Chemicals and standards

Unlabeled valemetostat, [2H6]-valemetostat, and reference standards for the metabolites CALZ-1808a, CALZ-1809a, and CALZ-1810a were synthesized by Daiichi Sankyo Co., Ltd. [2H6]-CALZ-1809a was synthesized by HepatoChem Inc. [14C]-valemetostat was manufactured by EAG Laboratories. All other chemicals and reagents were obtained from commercial suppliers and were of analytical grade.

Study design

This single-center, open-label, single-dose, phase 1 study (DS3201-A-U105) evaluated the ADME profile, PK, safety, and tolerability of [14C]-valemetostat in healthy male participants. The study was performed at Covance Clinical Research Unit (Madison, WI), and was conducted in compliance with the protocol, the ethical principles that have their origin in the Declaration of Helsinki, the International Conference on Harmonisation, consolidated Guideline E6 for Good Clinical Practice, and applicable regulatory requirements. The protocol, amendment, letter of administrative change, and informed consent were approved by the Institutional Review Board (IRB).

Participants

Healthy male participants 18–55 years of age with a body mass index of ≥ 19.0 and < 30.0 kg/m2 were enrolled. The health status was confirmed through medical history, physical examination, 12-lead electrocardiogram, and clinical laboratory evaluations. Exclusion criteria included use of selected drugs of abuse or any medications; positivity for the hepatitis panel and human immunodeficiency virus; history or current evidence of malignancies, cardiac, hepatic, renal, pulmonary, endocrine, neurologic, infectious, gastrointestinal, hematologic, or oncologic disease; and exposure to radiations.

Dose preparation/schedule

Each participant received a single oral dose of 200 mg valemetostat containing approximately 150 µCi of 14C-radiolabeled valemetostat. The selected 200-mg dose was established as the recommended phase 2 dose in the first-in-human phase 1 clinical trial (DS3201-A-J101) [10]. At this dose, valemetostat was shown to be safe and well tolerated among healthy participants prior to the initiation of this study [13].

The radioactive dose of approximately 150 µCi was considered an acceptable dose for humans in 14C-labeled drug metabolism studies of this type based on quantitative whole-body autoradiography studies using male Sprague Dawley and Long-Evans (pigmented) rats [16]. The overall whole-body radiation dose in human male participants following the administration of a single 150 µCi (5.55 MBq) dose of [14C]-valemetostat was calculated to be 26.6 mrem (0.266 mSv), which is well below the US Food and Drug Administration exposure limit of 3000 mrem (30 mSv) for a single dose in human isotope studies [17].

The final dose formulation for oral administration was prepared at the study site by dissolving the powdered [14C]-valemetostat in sterile water to a concentration of approximately 4 mg/mL (3 µCi/mL). The dose contained approximately 5.55 MBq (0.266 mSv) of radioactivity, which is within the acceptable range as set by the International Commission on Radiological Protection guidelines for category IIa studies (0.1–1 mSv) [18].

Eligible participants were admitted 24 h prior to the drug administration and were kept for overnight fasting lasting at least 8 h. Participants remained seated for 4 h after dosing and were restricted from caffeinated beverages, grape-fruit juice, alcohol, and concomitant medications. Participants were discharged after sample collections on study day 8 if≥ 90% of the radioactive dose was recovered and if urine and fecal sample collections over three consecutive 24-hour days each contained < 1% of the administered radioactivity. If these radioactivity recovery criteria were not met by study day 8, blood, urine, and fecal samples were collected daily, and participants were discharged from the clinic once radioactivity recovery criteria were met. Participants were discharged by day 22 even if the radioactivity recovery criteria were not met, with return visits to the clinic every 48 or 72 h thereafter until the remaining radioactivity in plasma was undetectable, or up to study day 31, whichever occurred first (Supplementary Fig. 1).

