This phase I, open-label single-dose study consisted of eight subjects with mild HI paired with eight healthy control individuals, and eight subjects with moderate HI paired with eight healthy control individuals, for a total of 32 subjects. For each level of HI, eight subjects were enrolled with the expectation that at least seven subjects would complete the study. As per the US Food and Drug Administration Guidance for Industry: Pharmacokinetics in Patients with Impaired Hepatic Function, eight subjects in the moderate HI arm and the control group is a sufficient number of subjects to provide evaluable data [
16]. The sample sizes were not based on statistical considerations, but rather the number of subjects considered sufficient to achieve the study objectives.
The subjects with HI and the healthy control individuals were matched in a 1:1 ratio according to sex, age (± 10 years), and body mass index (± 15%). The Child–Pugh score, a method of assessing the prognosis of chronic liver disease based on serum total bilirubin, albumin, prothrombin time, presence of ascites, and hepatic encephalopathy, was used to assess the severity of HI [
17]. A Child–Pugh classification of A (5–6 points) was considered mild HI, and a Child–Pugh classification of B (7–9 points) was considered moderate HI. The healthy matched control individuals were recruited and enrolled after all HI subjects received the study drug.
2.1 Study Population
Subjects for the study included adults 18 to 65 years of age, with a body mass index of 18 to 40 kg/m2, inclusive. All subjects had to be in good health at screening, outside of HI, as determined by medical history, physical examination, vital signs, and clinical laboratory tests. Subjects with HI were required to have a documented history of chronic liver disease or a history of chronic hepatitis B or hepatitis C infection, a Child–Pugh A or B classification score, and normal or non-clinically significant findings at physical examination with the exception of findings consistent with HI. Female subjects were required to either be of non-childbearing potential or agree to use an acceptable means of contraception. Male subjects had to agree to use an acceptable means of contraception and not donate sperm for at least 6 weeks’ post-dose.
Exclusion criteria for all subjects included any history of dizziness or vertigo or inner ear problems, history of clinically significant hypotension, or history of any psychotic mental illness. Other exclusion criteria included concomitant use of any central nervous system depressant drug within 7 days before dosing or the use of medications known to affect the elimination of serum creatinine or inhibitors of renal tubular secretion within 60 days. Any subject who had a positive response on the Columbia Suicide Severity Rating Scale or an estimated glomerular filtration rate of < 70 mL/min was also excluded from the study. Additional exclusion criteria for the matched healthy subjects included any clinically relevant abnormality identified on physical examination, electrocardiogram, vital signs, or clinical laboratory tests at screening; a clinically unstable, uncontrolled medical condition; liver function tests above the upper limit of normal at screening and on day −2; and the use of prescription or over-the-counter medications, with the exception of acetaminophen, stool softeners, and topical hydrocortisone cream, within 14 days prior to receiving the study drug.
2.2 Study Design
The screening visit was performed within 21 days prior to study drug administration, and all subjects were admitted to the clinical unit on study day −2. All subjects received 15 mg of mirogabalin orally on day 1 with approximately 240 mL of water after an overnight fast of at least 10 h. This dose was considered to be safe in subjects with HI because mirogabalin (Tarlige; Daiichi Sankyo, Co., Ltd., Tokyo, Japan) is mainly cleared by renal elimination, is minimally metabolized, and doses of 15 mg daily or twice daily are currently being tested in phase III studies.
2.3 Study Assessments
2.3.1 Blood Sampling
Serial samples of 4 mL of venous blood for pharmacokinetic assessments of A200-700 and A204-4455 were collected pre-dose and at 0.5, 1, 1.5, 2, 3, 4, 6, 9, 12, 24, 36, 48, and 72 h post-dose. An additional 4-mL venous blood sample to assess the binding of A200-700 to plasma proteins was collected 1 h post-dose.
Blood samples were collected into pre-chilled, 4-mL K2EDTA vacutainer tubes to the specified collection volume. The tube was gently inverted eight or more times to thoroughly mix with the anticoagulant, and then was placed in an ice bath. The samples were centrifuged within 30 min of collection, for approximately 10 min at 1500×g, to obtain plasma and then stored in darkness at – 20 °C until shipment, on dry ice, to Celerion MDS (Lincoln, NE, USA).
2.3.2 Bioanalytical Assay
All plasma samples were assayed for A200-700 and A204-4455 concentrations using a validated liquid chromatography-tandem mass spectrometry method (Celerion). A200-0700 and its internal standard were extracted from a 0.050-mL plasma sample, after mixing with 0.2 mL of 0.1% (v/v) formic acid (Mallinckrodt, Center Valley, PA, USA), using a 96-well solid-phase extraction plate conditioned with 0.5 mL of methanol (EMD Millipore, Burlington, MA, USA) and 0.5 mL of ultrapure water. The plate was washed with 0.5 mL of water and 0.75 mL of 5% methanol in water and eluted with mobile phase solution, 0.2 mL of an 80:20 (v/v) mixture of acetonitrile (EMD Millipore) and 20 nM ammonium formate in water (pH 2.5) [Acros, Bridgewater, NJ, USA]. Chromatographic separation was performed using a Zorbax 300-SCX (Agilent Technologies; Santa Clara, CA, USA) column (50 × 3.0 mm, 5-µm particle size) and isocratic elution at a 1.0 mL/min flow rate. Detection was performed by a Sciex API 4000 (Sciex, Framingham, MA, USA) tandem mass spectrometer with a TurboIonSpray source in the positive ion mode and multiple-reaction monitoring for mirogabalin and the internal standard. The lower limit of quantitation was 1.00 ng/mL.
