Participants were male healthy volunteers with overweight or obesity aged 18-55 years with a body mass index (BMI) ≥28 kg/m2 for participants which received subcutaneous AT biopsies (10-360 mg group), BMI≥25 kg/m2 for participants which did not receive AT biopsies (2.5-5 mg group). The main exclusion criteria were any evidence of a clinically relevant concomitant disease, and gastrointestinal, hepatic, renal, respiratory, cardiovascular, metabolic, immunological or hormonal disorders besides hyperlipidemia.
The study was undertaken according to the Declaration of Helsinki and Good Clinical Practice principles. The protocol was approved by local independent ethics committee (Ärztekammer Nordrhein, Düsseldorf, Germany). All participants provided written informed consent before participation. The clinical trial registry number is NCT01587417 (www.clinicaltrials.gov).
Study design and procedures
This was a randomized, double-blind, placebo-controlled, single rising dose study conducted at a single center in Germany. A total of 72 healthy male volunteers entered and 71 completed the study. Subjects were treated in 9 sequentially ascending dose groups of 2.5, 5, 10, 20, 40, 80, 160, 240, and 360 mg of BI 187004 or matching placebo. Within each dose group, 6 subjects were treated with a single dose of BI 187004, and 2 subjects received placebo (except for the 5 mg dose group, where only 1 subject received placebo). The subjects were admitted to the study center on day -2, 36 hours prior to the treatment and were discharged on day 2. Follow-up on an ambulatory basis was performed between 11 and 15 days after study drug administration. The active drug was provided as a powder for oral solution (PFOS). At the time of use the oral solution for dosing was prepared using the PFOS and a co-supplied aqueous solvent containing HP-beta-cyclodextrin 100 mg/mL. The lowest dose level solutions were prepared by further dilution of the lower concentration solution with additional solvent. Subcutaneous AT sample collection occurred at three different time points for each subject on day -1 (baseline), day 1 (10 h after single dose) and day 2 (24 h after single dose) via incision biopsy in the umbilical region.
Plasma samples for PK analyses were obtained from day 1 to day 2 (pre-dose, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 24, 36 hours), day 3 to day 5, day 11 to day 15 (in the morning). 24-hour urinary collection for PK and PD analysis was obtained from day -1 to day 4 in 4-hour to 12-hour intervals (relative to drug administration).
BI 187004 concentrations in plasma and urine samples were measured with validated bioanalytical methods using a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The assay method consists of a solid supported liquid-liquid extraction of human plasma, coupled with quantitative LC-MS/MS determination of the extracted samples and was validated for the concentration range of BI 187004 of 3 - 3,000 nmol/L. The BI 187004 plasma concentration data were generated from 11 analytical runs. For each accepted run, the acceptance criteria for the calibration standards and quality controls were met.
The BI 187004 concentrations in urinary samples were measured using a LC-MS/MS method which was validated for the concentration range of BI 187004 of 5 - 5,000 nmol/L. The BI 187004 urine concentration data were generated from 5 analytical runs. For each accepted run, the acceptance criteria for the calibration standards and quality controls were met.
The acceptance criteria for the plasma and urine assay were (i) a regression coefficient (R2) of ≥ 0.98; (ii) at least 75% of calibration standards with ≤ 15 %RE (relative error / accuracy) and (iii) each accepted curve contained at least 6 concentration levels.
Safety and tolerability of BI 187004 were assessed in a descriptive way using the following investigations: recording of adverse events, clinical laboratory parameters (haematology, coagulation, enzymes, substrates, electrolytes, hormones, and urinalysis), vital signs (blood pressure, puls rate), 12-lead ECG (electrocardiogram) with special attention to QTc prolongation, and physical examination (occurrence of findings).
Measurement of urinary corticosteroids
The urinary samples for determination of urinary free cortisol (UFF), urinary free cortisone (UFE), urinary total cortisol (UTF), urinary total cortisone (UTE), and their metabolites 5alpha-tetrahydrocortisol (5alpha-THF), 5beta-tetrahydrocortisol (5beta-THF), and tetrahydrocortisone (THE) were analyzed using a validated LC-MS/MS method.
Concentration measurement of BI 187004 in adipose tissue
The BI 187004 concentrations in human AT samples were measured using a protein-precipitation extraction of homogenized human AT, coupled with quantitative LC-MS/MS determination of the extracted samples. The assay was qualified for the BI 187004 concentration range of 2.5 ‑ 10,000 ng/g. The BI 187004 AT concentration data for study samples were generated from seven analytical runs. For each run, the acceptance criteria for the calibration standards and QC samples were met. Acceptance criteria for all sample analysis runs were ≤25% RSD (relative standard deviation / precision) and ±25% RE (relative error / accuracy) for calibration standards and quality control samples.
Ex vivo measurement of 11beta-HSD1 activity
Inhibition of 11beta-HSD1 in AT was measured by conversion of deuteriz ed (d2)-cortisone to d2-cortisol ex vivo in subcutaneous AT biopsies. Immediately after taking the biopsies with an open incision, tissue samples were cut into fragments, placed into 48-well tissue culture plates and incubated in assay buffer. A triplicate set of tissue fragments was incubated in media with d2‑cortisone and 1 μM test substance to establish the background level of d2-cortisol. After overnight incubation, about 80 μL of the supernatant were snap frozen and stored at ≤-60 °C. Then, d2-cortisone and d2-cortisol were measured using a validated LC-MS/MS method with the formation of d2-cortisol being an indirect indicator for enzyme activity.
11beta-HSD1 inhibition in AT was calculated as individual percent change from baseline to day 1 and day 2 of the absolute deuterized cortisol measurements and expressed in medians. Safety laboratory parameters including hormone measurements are given as mean ± standard deviation, PK parameters as geometric mean (gMean) ± geometric coefficient of variation (%gCV), except for tmax (time of maximum observed drug concentration), which is given as median ± range. Methods for the determination of PK parameters were described previously . Dose proportionality of BI 187004 was explored using a power model. The placebo corrected percentage change from baseline of the data shown in Supplementary Table 2 and 4 was analyzed statistically by an ANCOVA model with baseline as covariate on the logarithmic scale. Placebo correction was achieved by estimating mean differences between each dose of BI 187004 and placebo with the corresponding 90% confidence interval (CI). The results were back-transformed to the original scale resembling post dose/baseline ratios for each dose of BI 187004 model.