The investigational medicinal product (IMP) was 30 mg fasudil hydrochloride (ERIL®) and was imported from the manufacturer, Asahi Kasei Pharma Corporation, Tokyo, Japan, by the sponsor and provided to the study site. The ATC-code is C04AX32. For IV application, the IMP was diluted in 100 ml 5% glucose solution. It was applied via intravenous catheter at a constant rate of infusion using a CE-certified volumetric infusion pump over a period of 30 min in a reclined position (30°–45°). To allow a complete delivery IMP, the infusion system was flushed with 50 mL 5% glucose solution. For oral application, the IMP was diluted in 20 mL 5% glucose solution and applied with 250 mL of tap water.

This was a phase I, single-center, open-label, randomized, two period cross-over clinical trial in 14 healthy women and men aged 30–70 years (EudraCT-no. 2019-001805-26). Two treatments were investigated, separated by a wash out phase of at least 3 days. For treatment A (test), oral fasudil was dosed once on day 1 to establish the pharmacokinetic (PK) profile followed by three times at an interval of 8 ± 1 hours on day 2 of the respective period to assess safety and gastrointestinal tolerability. For treatment B (reference), IV fasudil was dosed on day 1 of the respective period (Fig. 1). Subjects were randomly assigned to one of the treatment sequences (A–B or B–A) according to a randomization list generated using the Statistical Analysis System (SAS, version 9.4, Cary, NC). A cross-over design is the preferred design for a clinical trial evaluating the absolute bioavailability, because it removes the inter-subject variability from the comparison between formulations/treatments. Each subject serves as their own control, meaning that the comparison between the formulations/treatments is based on intra-subject comparison. This explorative clinical trial was expected to generate PK data to establish the bioavailability of an oral administration for the further development program of an oral fasudil formulation. Relevant deviations in kidney and liver function were excluded so as not to interfere with the metabolism of IMP. A detailed description of inclusion and exclusion criteria can be found as supplementary material (Table S1). Fourteen healthy women and men were enrolled in this clinical trial. The subjects were recruited from the clinical trial center subject pool and by public advertisement. The approval of both, the ethics committee (no. PVN7203) and the German Federal Institute for Drugs and Medical Devices (BfArM) were obtained prior to the start of the clinical trial, and the study was performed in accordance with good clinical practice and the Declaration of Helsinki. The clinical trial was designed in line with the “Guideline on the Investigation of Bioequivalence” (CPMP/EWP/QWP/1401/98 Rev. 1 of 20 January 2010). The study was conducted from 25 February 2020 through 4 January 2021, at the CTC North study ward at the University Medical Center Hamburg-Eppendorf, Hamburg, Germany. All participants signed an informed consent form as evidence of consent. For reporting of this trial, the CONSORT guidelines were applied (Supplementary Table S2) [30].

Trial schedule (sequence A–B). Exemplary time and events of the SAFE-ROCK trial for a participant randomly allocated to sequence treatment A (oral) followed by treatment B (IV). ECG electrocardiogram, PK pharmacokinetic measurements

Screening procedures had to be completed between 28 days and 2 days prior to receiving the first dose of clinical trial medication. The following events were performed: acknowledging extended hygiene measures, filling out questionnaire for self-assessment of current state of health, measuring body temperature, SARS-CoV-2 polymerase chain reaction (PCR) test, inclusion/exclusion criteria check, obtaining medical history and demographics, physical examination, vital signs, height, and weight (BMI), ECG, clinical chemistry, hematology, serology, urine pregnancy test, urine drug screen, and an alcohol breath test. A complete list of screening procedures performed can be found in the supplementary material (Table S3). All laboratory assays were performed according to the laboratory’s normal procedures. Reference ranges were supplied by the laboratory and used to assess the laboratory data for clinical significance and out-of-range pathological changes. Out-of-range laboratory values were assessed for clinical significance by a physician. Abnormal laboratory values that were unexpected or not explained by the subject’s clinical condition may have been, at the discretion of the physician, principal investigator, or sponsor, repeated until confirmed, explained, or resolved as soon as possible. The following laboratory assessments were performed: blood samples (7.5 mL) for serum biochemistry were collected into a serum separator gel tube at the time points described in Table S3. The following parameters were assessed: aspartate transaminase (AST); creatinine clearance (GFR, using CKDEPI); alanine transaminase (ALT); alkaline phosphatase (AP); gamma glutamyl transferase (GGT); total bilirubin; creatinine; and lactate dehydrogenase (LDH). Blood samples (2.6 mL) for serum hematology were collected into tube a tube containing potassium ethylene diamine tetra-acetic-acid (EDTA) anticoagulant at the time points described. The following parameters were assessed: hemoglobin; hematocrit; red blood cells (RBC); mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); mean corpuscular hemoglobin concentration (MCHC); total and differential white blood cell (WBC) count (lymphocytes, neutrophils, eosinophils, basophils, monocytes); and platelet count. In all females of child-bearing potential regular pregnancy test (urine or serum) was performed as indicated in Table S3. During the screening period only, serum analyses for the presence of human immunodeficiency virus (HIV) antibody (HIV I and HIV II), HCV antibody, and HBsAg were performed. Drug and alcohol tests were performed as indicated or at the physician’s discretion. Blood samples of 9.8 mL (2 × 4.9 mL EDTA) for PK analysis were collected at the following time points (in hours after dosing): day 1: predose (within 30 min before dose administration), 0:15, 0:30, 00:45, 1:00, 1:30, 2:00, 3:00, 4:00, 5:00, 6:00, 8:00, 12:00, 16:00 h; day 2: 24:00 h. Within 90 min after sampling, samples were centrifuged at 1.100g (10 min, 4 °C) and stored in polypropylene tubes at −80 °C. During oral application, participants were fed 2, 6, and 12 h after dosing on day 1 and 2, 5.5, 10, and 12 h after dosing on day 2.

