Compound
The batches of MIV-711 (previously referred to as MV076159; [25]) used in nonclinical experiments were synthesized at Medivir (Huddinge, Sweden). In all nonclinical experiments in vivo, MIV-711 was suspended in 1% w/v methyl cellulose (Methocel 4AC premium, Sigma) and given as a suspension via oral gavage. The vehicle (1% w/v methyl cellulose) was administered to control animals. For the clinical study, MIV-711 was synthesized by NCK A/S (Farum, Denmark), and MIV-711 and placebo capsules were manufactured by Galenica AB (Malmö, Sweden).
Pharmacological characterization of MIV-711 in vitro
Enzyme assays
Recombinant cathepsin K enzymes from all species were expressed in E. coli, purified and activated. Human cathepsin F and cathepsin S were expressed in Baculovirus, purified and activated. Purified human cathepsins B and H (Athens Research Technology), cathepsin L (Calbiochem), and cathepsin V (R & D Systems) were purchased.
H-D-Ala_Leu-Lys-AMC was used as the substrate in assays of cathepsin K from non-rodent species (human, dog, rabbit and guinea pig), and Z-Leu-Arg-AMC was used as the substrate in assays of cathepsin K from rodent species (mouse and rat). For cathepsins S and V the substrate used was Boc-Val-Leu-Lys-AMC, for cathepsins F and L the substrate was H-D-Val-Leu-Lys-AMC, for cathepsin B the substrate was Z-Arg-Arg-AMC and for cathepsin H the substrate was H-Arg-AMC. All substrates were obtained from Bachem.
For cathepsin K the buffer was 100 mmol/L sodium phosphate, 5 mmol/L EDTA, 1 mmol/L DTT, 0.1% PEG 4000, pH 6.5. For cathepsin S the buffer was 100 mmol/L sodium phosphate, 100 mmol/L NaCl, 1 mmol/L DTT, 0.1% PEG 4000, pH 6.5. For cathepsin L the buffer was 100 mmol/L sodium acetate, 1 mmol/L EDTA, 1 mmol/L DTT, 0.1% PEG 4000, pH 5.5. For cathepsin B the buffer was 50 mmol/L sodium phosphate, 1 mmol/L EDTA, pH 6.25. For cathepsin F the buffer was 100 mmol/L sodium acetate, 1 mmol/L EDTA, 1 mmol/L DTT, pH 5.5. For cathepsin H the buffer was 100 mmol/L tris–acetate, 1% PEG4000, pH 7.5. For cathepsin V the buffer was 25 mmol/L sodium acetate, 2.5 mmol/L EDTA, pH 5.5.
Assays were carried out in white polystyrene 96-well plates in a final volume of 100 µL. Substrate concentrations were 10–100 µmol/L and enzyme concentrations were 0.1–5 nmol/L. Compounds were added in DMSO in the range of 1 nmol/L–100 µmol/L at a final DMSO concentration of 1%. Plates were read in a Fluoroskan Ascent (Thermo Labsystems, Helsinki, Finland) in kinetic mode, with excitation and emission filters of 390 and 460 nm, respectively. Rates were determined by linear regression of the fluorescence/time data in Excel. Rates were fitted by non-linear regression to either the competitive inhibition equation, with the substrate concentration fixed at the value in the assay and the KM fixed to the value previously determined, or the IC50 equation using GraphPad Prism (GraphPad, version 6, San Diego, USA) to obtain Ki or IC50 values, respectively.
The kinetics of MIV-711 inhibition of human recombinant cathepsin K were measured by progress curve analysis [26]. Briefly, 100 µmol/L substrate and 0.5 nmol/L cathepsin K were combined in 0.5 mL of buffer in a quartz cuvette and the fluorescence measured continuously in a spectrofluorometer (Hitachi F-4500, Hitachi Scientific Instruments, Woking, UK). Excitation wavelength was 385 nm and emission wavelength was 460 nm and slits were 5 and 10 nm for excitation and emission, respectively. PMT voltage was 700 V and the response time was 0.01 s. Once a linear rate was established, 5 µL MIV-711 (final assay concentration between 10 and 50 nmol/L) or 5 µL DMSO control was added and data collected until a new equilibrium rate was achieved. Data were imported into GraphPad Prism and then fitted to the Morrison equation [26] to obtain kobs. The kobs values were plotted against the inhibitor concentration and fitted to a straight line to obtain kon and koff.
