are median (minimum, maximum).
3.3.3. Cerebrospinal fluid
Quantifiable concentrations of PTC518 were present in the CSF on Day 7 following an LD of 90 mg PTC518 on Days 1–2, followed by 30 mg PTC518 QD on Days 3–7 (Figure 3). The mean (±SD) ratio of PTC518 concentrations in CSF to unbound free‐drug concentrations in plasma was 2.60 (±0.411). The mean (±SD) maximum concentration (C max) and area under the concentration–time curve from time 0.5–12 h (AUC0.5–12) of PTC518 in CSF vs. plasma (free‐drug concentration) were 4.69 (±1.13) vs. 2.08 (±0.244) ng mL−1 and 53.8 (±15.2) vs. 21.3 (±2.66) h ng mL−1, respectively.
FIGURE 3.
Mean (±SD) plasma (free‐drug concentration) and CSF PTC518 concentration–time profiles on Day 7 following multiple daily oral doses of 30 mg PTC518 under fasted conditions in healthy subjects (linear scale). CSF, cerebrospinal fluid; LD, loading dose; QD, once daily; SD, standard deviation. Subjects received an LD of 90 mg PTC518 on Days 1 and 2, followed by 30 mg PTC518 QD on Days 3 to 7 (cohort 3.1: n = 3). Note: plasma free‐drug concentrations were derived from the measured plasma total concentration using the percentage of unbound drug concentration in plasma from in vitro assays (18.5%).
3.3.4. Food effect
Comparison of PTC518 PK from two parallel cohorts (fasted and fed) at 15 and 45 mg PTC518 showed a 40%–55% increase of AUC when PTC518 was administered with a high‐fat, high‐calorie meal (Table 3).
TABLE 3.
PK parameters of PTC518 in plasma following a single Oral dose of 15 or 45 mg PTC518 under fasted and fed conditions in healthy subjects.
| Dose | PK Parameter a | Fasted condition b | Fed condition c | Fed condition (test)/fasted condition (reference) | |||
|---|---|---|---|---|---|---|---|
| n | GM | n | GM | GMR (%) | 90% CI | ||
| 15 mg | C max (ng mL−1) | 6 | 3.68 | 6 | 4.43 | 120.42 | (96.37–150.47) |
| AUC0‐t (h ng mL−1) | 6 | 157 | 6 | 221 | 141.11 | (116.08–171.54) | |
| AUC0‐inf (h ng mL−1) | 4 d | 178 | 3 e | 274 | 154.06 | (100.31‐236.61) | |
| 45 mg | C max (ng mL−1) | 6 | 8.41 | 5 | 15.9 | 188.74 | (156.36–227.81) |
| AUC0‐t (h ng mL−1) | 6 | 447 | 5 | 672 | 150.34 | (122.24–184.90) | |
| AUC0‐inf (h ng mL−1) | 3 e | 512 | 4 f | 780 | 152.44 | (109.74‐211.78) | |
Note: GMR and 90% CI: Reported as percentage.
Abbreviations: ANOVA, analysis of variance; AUC0‐inf, area under the concentration–time curve from time 0 to infinity; AUC0‐t , area under the concentration–time curve from time 0 to time t, where t is the time of the last measured (or measurable) concentration; CI, confidence interval; C max, maximum observed plasma concentration; GM, geometric least squares mean; GMR, geometric least squares mean ratio; PK, pharmacokinetics; R 2, square of the correlation coefficient; SAD, single ascending dose.
Backtransformed least squares mean and confidence interval from ANOVA model performed on log‐transformed values.
PTC518 PK data in the fasted state were taken from the 15 mg and 45 mg dose cohorts in the SAD part of the study (Cohorts 1.2 and 1.3 in which six subjects received PTC518 in each).
Two dose levels (15 mg and 45 mg) of PTC518 were administered 30 min after the start of a high‐fat, high‐calorie breakfast in two cohorts (Cohorts 4.1 and 4.2) of six subjects and five subjects, respectively.
n = 4, the AUC0‐inf values obtained from two subjects were not reportable and not included in the analysis due to AUC0‐t /AUC0‐inf ratio <0.80 or adjusted R 2 value <0.80.
n = 3, the AUC0‐inf values obtained from three subjects were not reportable and not included in the analysis due to AUC0‐t /AUC0‐inf ratio <0.80 or adjusted R 2 value <0.80.
n = 4, the AUC0‐inf value obtained from one subject was not reportable and not included in the analysis due to AUC0‐t /AUC0‐inf ratio <0.80 or adjusted R 2 value <0.80.
