FormalPara Key Points

Trofinetide is rapidly absorbed into the circulation whereupon it undergoes an initial rapid decline then a slower elimination phase.

Trofinetide undergoes minimal hepatic or intestinal metabolism, and excretion of trofinetide is primarily in the urine as parent compound.

1 Introduction

Trofinetide is the first drug to be approved for the treatment of Rett syndrome (RTT), a neurodevelopmental disorder caused by loss-of-function mutations in the MECP2 gene, which encodes methyl-CpG-binding protein 2 (MeCP2), resulting in ineffective or defective synaptic connections [1], neurological dysfunction, and lifelong disability [2, 3]. Trofinetide is a synthetic analog of glycine–proline–glutamate (GPE), a naturally occurring tripeptide in the brain that is enzymatically cleaved from insulin-like growth factor 1 [4, 5]. In the Mecp2-deficient mouse model of RTT, GPE partially reversed RTT-like symptoms, improved survival, and enhanced synaptic morphology and function [6].

Two Phase 2 studies have demonstrated a clinical benefit with trofinetide (70 and 200 mg/kg twice daily) in pediatric, adolescent, and adult females with RTT [7, 8]. In the Phase 3 LAVENDER study, weight-based dosing of trofinetide significantly improved caregiver- and clinician-rated efficacy measures over placebo in females with RTT aged 5–20 years [9]. The highest trofinetide dose, for participants who weighed >50 kg, was 12 g (60 mL) twice daily. Trofinetide showed linear pharmacokinetics across the dose range tested in pediatric RTT patients [7], which is consistent with population pharmacokinetic modeling in healthy adults following single and repeated ascending doses [10]. Hepatic metabolism was negligible, and renal excretion was the major route of trofinetide elimination. There was no accumulation upon multiple dosing, and no metabolic inhibition or induction was observed during treatment. A recent Phase 1 study in healthy subjects also indicated a negligible effect of food on the pharmacokinetics of trofinetide, excluded any potential diurnal variation in bioavailability, and demonstrated that approximately 70% of an oral dose of trofinetide is excreted unchanged in the urine [11].

An in vivo study in rats previously characterized the pharmacokinetics and metabolism of trofinetide (rat mass balance/ADME study number 616032; unpublished data). The current trial was designed to characterize the pharmacokinetic profile of trofinetide (absorption, metabolism, and excretion [AME]), mass balance of [14C]-trofinetide, and safety of trofinetide following administration of a single oral 12-g dose of trofinetide containing [14C]-trofinetide to healthy adult male subjects.

2 Participants and Methods

2.1 Trial Design

This was a Phase 1, open-label, single-dose trial in healthy male subjects that characterized the pharmacokinetics of trofinetide (AME), mass balance of [14C]-trofinetide, metabolic profile and metabolite identification, and safety profile of trofinetide following administration of an oral 12-g dose (the highest approved therapeutic dose of trofinetide).

The trial was performed at a specialist Phase 1 unit (Worldwide Clinical Trials in the USA) between August and November 2020. The maximum duration of participation for individual subjects was approximately 8 weeks. A trial schema is provided in Fig. 1.

Fig. 1
figure 1

Trial design. aDepending on the percentage of radioactivity in urine and fecal samples. bFor those subjects who completed their end-of-treatment procedures through Day 22, a follow-up clinic visit, and follow-up telephone call were completed. For those subjects who completed their end-of-treatment procedures on Day 23 or after, only the follow-up telephone call was needed

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Potential subjects were screened to assess eligibility within 28 days prior to trofinetide administration. Subjects were admitted to the clinical pharmacology unit on Day −2 for COVID-19 testing and for the collection of a fecal sample (if possible). On Day −1, baseline evaluations were performed, and urine and fecal samples were collected, as applicable. A 12-lead electrocardiogram (ECG) was completed in triplicate on Day 1 prior to dosing. Then, eligible subjects had blood, urine, and fecal samples collected, as applicable.