MIV-711 was analyzed against > 70 different ion channels, receptors, transporters and cytochrome P450 (CYP) enzymes at a concentration of 10 µmol/L at PanLabs (Taipei, Taiwan).
Osteoclast assay
The effect of MIV-711 on osteoclast-mediated bone resorption was evaluated using a complete kit from Cambrex Bio Science (Walkersville, MD, USA). Briefly, primary osteoclasts were generated from osteoclast precursors by incubation with RANK ligand and M-CSF in 96-well plates coated with human bone fragments, according to the instructions from the provider. After differentiation (minimum of 5 days), osteoclasts were exposed to different fixed concentrations of MIV-711 for 24 h. The resorption activity of the cultures was determined by quantifying CTX-I in the culture supernatants using a commercially available enzyme-linked immunosorbent assay (CrossLaps®, Nordic Bioscience Diagnostics A/S, Herlev, Denmark). Controls exposed to medium without inhibitor were set to 100%. CTX-I levels from MIV-711-treated cells were normalized and expressed as % of control. Curves were generated plotting the concentration of MIV-711 versus % of control. The curves were fitted to the Hill (four parameter) equation to generate an IC50 value using GraphPad Prism software. The IC50 value was defined as the concentration of MIV-711 that reduced CTX-I levels to 50% of control.
Nonclinical pharmacological characterization of MIV-711 in vivo
All animal studies were conducted according to the provisions of United Kingdom Law, in particular the Animals (Scientific Procedures) Act, 1986.
Monkeys: plasma CTX-I experiments—single dose
Eight cynomolgus monkeys (Macaca fascicularis) were used for measuring plasma levels of MIV-711 and CTX-I after a single dose. The animals were healthy young males, approximately 2–4 years old and weighing 3–4 kg. Experiments were performed at Covance Ltd (Harrogate, UK). The monkeys were used over an extended period of time (up to a year) and several cathepsin K inhibitors were evaluated during this time. At least 1 week was allowed for washout in between experiments. This was not believed to affect the results with MIV-711, and vehicle controls were run on a regular basis when appropriate. Each animal received at least one dose of MIV-711 and one dose of vehicle with at least 1 week of washout in between treatments.
In single dose experiments, animals were dosed with vehicle or MIV-711 (3–30 µmol/kg) via oral gavage at a dose volume of 4 mL/kg between 7:30 and 9:00 a.m. Blood samples were collected at baseline, 30 min, 1, 2, 4, 8, 12, 24 and 48 h after dose from the femoral vein and transferred into lithium heparin anticoagulant tubes. The samples were centrifuged and the two aliquots of the resultant plasma were frozen at − 70 °C. All samples were processed and stored within 30 min of collection. The aliquots were used to determine the MIV-711 concentration and CTX-I levels, respectively.
Each animal treated with MIV-711 was also treated with vehicle and sampled in an identical manner either within 2 weeks prior to MIV-711 administration, or after a washout period of between 1 and 2 weeks to enable each animal to serve as its own control in the analysis of the effects of MIV-711 on CTX-I. The area under the curve (AUC) of CTX-I during treatment with MIV-711 was divided by the AUC of CTX-I during treatment with vehicle, thus giving a % CTX-I inhibition value over 24 h. Since the concentration of MIV-711 was measured in each animal it was possible to relate the MIV-711 exposure over 24 h to the % CTX-I inhibition over 24 h in each animal.
Monkeys: plasma CTX-I experiments—multiple doses
Repeat dosing experiments were performed in four male monkeys from the group outlined above (Covance). Vehicle or MIV-711 (30 µmol/kg) was given via oral gavage once daily for 5 days between 7:30 and 8:30 a.m. Plasma for CTX-I measurements was collected on Day 1 at baseline, 1, 2, 4, 8 and 12 h after dose, on Days 2, 3 and 4 early in the morning before next dose (i.e. at trough), and on Day 5 at trough, 1, 2, 4, 8, 12 and 24 h after dose.
Monkeys: urine biomarker experiments—multiple doses
Biomarkers such as NTX-I (bone resorption) and CTX-II (cartilage degradation) are typically measured in urine. Additional experiments in which urine was collected were therefore performed. In these experiments, four cynomolgus monkeys (Macaca fascicularis) were used (Aptuit, Edinburgh, UK). The animals were healthy young females weighing 3.5–4 kg. Animals were dosed with vehicle or MIV-711 via oral gavage at a dose volume of 5 mL/kg between 7:30 and 8:30 a.m. Animals first received vehicle via oral gavage for five consecutive days. After a washout period, the animals then received MIV-711 (30 µmol/kg) via oral gavage for five consecutive days.