3.4. PTC518 pharmacodynamics
3.4.1. HTT mRNA
PTC518 showed a dose‐dependent effect on HTT pre‐mRNA splicing in healthy adult subjects after a single dose. The time to maximum effect was reached at 24 h postdose. The highest tested dose of 135 mg led to a 50% decrease from baseline (predose level prior to the first dose) in full‐length HTT mRNA levels (Figure 4A).
FIGURE 4.
Dose‐dependent decrease in full‐length HTT mRNA at 24 h following single ascending doses (panel A) and following multiple ascending doses for 14 days (panel B) of PTC518. HTT, huntingtin. Figures show the mean (± standard deviation) percent of baseline (predose level prior to the first dose) full‐length HTT mRNA at the time of maximum effect, 24 h postdose. The horizontal dotted lines show reductions from baseline of 30% and 50%. Single ascending doses (panel A): placebo (n = 10 [all five cohorts combined]), cohort 1.1 (5 mg: n = 6), cohort 1.2 (15 mg: n = 6), cohort 1.3 (45 mg: n = 6), cohort 1.4 (90 mg: n = 5), cohort 1.5 (135 mg: n = 6). Multiple ascending doses (panel B): placebo (n = 4 [cohorts 2.1 and 2.2 combined]), 15 mg for 14 days (cohort 2.1: n = 5), and 30 mg for 14 days (cohort 2.2: n = 6).
PTC518 showed a dose‐dependent effect on HTT splicing in healthy adult subjects after QD dosing of 15 mg PTC518 over 14 days, 30 mg PTC518 over 14 days, and 30 mg PTC518 over 21 days. After 14 days of dosing, the full‐length HTT mRNA levels in the blood decreased by a mean of approximately 40% from baseline in the 15 mg group and approximately 60% from baseline in the 30 mg group (Figure 4B).
3.4.2. HTT protein
PTC518 showed a dose‐dependent and time‐dependent effect on HTT protein levels in healthy adult subjects after multiple doses (Appendix 4 in the Supporting Information). While blood levels of tHTT were reduced in subjects who received 30 mg PTC518 for 14 days compared to those who received placebo, a significant treatment effect was observed following 21 days of dosing with 30 mg PTC518 (100 mg LD on Days 1–2 followed by 30 mg QD for 19 days). Maximum reduction was observed after the last dose (mean [SD] percent of baseline tHTT was 65% [10.6%] in subjects who received PTC518 vs. 97% [0.9%] in subjects who received placebo; P = .0068; Figure 5). The reduction in HTT protein was delayed relative to the mRNA, reflecting a longer half‐life of the protein compared to the mRNA. Given the long half‐life of HTT protein, steady state maximal reduction of the concentration of HTT in whole blood was not reached during the 21‐day treatment period.
FIGURE 5.
Decrease in total HTT protein in subjects (30 mg group) receiving PTC518 once daily for 21 days. HTT, huntingtin. Figure shows the mean (± standard deviation) percent of baseline (predose level prior to the first dose) HTT protein. The horizontal dotted lines show reductions from baseline of 30% and 50%. * P = .0068 calculated using two sample t‐test. Figure shows placebo (cohort 2.3: n = 2) and PTC518 30 mg for 21 days with 100 mg loading dose for 2 days (cohort 2.3: n = 6).
4. DISCUSSION
Single oral doses of PTC518 up to 135 mg and multiple oral doses up to 30 mg for 21 days were generally well tolerated in healthy subjects. TEAEs and laboratory data, as well as all other safety parameters were comparable across the treatment groups and placebo groups. Furthermore, food had no effect on the safety and tolerability of a single oral dose of PTC518. Exposure of PTC518 increased with dose and quantifiable concentrations of PTC518 were present in the CSF, supportive that PTC518 penetrates the blood–brain barrier in humans. Following 21 days of dosing with PTC518 (100‐mg LD on Days 1–2 followed by 30 mg QD for 19 days), reductions of approximately 60% in HTT mRNA and up to 35% in HTT protein levels were observed. The maximal reduction of HTT protein was not attained after 21 days of PTC518 treatment, indicating a potentially higher than 35% reduction at steady state.