On the morning of Day 1, after an overnight fast of approximately 10 h, a single 12-g dose of trofinetide containing [14C]-trofinetide was administered as an oral solution with ≤250 mL of water. Subjects continued to fast for 4 h after dosing; water was restricted for 1 h after dosing, then it was allowed ad libitum. To avoid any potential retention of drug-associated radioactivity in the elimination organs, subjects received a high-fiber diet (at least 25 g/day) and adequate fluid intake (at least 2 L/day) on Day 1 through Day 8, or up to the last day of sampling. Prior to pharmacokinetic sampling at 2 h after dosing, a 12-lead ECG was completed in triplicate. Blood, urine, and fecal samples were collected at regular intervals on Day 1 through Day 8, and collection could have continued up to Day 28 if recovery of radioactivity in excreta did not meet the protocol-specified minimum at the end of Day 8. The actual collection period did not exceed 10 days. Radioactivity measurements were made on blood and plasma samples collected on Day 1 through Day 8 and on urine and fecal samples collected on Day 1 through Day 8, and up to Day 10, as applicable.

Subjects were required to stay in the clinical pharmacology unit through Day 8 and were discharged on Day 8 if they met the discharge criteria (radioactivity levels in two consecutive urine and fecal samples were ≤1% of the administered dose). For subjects who did not meet the discharge criteria on Day 8, urine and fecal samples were collected daily until the discharge criteria were met or up to Day 28, whichever occurred first. Actual discharge of the final subject was on Day 10. End-of-treatment procedures occurred once the discharge criteria were met.

There were two scenarios for follow-up visits to assess safety, depending on when the end-of-treatment procedures occurred (i.e., when discharge criteria were met or up to Day 28). Subjects who completed their end-of-treatment procedures on Day 8 through Day 10, had two follow-up visits to assess safety: a follow-up clinic visit was completed approximately 1 week after the end-of-treatment procedures, and a follow-up telephone call was completed approximately 30 days after treatment administration.

Subjects had the right to withdraw from the trial at any time and for any reason but may have been discontinued for the following reasons: adverse event, death, lost to follow-up, noncompliance with treatment, physician decision, protocol violation, termination of the study by the sponsor, or withdrawal of consent.

2.2 Participants

The trial enrolled male subjects aged ≥18 to ≤45 years who had a body mass index ≥18 to ≤30 kg/m2 and body weight >60 kg and <100 kg. In the opinion of the investigator, subjects were in good health (defined by the absence of evidence of any active or chronic disease), as determined based on screening medical history, physical examination, laboratory test profile, vital signs, ECG, and the experience of regular bowel movements.

Subjects were excluded from study participation if they had a history or presence of a predefined cardiac conduction abnormality upon ECG at screening or on Day −1; they had a positive COVID-19 test result at baseline or screening; they had a recent history of incomplete bladder emptying with voiding or of waking more than once at night to void; they had a usual habit of less than one bowel movement every 2 days or more than three bowel movements per day; they had been exposed to radiation for therapeutic or diagnostic reasons, they had been exposed to radiolabeled substances, they were an occupationally exposed worker within the past 12 months prior to dosing of [14C]-trofinetide, or they anticipated exposure to radiation or radioisotopes in the next 12 months.

The use of caffeine- and xanthine-containing products was not permitted during the confinement period. Subjects were also required to abstain from alcohol, grapefruit, or Seville orange containing foods (e.g., orange marmalade) or beverages from 48 h prior to Day −1 through to the end of the trial.

2.3 Materials

The sponsor supplied trofinetide oral solution (200 mg/mL) containing [14C]-trofinetide. The trofinetide oral solution was supplied as an aqueous, ready-to-use, strawberry-flavored liquid in 500-mL, round, high-density polyethylene bottles that had child-resistant closures. Each 12-g dose of trofinetide contained 76.05 μCi of [14C]-trofinetide. Reference and internal standards for trofinetide were supplied by Acadia Pharmaceuticals Inc. (San Diego, CA, USA).

The planned target radioactivity had been 100 μCi, but this was not administered due to the improper calibration of the instrument used to measure the volume of radioactive material. Thus, the actual amount of radioactivity administered to all subjects was 76.05 μCi. An adjustment of 76.05 μCi instead of 100 μCi was applied to the calculation of all pharmacokinetic parameters to account for the reduced radioactive trofinetide dose administered.