Sample collection

Blood samples (8 mL) were collected for determination of total radioactivity and PK analyses of valemetostat and CALZ-1809a (a major oxidized metabolite of valemetostat) at pre-dose and at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 24, 36, 48, 72, 96, 120, 144, and 168 h post-dose. Approximately 1 mL from each blood collection was used for determination of whole blood radioactivity, and the remaining blood was used to separate plasma. Blood samples (10 mL) for metabolite identification were drawn at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, and 24 h post-dose. Urine was collected before dosing and at 0–12, 12–24, 24–48, 48–72, 72–96, 96–120, 120–144, and 144–168 h post-dose. One 10-mL aliquot was used for determination of radioactivity, and another two 10-mL aliquots were used for metabolite identification. Fecal samples were collected pre-dose, then at 0–24, 24–48, 48–72, 72–96, 96–120, 120–144, and 144–168 h post-dose. Feces samples of each participant were combined at 24-hour intervals, weighed, and homogenized with water. Duplicate weighed aliquots (approximately 0.2 g) were used for determination of radioactivity and duplicate 50 g subsamples were retained for metabolite identification. All aliquots and/or subsamples were stored at − 70 ℃ until used for further analysis. Additional collections were performed beyond 168 h if radioactivity recovery criteria for discharge had not been met.

Determination of total radioactivity

Blood, plasma, urine, and feces samples were mixed, and duplicate weighed aliquots (approximately 0.2 g) were analyzed for content of radioactivity by liquid scintillation counter (LSC), with blood and feces combusted prior to analysis.

Determination of valemetostat and CALZ-1809a in plasma

Plasma concentrations of valemetostat and CALZ-1809a were determined by ultra-high performance liquid chromatography (HPLC) equipped with an AB SCIEX triple quadrupole mass spectrometer, API 4000™ for detection of valemetostat, and API 5000™ for detection of CALZ-1809a. An aliquot of human plasma (K2EDTA) containing the analyte and internal standard was extracted using a protein precipitation procedure. Quantitation was done using a weighted linear regression analysis (1/concentration2) of peak area ratios of the analyte and internal standard. The lower limit of quantitation was 20 ng/mL and 1 ng/mL for valemetostat and CALZ-1809a, respectively.

PK assessments

The cumulative percentage of the dose excreted in the urine and feces was assessed based on the radioactivity measured in each sample. The PK parameters for total radioactivity in whole blood and plasma included estimation of Cmax, Tmax, AUC from time 0 to 24 h post-dose (AUC0 − 24 h), AUC from time 0 to the time of last measurable concentration (AUClast), AUCinf, and whole blood to plasma ratios for total radioactivity with respect to Cmax, AUClast, and AUCinf.

The PK parameters for valemetostat and CALZ-1809a in the plasma included, estimation of Cmax, Tmax; AUC0 − 24 h; AUClast; AUCinf; t1/2; apparent total body clearance and apparent volume of distribution based on the terminal phase (valemetostat only); plasma ratios of metabolite CALZ-1809a to valemetostat with respect to Cmax, AUClast, and AUCinf; and ratios of valemetostat to total radioactivity with respect to Cmax, AUClast, and AUCinf. The noncompartmental PK analyses for total radioactivity, valemetostat and its metabolites were performed using WinNonlin, version 8.1 (Certara, US, Inc.).

Quantification and characterization of metabolites

Biological samples were analyzed for identification, quantification, and structural elucidation of metabolites using HPLC + LSC and liquid chromatography–tandem mass spectrometry (LC–MS/MS) systems. As plasma samples around Cmax were missing for 1 participant (1001-0001) due to limited volumes obtained by venipuncture, two different samples were prepared for metabolite analysis described in Supplementary Table 1; an equal volume of plasma samples at various time points from participant 1001-0001 were pooled (Plasma 1), and plasma samples at Cmax from various participants (1001-0002 to 0008) were equally pooled (Plasma 2). For metabolite analysis in urine and feces, each individual samples were pooled at the time intervals shown in Supplementary Table 1. Exactly 2 mL of each pooled sample was then weighed, mixed with 3-fold volume of acetonitrile, shaken (10 min), centrifuged (1800 × g, 4℃, 10 min), and the supernatant was collected. The residue was extracted twice by suspending in water (the same volume of the sample), mixed with 3-fold volume of acetonitrile, shaken, and centrifuged in the same manner. All the three supernatants were combined and subjected to radiochromatographic analyses. The recovery of radioactivity (%) through sample processing, the recovery of radioactivity (%) during HPLC analysis of each sample, and the excretion (% of dose) in the urine and feces were calculated.

Radiochromatographic analyses of processed biological samples were performed using an HPLC system (Shimadzu; Kyoto, Japan). The HPLC column of 3 μm was passed with mobile phase A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) in a gradient manner at a flow rate of 1 mL/min.