These data support PTC518 as a potential first disease‐modifying treatment for HD, a devastating disorder with no approved disease‐modifying treatment. Although the objectives were met in this first‐in‐human study, further investigation of PTC518 in patients with HD is needed to assess the reduction in HTT in the disease setting. Several preclinical studies support the hypothesis that targeting the expression of HTT may prevent and/or slow disease progression. 7 , 13 , 14 , 15 Although the potential consequences of persistent reductions in wild‐type HTT levels are notable theoretical risks, available data suggest that targeting mutant HTT is likely to be essential for any treatment of the underlying mechanisms associated with HD and that a partial reduction (30%–50%) is associated with a meaningful benefit on disease phenotype and trajectory. 7
Currently, treatments approved for HD only alleviate symptoms. CSF or CNS delivery of antisense oligonucleotides (ASOs) or virus vectors expressing RNA‐induced silencing (RNAi) moieties designed to induce mHTT mRNA lowering have progressed to clinical trials. 6 , 16 , 17 , 18
Since the whole brain is affected in HD, there are challenges for these HTT‐lowering modalities, which do not cross the blood–brain barrier, are unevenly distributed and require invasive lumbar puncture or brain surgery for delivery to the spinal fluid or brain. 17 Small molecules with desirable PK properties are preferred modalities for development and have many advantages, including their defined structure, relative ease of manufacture, oral administration and the ability to cross the blood–brain barrier.
PTC518 modulates splicing of the HTT pre‐mRNA, resulting in the inclusion of a pseudo exon (from within an intron) with a premature translation termination codon, leading to HTT mRNA degradation and reduction in HTT protein levels. In the transgenic bacterial artificial chromosome HD (BACHD) mouse model that expresses a full‐length mutant human HTT gene, PTC518 demonstrated dose‐dependent human HTT (hHTT) protein lowering in brain, muscle, whole blood and white blood cells isolated from whole blood (unpublished data on file, PTC Therapeutics). In this first‐in‐human study, the reduction in HTT mRNA and HTT protein after multiple dose administration of PTC518 demonstrates the proof of mechanism of this oral splicing modifier and supports PTC518 as a potential first disease‐modifying treatment for HD.
The PK of PTC518 were well characterized and were consistent with projections based on preclinical data. PTC518 was absorbed slowly in humans after oral administration, with a median T max of 6–7 h after single and multiple doses and exhibited a long terminal T 1/2. Increased PTC518 exposures (C max and AUC) were observed with increased doses.
The terminal T 1/2 was significantly longer following multiple doses (range 336–481 h) compared to the single dose (range 54.0–75.3 h). This could be attributed to changes in absorption extent and the reduced clearance of PTC518 after repeated doses as confirmed by decreased slopes when overlaying dose‐normalized concentration–time profiles following single and multiple doses. In addition, the difference in sampling window could potentially exaggerate such a difference further (up to 168 h postdose following a single dose and up to 336 h following the last dose in the MAD study). While the pilot assessment of FE using two parallel cohorts showed a minimal food effect, a future study using a crossover design will enable a definitive assessment of the effect of food on PTC518 PK.
Measuring drug concentrations in the CSF is often used as a surrogate for brain penetration. 19 , 20 , 21 Quantifiable concentrations of PTC518 were present in the CSF on Day 7 after 30 mg QD (with 90 mg LD on Days 1–2), supporting that PTC518 penetrates the blood–brain barrier, as observed in preclinical models (mice and monkeys). The PTC518 concentrations in CSF were approximately 2.6‐fold higher than the unbound free‐drug concentrations in plasma.
In addition to significant correlation between HTT levels in the brain and CSF, correlation between HTT levels in the CSF and systemic circulation has been observed preclinically with similar molecules. 8 In this study, data analysis showed a significant reduction in both HTT mRNA and HTT protein levels after PTC518 treatment across all cohorts in both SAD and MAD parts of the study. A dose‐dependent decrease in full‐length HTT mRNA levels of up to 60% was observed in the blood of PTC518‐treated subjects. Following 21 days of dosing with 30 mg PTC518 (100 mg LD on Days 1–2 followed by 30 mg QD for 19 days), significant reductions of approximately 35% (P = .0068) in HTT protein levels were observed compared to placebo (approximately 3%), in line with data suggesting that a partial (30%–50%) reduction is associated with a positive risk–benefit profile. 7 Given the long half‐life of HTT protein, steady‐state maximal reduction of the concentration of HTT in whole blood was not reached during the 21‐day treatment period but is expected to be reached by the end of 3‐month dosing.
Reduction in HTT protein was delayed relative to the mRNA, reflecting a longer half‐life of protein compared to mRNA. This is in agreement with preclinical data, which showed a strong correlation between levels of HTT pre‐mRNA splicing and the degree of protein lowering in an HD mouse model (unpublished data on file, PTC Therapeutics). It is anticipated that the observed HTT mRNA changes in blood will result in similar decreases in HTT protein levels in HD patients when a steady‐state decrease in HTT is attained over time with continued PTC518 treatment.