2.4 Dose Selection

In this trial, trofinetide was administered as a mixture of trofinetide and [14C]-trofinetide. The [14C]-trofinetide was radiolabeled on the methyl group of the 2-methyl-proline amino acid. Per U.S. Food and Drug Administration (FDA) regulations, the amount of radioactive material to be administered was the smallest radioactive dose with which it was practical to perform the study and fulfill its objectives.

The radiochemical dose selected for the current trial was informed by a prior nonclinical study in rats that investigated the distribution of [14C]-trofinetide–derived radioactivity to selected tissues (rat mass balance/ADME study number 616032; unpublished data). The 12-g dose of trofinetide reflects the approved dose for trofinetide, which was confirmed by modeling and simulation that showed the weight-based dosing regimen used in the LAVENDER study achieved the target exposure range [12].This informed the selection of a single 12-g dose of trofinetide (containing [14C]-trofinetide) in the current trial, as this dose was expected to achieve the target exposure required to study the AME of trofinetide and is the highest approved clinical dose.

2.5 Sample Collection

Blood samples (4 mL) for pharmacokinetic analysis of trofinetide and [14C]-trofinetide–derived radioactivity were taken before dosing (within 1 h) and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 18, 24, 36, 48, 72, 96, 120, 144, and 168 h after dosing. A 5-min window around each nominal timepoint up to 12 h, inclusive, was permitted for each pharmacokinetic measurement, and a 15-min window was permitted for each pharmacokinetic measurement at 18 h and beyond. Additional blood samples (8 mL) were collected at each timepoint for total radioactivity assessment, for determination of the blood-to-plasma ratio, and for metabolite profiling and identification.

Urine samples for pharmacokinetic analysis of trofinetide and [14C]-trofinetide–derived radioactivity were collected at the following timepoints: Day −1 or on the morning of Day 1 before dosing, pooled over 2-h intervals for the first 12 h after dosing on Day 1, pooled over 6-h intervals during the 12- to 48-h period after dosing, and pooled daily up to Day 8 during each subsequent 24-h period. At each specified time interval, samples were pooled and aliquoted from each urine specimen. Additional samples were taken as needed. A portion of the pooled urine sample was centrifuged at approximately 10,000g at 4 °C for 10 min. Duplicate aliquots were radio-assayed before and after centrifugation. The resultant data were used to determine the recovery of radioactivity from centrifugation. The supernatants were subjected to liquid chromatography radio-profiling.

Fecal samples for pharmacokinetic analysis of trofinetide and [14C]-trofinetide–derived radioactivity were collected as a single fecal sample prior to dosing (on Day −2, Day −1, or Day 1) and as voided, beginning after dosing and lasting through Day 8 (168 h after dosing). Following collection, fecal specimens were weighed and the weights recorded. Samples collected within the same 24-h period were combined and processed for measurement of total radioactivity.

Additionally, the study assessed total radioactivity in whole blood and plasma over time, blood-to-plasma ratio at each collection time, total radioactivity in urine and in feces over time, percentage of total radioactivity in urine and in feces to total recovery, and percentage of radioactive dose recovered in urine and feces. Metabolite profiling and identification of trofinetide-derived materials in circulation and excreta were also performed.

All human plasma, blood, and urine were received frozen at WuXi AppTec from Worldwide Clinical Trials, San Antonio, TX on October 14 and 21, 2020, and feces on October 27 and 28, 2020. Plasma, blood, urine, and feces samples were stored immediately in a freezer at approximately 70 °C, except during subsampling.

2.6 Bioanalysis and Pharmacokinetic Assessment

2.6.1 Bioanalysis of Total Radioactivity

Blood, plasma, feces, and urine were analyzed for total radioactivity by Worldwide Clinical Trials Early Phase Services/Bioanalytical Sciences, LLC (Austin, TX, USA) using either a Hidex Model 600SL (Hidex, Okegawa, Saitama, Japan) or a Beckman Coulter Model LS65000 (Beckman Coulter, Brea, CA, USA) liquid scintillation counter. Urine and plasma were directly analyzed by mixing the samples with scintillation cocktail and counting for up to 10 min or to 2-sigma statistical error. Blood and feces homogenates were aliquoted into combustion cones, allowed to dry overnight, and then oxidized using a Packard Model 307 Sample Oxidizer with capture of the CO2 in CarboSorb® E absorbent fluid and subsequent liquid scintillation counting. Quality control samples were prepared in each of the four biomatrices and analyzed in parallel with the study samples in order to monitor the accuracy of the assay.