The HPLC eluate of the biological samples were fractionated at 18 s intervals using a fraction collector (RETRIEVER IV; Teledyne Isco, Lincoln, NE). The radioactivity of each fraction was measured by LSC (2500 TR, 2700 TR, 3100 TR, or 3110 TR; PerkinElmer; Waltham, MA). Radiochromatograms of each sample were reported using Microsoft Office Excel (versions 2010 and 2016, Microsoft, Redmond, WA). Radioactive peaks corresponding to [14C]-valemetostat and its metabolites were identified and quantified from the radiochromatograms. Radioactivity concentrations were converted to valemetostat (ng eq. of valemetostat/mL). The lower limit of quantification was designated as 1.0%, values below this were considered not detectable. Metabolite profiling was performed using LTQ Orbitrap XL system (Thermo Fisher Scientific, Waltham, MA). The structure of each metabolite was identified or estimated using the data obtained from analysis of each sample. The LC–MS/MS system was equipped with an electrospray interface and was operated using the positive ion detection mode. Chemical structures of [14C]-valemetostat and its metabolites detected as radioactive peaks were identified by comparing the respective retention times, molecular weights, molecular formulae, and tandem mass spectrometry (MS/MS) spectra to those of the authentic standards. Ultraviolet and mass chromatograms of a mixed authentic standard solution were reported using Xcalibur v2.1.0 (Thermo Fisher Scientific; Waltham, MA). Chemical structures of metabolites that did not match with any authentic standard were elucidated from the MS (MS/MS) spectra. Representative MS (MS/MS) spectra of each metabolite were reported using Xcalibur.

Safety

Safety assessments were conducted by recording treatment-emergent adverse events (TEAEs), clinical laboratory assessments (clinical chemistry, hematology, coagulation parameters, and urinalysis), vital signs (pulse rate, blood pressure, oral body temperature, and respiratory rate), electrocardiograms, and physical examinations. TEAEs were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0.

Statistical assessments

The sample size of eight participants was not based on any statistical consideration but was considered sufficient to assess the recovery of [14C]-valemetostat. Data are summarized statistically and represented as mean ± SD.

In vitro protein binding assessment

Plasma, 4% human serum albumin (HSA), and 0.1% AAG were each fortified with valemetostat to reach a final concentration of 600, 1000, 3000, 5000, and 10,000 ng/mL. Solutions were ultracentrifuged (OptimaTM MAX, Beckman Coulter, Inc.) at 627,000 × g for 160 min at 4 ℃ and the middle supernatant layer was retained and analyzed by LC–MS/MS for valemetostat concentration. The binding percentages of valemetostat to the proteins were determined by using the equation:

$$\:Protein\:binding\:\left(\%\right)=\:\frac\times\:\:100$$

Where Cp represents the total concentration of valemetostat in plasma, 4% HSA and 0.1% AAG, and Cs represents the free concentration of valemetostat in the corresponding sample.

The unbound fraction of valemetostat is determined by the equation:

$$\:Unbound\:fraction\:=\:\frac$$

Each experiment was done in triplicate.

For total concentration measurement, samples were prepared for LC–MS/MS by mixing an aliquot of plasma, 4% HSA or 0.1% AAG; acetonitrile; and an internal standard (500 ng/mL [2H6]-valemetostat) and centrifuged at 13,000 × g for 2 minutes at 4 ℃. The supernatant was collected and mixed with 5 mmol/L ammonium formate containing 0.01% formic acid. For unbound concentration measurement, samples were prepared for LC–MS/MS by mixing an aliquot of plasma, 4% HSA or 0.1% AAG supernatant; internal standard (25 ng/mL [2H6]-valemetostat); 5 mmol/L ammonium formate containing 0.01% formic acid; and acetonitrile. Valemetostat was separated on an OSAKA SODA CO., LTD. C18 column (2.0 mm 2.0 mm × 50 mm internal diameter × 50 mm, particle size 3 μm) in a mobile phase of 5 mmol/L ammonium formate containing 0.01% formic acid (phase A) and acetonitrile (phase B). Samples were eluted with a gradient increasing of phase B/phase A from 80:20 to 5:95, then back to 80:20. The total analytical run time was 5 min, and the flow rate was 0.5 mL/min. Detection was performed by tandem mass spectrometer (API5000; AB SCIEX), which was operated in the positive ion detection mode. Multiple reaction monitoring was used for quantification.