For A204-4455, plasma samples containing an analyte and internal standard were mixed with equal-volume ammonium acetate (250 mM) and extracted with n-Butyl chloride in a 96-well plate. Extracted samples were analyzed on an ACE C18 column (50-mm length, 3.0-mm internal diameter, 5-μm particle size; Advanced Chromatography Technologies, Aberdeen, Scotland, UK) at an ambient temperature, with a mobile phase of 40:60:0.1 (v/v) acetonitrile:water:formic acid and a flow rate of 1.0 mL/min. The lactam metabolite of mirogabalin and the internal standard were detected using an AB Sciex API 4000™ triple quadrupole mass spectrometer (Sciex). The calibration curve for the analyte (1/concentration2 weighted linear regression) ranged from 0.1 to 50 ng/mL. The intra- and inter-assay precision (coefficient of variation) values in validation were 1.9% to 17.3% and 3.4% to 14.2%, respectively; the intra- and inter-assay accuracy values were − 9.2% to 10.0%, and −4.7% to 1.3%, respectively. Dilution integrity was verified at a concentration of 200 ng/mL. The lower limit of quantitation was 1.00 ng/mL.
For the protein-binding analysis, equilibrium dialysis was performed to measure the amount of A200-0700 bound to a plasma protein. Free A200-0700 was dialyzed using a rapid equilibrium dialysis device (Pierce Biotechnology, Waltham, MA, USA) through a membrane until its concentration across the membrane was at an equilibrium between the plasma and buffer chambers. The time to equilibrium was approximately 12 to 20 h on an orbital shaker at 500 rpm at 37 °C. Following dialysis, the plasma chamber sample was diluted 1:1 with blank buffer (phosphate-buffered saline) and the buffer chamber sample was diluted 1:1 with control plasma (i.e., pooled blank plasma).
2.3.3 Pharmacokinetic Analysis
Pharmacokinetic parameters were estimated from the plasma concentrations using standard methods of non-compartmental analysis using WinNonlin software version 6.3 (Pharsight Inc., Mountain View, CA, USA). Estimated parameters for A200-700 and A204-4455 included maximum observed concentration (Cmax), Tmax, area under the concentration–time curve until the last quantifiable concentration (AUClast) as calculated by the logarithm-linear trapezoidal method, area under the concentration–time curve from time 0 to infinity (AUCinf), elimination half-life, total apparent clearance, and volume of distribution in the terminal phase.
Actual time was used for pharmacokinetic parameter estimation. If the first value below the level of quantitation was at the beginning, it was considered as a zero. If a below the level of quantitation value was flanked by two non-zero concentrations, it was treated as a missing value. All other cases of values that were below the level of quantitation were treated as zero.
2.3.4 Safety Assessment
The safety measures assessed include laboratory tests, physical examination, vital signs, 12-lead electrocardiogram, Columbia Suicide Severity Rating Scale, and evaluation of any suspected treatment-emergent adverse events (AEs). All AEs recorded were coded according to the Medical Dictionary for Regulatory Activities (Version 17.1) terminology. Increased suicidal behavior and ideation or increased hepatic transaminases were treated as AEs of special interest.
2.3.5 Statistical Analysis
The safety analysis set included all subjects who received one dose of the study medication. The pharmacokinetic analysis set included all subjects who received one dose of the study drug and had enough plasma concentrations over time to characterize pharmacokinetic parameters. The pharmacokinetic parameters were summarized by subject group, analyte, and nominal time point using descriptive statistics.
The geometric least-square means and geometric mean coefficient of variation were calculated for Cmax, AUClast, and AUCinf. For A200-700 Cmax, AUClast, and AUCinf, each HI group was compared with the corresponding matched control group using a mixed-effect analysis of variance with subject groups as fixed effects on the logarithm-transformed Cmax, AUClast, and AUCinf. For A204-4455, the Cmax, AUClast, and AUCinf were assessed by descriptive statistics for each HI group and matched control group. Although the study was not powered as a bioequivalence trial, the 90% confidence interval of the ratio of the log-transformed AUC and Cmax of each HI group to the matched control individuals had to fall completely within the range of 80% to 125% to be considered bioequivalent. Statistical significance of protein-binding differences between the mild and moderate HI groups was determined using a student’s t test.