2.6.2 Bioanalysis of Blood Samples

Lithium heparinized blood samples were analyzed for concentrations of trofinetide by Worldwide Clinical Trials using a validated liquid chromatographic-tandem mass spectrometric bioanalytical method in a sample volume of 50 µL. Sample processing was performed by solid phase extraction of trofinetide and [14C]-trofinetide, along with the internal standard [13C5,15N]-trofinetide, at alkaline pH using an Oasis® MAX plate (Waters Corporation, Milford, MA, USA). High-performance liquid chromatography (HPLC) was performed using a Restek™ Ultra PFPP column (100 × 3.0 mm, 5 µm) (Bellefonte, PA, USA) and analyzed using a Sciex API 4000 mass spectrometer with Turbolonspray ionization source (Sciex, Framingham, MA, USA) and Analyst™ (version 1.6.1) control software (AB Sciex, Concord, Ontario, Canada). The assay range was 0.100–100 µg/mL for trofinetide.

The precision (coefficient of variation [%CV]) and accuracy (relative error [RE%]/mean % different [Bias%]) of the HPLC method were acceptable for trofinetide (≤15% [≤20% at the lower limit of quantification]). Mean recoveries of trofinetide were 34.44–58.91%, and for its 13C5,15N-labeled internal standard, mean recoveries were 34.18–56.51%. The internal standard-normalized recoveries ranged from 99.12% to 101.37%. Validation and acceptance criteria were based on FDA guidance for industry on bioanalytical method validation [13].

2.6.3 Bioanalysis of Urine Samples

Urine samples were analyzed for concentrations of trofinetide by Worldwide Clinical Trials using a validated liquid chromatographic-tandem mass spectrometric bioanalytical method in a sample volume of 20 µL. Urine containing trofinetide and [14C]-trofinetide, along with the internal standard [13C5,15N]-trofinetide, was extracted using direct dilution. High performance liquid chromatography was performed using a Restek™ Ultra PFPP column (100 × 3.0 mm, 5 µm) and analyzed using a Sciex API 4000 mass spectrometer with Turbolonspray ionization source and Analyst™ (version 1.6.1) control software.

The assay range was 0.0500–50.0 mg/mL for trofinetide. The precision (%CV) and accuracy (RE%/Bias%) of the HPLC method were acceptable for trofinetide (≤15% [≤20% at the lower limit of quantification]). Mean recoveries of trofinetide were 76.87–84.39%, and for its 13C5,15N-labeled internal standard, mean recoveries were 79.14–88.95%. The internal standard-normalized recoveries ranged from 92.48% to 97.37%. Validation and acceptance criteria were based on FDA guidance for industry on bioanalytical method validation [13].

2.6.4 Metabolite Profiling and Identification

For metabolite profiling, an additional blood sample (8 mL) was collected at each timepoint, and urine and feces were collected as voided beginning after dosing and lasting through Day 8 (168 h after dosing) or until criteria for release from the clinic had been met. Levels of radioactivity in human blood, plasma, and urine were determined by direct liquid scintillation counting. Levels of radioactivity in fecal samples were determined by solubilization prior to liquid scintillation counting.

Plasma and blood samples were prepared for radio-profiling by two pooling methods:

  1. (1)

    Pooling across timepoints: Equal volumes of plasma or blood from each subject were pooled for each sampling timepoint through 12 h after dosing (i.e., the time after which radioactivity was no longer quantifiable). This pool was utilized for assessment of the time courses for the individual trofinetide-derived components.

  2. (2)

    Area under the concentration-time curve (AUC) pooling: An AUC pool was produced for each subject using the Hamilton [14] pooling method, in which the volume of the sample from each timepoint in the pool was proportional to the intervals between successive timepoints for that subject. This pool was utilized for assessment of the relative systemic exposures to the individual [14C]-trofinetide-derived components.

Portions of each plasma or blood pool were analyzed for total radioactivity by liquid scintillation counting as a baseline against which to measure extraction efficiency. Prior to radio-profiling, the pooled plasma samples were extracted with acetonitrile and run through solid phase extraction with elution using methanol. The extraction efficiencies of both steps for all plasma and blood samples were at least 90% and 93%, respectively.