In conclusion, this study demonstrated the ability of PTC518, an orally bioavailable, small‐molecule splicing modifier to predictably and reversibly lower HTT mRNA and protein levels in healthy human subjects. The effect of PTC518 was dose‐dependent, and the drug was well tolerated. The results of this first‐in‐human study demonstrate the potential for PTC518 as a breakthrough treatment for HD and support further studies in patients with HD.
AUTHOR CONTRIBUTIONS
Lan Gao, Anuradha Bhattacharyya, Brian Beers, Diksha Kaushik, Allan Kristensen, Lee Golden and Ronald Kong contributed to the design of the study. Khalid Abd‐Elaziz, Lan Gao, Anuradha Bhattacharyya, Brian Beers, Diksha Kaushik, Lee Golden and Ronald Kong contributed to conduct of the study. Diksha Kaushik and Anuradha Bhattacharyya acquired the PK and PD data. Lan Gao, Anuradha Bhattacharyya, Brian Beers, Amy‐Lee Bredlau, Allan Kristensen, Lee Golden and Ronald Kong analysed and interpreted the data. Richard Grant, Ronald Kong, Lan Gao and Anuradha Bhattacharyya contributed to the manuscript draft. All authors contributed to revision, editing and approval of the manuscript.
CONFLICT OF INTEREST STATEMENT
Lan Gao, Anuradha Bhattacharyya, Brian Beers, Diksha Kaushik, Amy‐Lee Bredlau, Allan Kristensen, Richard Grant, Lee Golden and Ronald Kong are employees and stock owners of PTC Therapeutics.
Khalid Abd‐Elaziz is an employee of the University Medical Center Groningen, University of Groningen, Groningen, Netherlands.
Supporting information
Appendix 1. Study design.
Appendix 2. Bioanalytical methods.
Appendix 3. Summary of subject demographics.
Appendix 4. Mean percent of baseline HTT protein levels after multiple doses of PTC518 or placebo (measured at 24 h after last dose).
Gao L, Bhattacharyya A, Beers B, et al. Pharmacokinetics and pharmacodynamics of PTC518, an oral huntingtin lowering splicing modifier: A first‐in‐human study. Br J Clin Pharmacol. 2024;90(12):3242‐3251. doi: 10.1111/bcp.16202
The authors confirm that the Principal Investigator for this paper is Khalid Abd‐Elaziz, MD, and that he had direct clinical responsibility for the subjects enrolled.
Funding information The work was funded by PTC Therapeutics.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
REFERENCES
- 1. Caron NS, Wright GEB, Hayden MR. Huntington disease. In: Adam MP, Ardinger HH, Pagon RA, et al., eds. GeneReviews® [internet]. University of Washington, Seattle; 1998. Oct 23 [updated 2020 Jun 11]:1993‐2022. [Google Scholar]
- 2. Ross CA, Aylward EH, Wild EJ, et al. Huntington disease: natural history, biomarkers and prospects for therapeutics. Nat Rev Neurol. 2014;10(4):204‐216. doi: 10.1038/nrneurol.2014.24 [DOI] [PubMed] [Google Scholar]
- 3. Gatto EM, Rojas NG, Persi G, Etcheverry JL, Cesarini ME, Perandones C. Huntington disease: advances in the understanding of its mechanisms. Clin Park Relat Disord. 2020;3:100056. doi: 10.1016/j.prdoa.2020.100056 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Jimenez‐Sanchez M, Licitra F, Underwood BR, Rubinsztein DC. Huntington's disease: mechanisms of pathogenesis and therapeutic strategies. Cold Spring Harb Perspect Med. 2017;7(7):a024240. doi: 10.1101/cshperspect.a024240 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Li S, Li XJ. Multiple pathways contribute to the pathogenesis of Huntington disease. Mol Neurodegener. 2006;1(1):19. doi: 10.1186/1750-1326-1-19 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Tabrizi SJ, Flower MD, Ross CA, Wild EJ. Huntington disease: new insights into molecular pathogenesis and therapeutic opportunities. Nat Rev Neurol. 2020;16(10):529‐546. doi: 10.1038/s41582-020-0389-4 [DOI] [PubMed] [Google Scholar]