As primary endpoints of absolute bioavailability we investigated the Area under the concentration time curve from zero up to the last measured concentration (AUC0–tz), Area under the concentration time curve from zero extrapolated to infinity (AUC0-∞) and highest measured concentration determined in the measuring interval (Cmax) of fasudil and its active metabolite hydroxyfasudil. Blood levels and further PK characteristics of fasudil and hydroxyfasudil after the oral or the IV administration were analyzed as secondary objectives. Tolerability was assessed as proportion of subjects without significant drug intolerance during the treatment (oral or IV) period, and safety was assessed by the proportion of subjects without treatment-associated serious adverse event (SAE). Subjects were questioned in a general way to ascertain if AEs have occurred (e.g., “Have you had any health problems since the last time you came to the clinic/since you were last questioned?”). This open, standardized questioning was done discretely to prevent subjects from influencing each other. Spontaneous reports of AEs were also recorded as well as AEs that were observed by the investigator, the physician, or a staff member. All AEs were reviewed, confirmed, and classified by a qualified, designated physician. In additionally, the gastrointestinal symptom rating scale (GSRS) was applied, a validated disease-specific questionnaire used to evaluate common symptoms of gastrointestinal disorders [31]. It contains 15 items related to signs and symptoms experienced by the subject, each rated on a seven-point Likert scale from no discomfort (score = 1) to very severe discomfort (score = 7). In this way, the total score is comprised between 15 and 105.

For pharmacokinetic analyses, plasma samples were collected as described. Simultaneous quantification of fasudil and hydroxyfasudil was carried out in plasma matrix by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), following a previously published protocol. [32] For this, a QTRAP® 5500 LC-MS/MS System (Sciex, Framingham, MA) with a HPLC pump 1260 (Agilent Technologies, Santa Clara, CA) and a CTC Autosampler PAL HTCxt (Axel Semrau GmbH, Sprockhövel, Germany) was used. This method is based on protein precipitation as sample preparation followed by reverse phase chromatography coupled to tandem mass spectrometry (triple quadrupole). Chromatographic separation of matrix components and analytes was carried out with a C18-reversed-phase column using a solvent gradient. Electrospray ionization in positive mode (ESI / +) generates and detects selected ion fragments in the mass spectrometer. The quantification was carried out via a matrix replenishment with a standard solution. The calibration standards and quality control samples, containing low, medium, and high concentrations of fasudil or hydroxyfasudil, were prepared by spiking blank human K2-EDTA plasma using two different stock solutions. For each analytic run, remeasured quality control samples needed to be within ± 20% for the low quality control and ± 15% for medium and high quality control. Method validation comprised of the determination of the linear range, the limit of detection (LOD), and the precision. The linear range has been validated from 1–500 µg/L for both fasudil and hydroxyfasudil with a lower limit of detection (LLOD) of 1 µg/L. The intra-assay coefficient of variation at 50 µg/L (N = 10) was 3.6% for fasudil and 3.7% for hydroxyfasudil. The recovery rates from plasma were 118% for fasudil and 124% for hydroxyfasudil. Sample stability (human plasma) under different storage conditions [repeated freeze/thaw cycles, short- (24 h) and long-term (30 day) storage] for fasudil and hydroxyfasudil has been reportedly very good (> 94%). [32] The measurement was done in a single batch.

All statistical calculations were carried out using SAS language and procedures (SAS 9.4 version SAS-Institute, Cary NC) and R Version 4.1.0 (R Core Team, Vienna, Austria). WinNonlin version 8.1.0.3530 (Phoenix 64) was used for the derivation of the PK parameters. The individual subject values for concentrations and as well as secondary and subsidiary PK parameters are tabulated with descriptive statistics. Two-sample t-tests or paired Wilcoxon–Mann–Whitney tests were performed for comparison of metric variables between groups, whereas Fisher’s exact tests were performed for the analysis of categorical data. Absolute bioavailability of fasudil and hydroxyfasudil was estimated by the geometric mean ratios resulting from estimates of analysis of variance (ANOVA) model for each primary endpoint on the log-scale. Ratios of the geometric means (test/reference) as well as their two-sided 90% confidence intervals (CIs) were presented. For each endpoint, the difference between the means [log(test)-log(reference)] were derived from the ANOVA as least squares means (LS-MEANS), whereas the residual error and the quantiles from the t-distribution were used to calculate the two-sided 90% CIs. These values were back transformed to the original scale. Additionally, the two-sided 90% CIs were calculated based on the residual error from the ANOVA. This corresponds to the two one-sided t-test procedures, each at a significance level of 5%. Since the clinical trial is of exploratory nature, no formal hypothesis test and associated acceptance range are specified. In a post hoc analysis, terminal half-life (t½) and time to peak (tmax) were analyzed on the group level by unpaired t-tests.