Urine samples were prepared for radio-profiling by pooling an equal percentage of volume of individual urine samples across subjects and timepoints to create one pooled sample for analysis. Sampling times for pooling were selected to reach >95% of the cumulative percent of dose recovery in urine for each subject. For subjects 1–5, samples up to 36 h were pooled; for subject 6, samples up to 30 h were pooled; for subjects 7 and 8, samples up to 48 h were pooled. A portion of each pooled sample, which represented 95.5% of the dose in urine, was centrifuged to remove solids, and the supernatant was analyzed via HPLC with radiochemical detection.

Fecal samples from up to 192 h after dosing were prepared for radio-profiling by pooling an equal percentage of weight of individual fecal samples across subjects and timepoints to create one pooled sample for analysis. Sampling times for pooling were selected to cover >95% of the cumulative percent of dose recovery in feces for each subject. A portion of the pooled sample, which represented 97.5% of the dose recovered in feces, was extracted with acetonitrile, centrifuged, evaporated to dryness, and reconstituted for analysis via HPLC with radiochemical detection. Total recovery after processing was 94.6%.

Blood, plasma, urine, and feces extracts were initially analyzed using a Waters® 2695 Separations Module and Waters® 2489 UV/vis detector (Waters Corporation, Milford, MA, USA) on an Ace 3, C18 (150 × 4.6 mm, 3 µm) (Avantor, Allentown, PA, USA) column with fractions of column effluent collected every 15 s into Deepwell LumaPlate™-96 plates (PerkinElmer, Waltham, MA, USA). Radioactivity in each fraction was determined by Packard TopCount® NXT™ Microplate Scintillation and Luminescence Counter technology (PerkinElmer, Waltham, MA, USA).

Metabolite characterization and identification for all four biomatrices were conducted using the same HPLC system and column but with mass spectrometric analysis using an accurate mass Thermo Q Exactive™ Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with electrospray ionization in positive ion mode.

2.6.5 Pharmacokinetic Data Analysis

Concentration/amount-time data were analyzed using noncompartmental methods in Phoenix™ WinNonlin® version 8.1 (Certara, Princeton, NJ, USA) in conjunction with the internet-accessible implementation of Pharsight® Knowledgebase Server™ version 4.0.4 (Certara).

Pharmacokinetic parameters in blood for trofinetide and [14C]-trofinetide–derived radioactivity included maximum (peak) observed drug concentration (Cmax), time to maximum (peak) observed drug concentration (Tmax), time of last quantifiable drug concentration (Tlast), AUC from time 0 to Tlast (AUC0–t), AUC from time 0 to infinity (AUC0–∞), percentage of AUC extrapolated from Tlast to infinity (%AUCext) calculated as 100 × (AUC0–∞−AUC0–t)/AUC0-∞, apparent terminal elimination half-life (t½), terminal phase elimination rate constant (λz), last quantifiable drug concentration (Clast), apparent systemic clearance following non-intravenous (e.g., oral) administration (CL/F), and apparent volume of distribution following non-intravenous (e.g., oral) administration (Vz/F).

Pharmacokinetic parameters in urine for trofinetide and [14C]-trofinetide-derived radioactivity included maximum urinary excretion rate, renal clearance of drug (CLr), non-renal clearance of drug (CLnr), ratio of renal clearance to systemic clearance (CLratio) calculated as CLr/CL, amount of unchanged drug excreted into urine (Aeu), and percentage of dose excreted in urine as unchanged drug.

Mass balance was assessed by calculating the sum of total radioactivity collected in urine and feces relative to the amount of radioactive dose administered.

2.7 Safety Assessments

Safety and tolerability were evaluated by treatment-emergent adverse event (TEAE) review, physical examinations, clinical laboratory evaluations, vital sign measurements, and ECGs. Severity of TEAEs were graded by the investigator as mild (awareness of sign or symptom but easily tolerated, causing minimal discomfort, and not interfering with normal everyday activities), moderate (sufficiently discomforting to interfere with normal everyday activities) or severe (incapacitating and/or preventing normal everyday activities).