- 7. Leavitt BR, Kordasiewicz HB, Schobel SA. Huntingtin‐lowering therapies for Huntington disease: a review of the evidence of potential benefits and risks. JAMA Neurol. 2020;77(6):764‐772. doi: 10.1001/jamaneurol.2020.0299 [DOI] [PubMed] [Google Scholar]
- 8. Bhattacharyya A, Trotta CR, Narasimhan J, et al. Small molecule splicing modifiers with systemic HTT‐lowering activity. Nat Commun. 2021;12(1):7299. doi: 10.1038/s41467-021-27157-z [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Ratni H, Ebeling M, Baird J, et al. Discovery of risdiplam, a selective survival of motor neuron‐2 (SMN2) gene splicing modifier for the treatment of spinal muscular atrophy (SMA). J Med Chem. 2018;61(15):6501‐6517. doi: 10.1021/acs.jmedchem.8b00741 [DOI] [PubMed] [Google Scholar]
- 10. Dhillon S. Risdiplam: first approval. Drugs. 2020;80(17):1853‐1858. doi: 10.1007/s40265-020-01410-z [DOI] [PubMed] [Google Scholar]
- 11. Paik J. Risdiplam: a review in spinal muscular atrophy. CNS Drugs. 2022;36(4):401‐410. doi: 10.1007/s40263-022-00910-8 [DOI] [PubMed] [Google Scholar]
- 12. Cattaneo E, Zuccato C, Tartari M. Normal huntingtin function: an alternative approach to Huntington's disease. Nat Rev Neurosci. 2005;6(12):919‐930. doi: 10.1038/nrn1806 [DOI] [PubMed] [Google Scholar]
- 13. Garriga‐Canut M, Agustin‐Pavon C, Herrmann F, et al. Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice. Proc Natl Acad Sci U S A. 2012;109(45):E3136‐E3145. doi: 10.1073/pnas.1206506109 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Kordasiewicz HB, Stanek LM, Wancewicz EV, et al. Sustained therapeutic reversal of Huntington's disease by transient repression of huntingtin synthesis. Neuron. 2012;74(6):1031‐1044. doi: 10.1016/j.neuron.2012.05.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Stanek LM, Sardi SP, Mastis B, et al. Silencing mutant huntingtin by adeno‐associated virus‐mediated RNA interference ameliorates disease manifestations in the YAC128 mouse model of Huntington's disease. Hum Gene Ther. 2014;25(5):461‐474. doi: 10.1089/hum.2013.200 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Tabrizi SJ, Leavitt BR, Landwehrmeyer GB, et al. Targeting huntingtin expression in patients with Huntington's disease. N Engl J Med. 2019;380(24):2307‐2316. doi: 10.1056/NEJMoa1900907 [DOI] [PubMed] [Google Scholar]
- 17. Estevez‐Fraga C, Flower MD, Tabrizi SJ. Therapeutic strategies for Huntington's disease. Curr Opin Neurol. 2020;33(4):508‐518. doi: 10.1097/WCO.0000000000000835 [DOI] [PubMed] [Google Scholar]
- 18. Wild EJ, Tabrizi SJ. Therapies targeting DNA and RNA in Huntington's disease. Lancet Neurol. 2017;16(10):837‐847. doi: 10.1016/S1474-4422(17)30280-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Liu X, Smith BJ, Chen C, et al. Evaluation of cerebrospinal fluid concentration and plasma free concentration as a surrogate measurement for brain free concentration. Drug Metab Dispos. 2006;34(9):1443‐1447. doi: 10.1124/dmd.105.008201 [DOI] [PubMed] [Google Scholar]
- 20. Caruso A, Alvarez‐Sanchez R, Hillebrecht A, et al. PK/PD assessment in CNS drug discovery: prediction of CSF concentration in rodents for P‐glycoprotein substrates and application to in vivo potency estimation. Biochem Pharmacol. 2013;85(11):1684‐1699. doi: 10.1016/j.bcp.2013.02.021 [DOI] [PubMed] [Google Scholar]
- 21. Nagaya Y, Nozaki Y, Kobayashi K, et al. Utility of cerebrospinal fluid drug concentration as a surrogate for unbound brain concentration in nonhuman primates. Drug Metab Pharmacokinet. 2014;29(5):419‐426. doi: 10.2133/dmpk.DMPK-14-RG-026 [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Appendix 1. Study design.
Appendix 2. Bioanalytical methods.
Appendix 3. Summary of subject demographics.
Appendix 4. Mean percent of baseline HTT protein levels after multiple doses of PTC518 or placebo (measured at 24 h after last dose).
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.