2.8 Statistical Analysis

The safety analysis set included all subjects who received at least one dose of trofinetide. The pharmacokinetic analysis set included all subjects who received at least one dose of trofinetide and provided sufficient blood and urine concentration data to calculate at least one pharmacokinetic parameter.

No formal statistical analyses were performed. Plasma, blood, urine and fecal concentrations and radioactivity-time curves were presented as mean and standard error of the mean.

Pharmacokinetic parameters were summarized by descriptive statistics. Assuming a log-normal distribution for the Cmax and AUC values, two-sided 95% confidence intervals were provided for the geometric means of Cmax, AUC0-t, and AUC0-∞.

The trial intended to enroll eight male subjects to ensure that six subjects completed the trial. The sample size was determined empirically based on clinical considerations.

3 Results

3.1 Demographic and Baseline Characteristics

Eight subjects were enrolled at one site, all of whom received a single 12-g dose of trofinetide containing [14C]-trofinetide. All eight subjects met the discharge criteria (i.e., consecutive urine/fecal samples had radioactivity levels ≤ 1% of administered dose) by Day 10 (216 h), completed the trial as planned, and were included in both the safety and pharmacokinetic analysis sets. Subject characteristics are presented in Table 1. All subjects were male, and most were White (75.0%). The mean age was 31.9 years. The mean body mass index was 26.73 kg/m2, and the median weight (minimum, maximum) was 78.25 kg (72.6 kg, 90.8 kg). One subject (12.5%) received a concomitant medication of ciprofloxacin for a TEAE of bilateral conjunctivitis on Day 5.

Table 1 Subject (N = 8) demographic and baseline characteristics; safety analysis set
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3.2 Pharmacokinetics (Absorption, Metabolism, Excretion)

Blood concentration-time profiles of trofinetide and [14C]-trofinetide–derived total radioactivity are displayed in Fig. 2, and pharmacokinetic parameters are presented in Table 2. The blood concentration-time profiles of trofinetide and [14C]-trofinetide-derived total radioactivity were almost superimposable up to approximately 12 h (the time of the last measurable concentration of radioactivity). The last measurable concentration of trofinetide was at 120 h. The Cmax values for trofinetide and total radioactivity were comparable, with trofinetide being approximately 10% higher. Similarly, the AUCs were comparable, with trofinetide being approximately 14% higher due to the low measurable blood concentrations in the terminal phase through 120 h. The t½ for total radioactivity was approximately 2.6 h and is representative of the early decline (t½ alpha); the t½ for trofinetide was approximately 20 h and is representative of the terminal elimination phase (t½ beta).

Fig. 2
figure 2

Mean (± SE) blood concentration-time profiles of trofinetide and [14C]-trofinetide total radioactivity after a single 12 g dose up to 168 h on a linear and b semilogarithmic scales and truncated to 24 h on c linear and d semilogarithmic scales; pharmacokinetic analysis set. Note: An adjustment factor of 76.05% was applied to account for the reduced radioactive trofinetide amount administered (76.05 μCi instead of the approximately 100 μCi planned). SE standard error of the mean

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Table 2 Summary of blood and urine pharmacokinetic parameters after a single 12-g dose of trofinetide containing [14C]-trofinetide; pharmacokinetic analysis set
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Urine excretion rates of trofinetide and [14C]-trofinetide-derived total radioactivity are displayed in Fig. 3, and pharmacokinetic parameters are presented in Table 2. The urine concentration-time profiles of trofinetide and total radioactivity were similar. The last measurable concentrations were at 96 and 144 h, respectively. The excretion rates of trofinetide and [14C]-trofinetide-derived radioactivity in urine peaked rapidly then declined over the first 12 h after dosing; the rate was significantly reduced thereafter until the end of sample collection. Pharmacokinetic parameters in urine for trofinetide and total radioactivity were similar. The excretion of trofinetide and total radioactivity in urine reached a maximum rate with a median midpoint time of 3.00 h after dosing. The amount of trofinetide excreted unchanged in urine was approximately 10 g (80.6%), which was comparable with the total percentage of non-radiolabeled and radiolabeled dose excreted unchanged (83.8%).

Fig. 3
figure 3

Mean (± SE) urine excretion rate of trofinetide and [14C]-trofinetide total radioactivity on a linear and b semilogarithmic scales; pharmacokinetic analysis set. Note: An adjustment factor of 76.05% was applied to account for the reduced radioactive trofinetide amount administered (76.05 μCi instead of the approximately 100 μCi planned). SE standard error of the mean

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3.3 Mass Balance

Mean blood and plasma total radioactivity concentration-time curves are presented in Fig. 4. Concentrations of [14C]-trofinetide-derived radioactivity in blood and plasma were qualitatively similar, with concentrations in plasma consistently higher at all timepoints. The blood-to-plasma total radioactivity ratios at all timepoints were consistent, ranging between 0.529 and 0.592.

Fig. 4
figure 4

Mean (± SE) [14C]-trofinetide concentrations (µg-eq/mL) in blood and plasma at each nominal timepoint on a linear and b semilogarithmic scales; pharmacokinetic analysis set (all available concentration–time data within collection windows were included in the plots). SE standard error of the mean

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Mean cumulative amounts of [14C]-trofinetide-derived radioactivity recovered in urine, feces, and total recovery (percentage of administered dose) were also determined and are presented in Fig. 5. Total radioactivity recovered in urine and feces collectively increased rapidly in the first 24 h, with approximately 80% of the radiochemical dose recovered, then gradually increased in small increments thereafter until the end of the trial, with 99% recovered overall.

Fig. 5
figure 5

Mean (± SE) cumulative amount of [14C]-trofinetide recovered in urine and feces, and total recovery (percentage of administered dose) on a linear scale; pharmacokinetic analysis set. Note: An adjustment factor of 76.05% was applied to account for the reduced radioactive trofinetide amount administered (76.05 μCi instead of the approximately 100 μCi planned). Urine and feces were collected before dosing and up to 216 h after oral administration of 12 g of trofinetide containing 76.05 µCi of [14C]-trofinetide. Mean total recovery of the radiochemical dose at the time of the last collection of excreta was 98.97%, with 83.84% in urine and 15.13% in feces. SE standard error of the mean

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The average total fraction of the radiochemical dose recovered in excreta was approximately 80% after 24 h, >85% after 48 h, >90% after 72 h, approximately 95% after 96 h, >95% after 120 h, >95% after 144 h, and approximately 99% after 168 h. The percentages of the radiochemical dose recovered in urine and feces were 83.8% and 15.1%, respectively.

3.4 Metabolite Profiling

The pharmacokinetic data indicated that the parent compound is the primary trofinetide-related component in human blood, plasma, urine, and feces. In the 0.5- to 12-h timepoint pools, parent [14C]-trofinetide represented 87.2–98.3% of total radioactivity in blood and 73.1–97.5% of total radioactivity in plasma. In the 0- to 12-h AUC pools of blood and plasma, parent [14C]-trofinetide accounted for an average of 88.4% and 93.1%, respectively, of total radioactivity. Metabolites formed by hydrolytic loss of the glutamic acid moiety either in the absence (M186) or presence (M168) of cyclization of the product dipeptide represented an average of 1.25% and 0.42%, respectively, of total radioactivity AUC in blood and 1.61% and 2.24%, respectively, of total radioactivity in plasma. In the 0- to 48-h pooled urine sample, 91.5% of total radioactivity was accounted for by parent [14C]-trofinetide, while M186 and M168 represented 1.79% and 1.46%, respectively. In the 0- to 192-h pooled fecal sample, 52.7% of total radioactivity was accounted for by parent [14C]-trofinetide, while M186 and M168 represented 16.3% and 4.01%, respectively. M129, a product of hydrolysis of both the glutamic acid and glycine amino acids, was found only in feces and represented 5.65% of fecal radioactivity. Mean radioactivity contributions of [14C]-trofinetide and metabolites after oral administration (0–48 h) in plasma, blood, urine, and feces are shown in Table 3. In summary, in blood and plasma, M168 and M186 accounted for ≤ 2.24% of the AUC0-12 pools, while M168 accounted for 1.17% and 0.59% and M186 accounted for 1.43% and 2.41% of the total dose in urine and feces, respectively. M129, which was not detected in plasma, blood, or urine, accounted for 0.84% of the total dose in feces.

Table 3 Mean radioactivity contributions of [14C]-trofinetide and metabolites in humans after oral administration (0 to 12 h [plasma and blood] or 0 to 48 h [urine and feces])
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3.5 Safety and Tolerability

A single 12-g dose of trofinetide containing [14C]-trofinetide was well tolerated in this study. During the trial, TEAEs were reported in two subjects: conjunctivitis (n = 1) and musculoskeletal chest pain (n = 1). Both TEAEs were mild in severity, resolved, and were not considered related to treatment. There were no deaths or serious TEAEs and no TEAEs leading to discontinuation from the trial. No subjects had QT-interval corrected using Fridericia’s formula (QTcF) ≥450 ms or a QTcF change from baseline >30 ms. There were no clinically meaningful changes in individual laboratory parameters, vital signs, physical findings, or ECG results.

4 Discussion

This trial assessed the AME of trofinetide and [14C]-trofinetide, the mass balance of [14C]-trofinetide, and metabolite profiling and identification in healthy male subjects. The results have been useful in guiding the clinical development of trofinetide.

Following a single 12-g oral dose of trofinetide containing [14C]-trofinetide in healthy male subjects, trofinetide and total radioactivity pharmacokinetic profiles were similar in both blood and urine. Trofinetide was the major entity circulating in blood and excreted in urine with only small numbers of metabolites present. Trofinetide had a Tmax of approximately 2–3 h and was rapidly absorbed into the circulation with an initial rapid decline (t½ alpha of approximately 2.6 h, which is considered to be the effective half-life), followed by a relatively slow elimination phase (t½ beta of approximately 20 h). The blood-to-plasma total radioactivity ratios were consistent, ranging from 0.529 to 0.592, indicative of a lack of affinity for the cellular portion of blood. Renal excretion was the major elimination route of trofinetide‐related radioactivity and accounted for 83.8% of the administered radiochemical dose. Only 15.1% of the administered radioactivity was recovered in feces, indicating that fecal excretion was a minor route of elimination for trofinetide and its metabolites. Combined, urine and fecal recovery of radioactivity accounted for 99% of the administered dose at 168 h after dosing. There was minimal metabolism of trofinetide with < 4% of metabolites (M168 and M186) identified in the AUC0-12 pool in plasma or blood. The same metabolites and an additional metabolite (M129) represented ≤ 6.5% of the radiochemical dose in both urine and fecal samples at 48 h after dosing. The minimal metabolism of trofinetide suggests there will be a low potential for drug-drug interactions. This was corroborated in a recent physiologically based pharmacokinetic modeling study in which single and multiple therapeutic doses of trofinetide had no impact on hepatic cytochrome P450 3A4 (CYP3A4) enzyme metabolism and a weak inhibitory effect on intestinal CYP3A4 metabolism [15]. The pharmacokinetic parameters in whole blood and urine are comparable with results from the Phase 1 food effect trial whereby trofinetide was administered under morning fasted, morning fed, and evening fasted conditions [11], and exposure parameters were comparable with those reported in a population pharmacokinetic modeling study based on the approved weight-based dosing regimen in the LAVENDER study [12].

Trofinetide containing [14C]–trofinetide was well tolerated with no deaths, no serious TEAEs, and no TEAEs leading to discontinuation from the trial. There were only two TEAEs during the trial, neither of which were treatment related, and both were mild in severity. In addition, there were no clinically meaningful changes in individual laboratory parameters, vital signs, physical findings, or ECG results.

4.1 Limitations

The inclusion of healthy adult subjects in Phase 1 human mass balance trials is typical [16,17,18]; nevertheless, the population studied in this analysis does not represent the target population: pediatric and adult patients with RTT, who may have gastrointestinal confounders and are typically of lower-than-average weight [19].

5 Conclusions

The pharmacokinetic profiles of trofinetide and total radioactivity in whole blood and urine indicate that trofinetide is rapidly absorbed into the circulation whereupon it undergoes an initial rapid decline then a slower elimination phase, consistent and in agreement with the pharmacokinetic parameters reported for trofinetide in previous studies. Metabolic and excretion profiles confirm that trofinetide undergoes minimal hepatic or intestinal metabolism and is primarily excreted in the urine. Trofinetide containing [14C]–trofinetide was well tolerated.