Urine was collected between 8–12 a.m., 12 a.m.–4 p.m. and 4 p.m.–8 a.m. on Day 1 and Day 5 of respective treatment. Biomarkers were measured in each urine sample, corrected for creatinine, and the mean value of the three samples on Day 1 and Day 5 was calculated. The effect of MIV-711 was evaluated by comparing biomarkers in urine samples collected on Day 1 and Day 5 with respective vehicle sample.
Single ascending dose study in healthy subjects
The protocol and informed consent documentation for the clinical study was reviewed and approved by the independent ethics committee specific for the study center. The study was conducted in accordance with the International Conference on Harmonisation and Good Clinical Practice regulations and guidelines. Prior to the study, all subjects signed informed consent forms and volunteer information documents.
Subjects
Twenty-seven healthy male and female subjects (18 male and 9 female) of any ethnic origin (24 White, 1 Asian, 1 Black, 1 Black/Caribbean White), aged between 19 and 64 years, and with a body mass index (BMI) between 18.0 and 32.0 kg/m2, were selected for this study. Inclusion criteria for enrollment followed the standard practice for first-in-man studies.
Study design
This was a randomized, double-blind, placebo-controlled, sequential, single-ascending dose study conducted at a single site with the objective of evaluating the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics of MIV-711. Subjects were randomized into 3 groups using a computer-generated pseudo-random permutation procedure. Each group consisted of 9 subjects with at least 3 subjects of each gender per group and subjects participated in 1–2 treatment periods. There was a minimum of 10 days between dose escalations and a minimum of 20 days between dosing occasions for individual subjects. For each subsequent dosing occasion, the decision to escalate the dose to the next level was taken after a thorough review of the safety, tolerability and PK data. In each treatment period, 7 subjects received MIV-711 and 2 received placebo (comprising of 1 subject of each gender). Dosing occurred on the morning of Day 1 following an overnight fast and subjects were kept in the fasted state until approximately 4.5 h after dosing. MIV-711 was given as a combination of 10 or 40 mg hard gelatin capsules, while placebo was given in similar capsules of identical appearance.
Assessments
Safety
Standard safety assessments were performed throughout the study and included physical examination, vital signs, 12-lead electrocardiogram (ECG), continuous ECG Holter monitoring, hematology, biochemistry, urinalysis and adverse event monitoring.
PK
Blood samples were collected at pre-dose, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, 24, 36 and 48 h post-dose and processed into plasma for the measurement of concentrations of MIV-711. The PK analysis was conducted using WinNonlin Enterprise Version 5.2 (Pharsight Corporation, Mountain View, CA, USA).
Pharmacodynamic
Blood samples were collected at pre-dose, 4, 8, 24 and 48 h post-dose and processed into serum for the measurement of CTX-I concentrations.
Bioanalysis of MIV-711
The quantification of MIV-711 in monkey plasma was performed by taking 10–50 µL of plasma and mixing with 3 volumes of acetonitrile (10 s, Vibrofix). The sample was centrifuged (10 min, 20,000×g, 7 °C) and 5 µL of the supernatant was injected onto the liquid chromatography with tandem mass spectrometric detection (LC–MS/MS) system. The lowest concentration of the standard curves and the lower limit of quantification (LLOQ) was 1 nmol/L of MIV-711. The human plasma samples were prepared by protein precipitation for sample extraction followed by LC–MS/MS. The lowest concentration of the standard curves and the LLOQ was 4 nmol/L of MIV-711 using 25 µL of plasma.
Biomarker measurements
The quantification of CTX-I in monkey plasma samples and human serum samples was performed using a commercially available kit (Serum CrossLaps ELISA, IDS Nordic, Herlev, Denmark). The levels of CTX-I in monkey urine samples were measured using a commercially available kit (Urine BETA CrossLaps ELISA, IDS Nordic). The quantification of NTX-I in monkey samples was performed using a commercially available kit (Osteomark NTx Urine, Wampole Laboratories, Inc, Princeton, USA). Urinary levels of CTX-II in monkey samples were measured using a commercially available kit (Urine CrossLaps EIA, IDS Nordic). The concentrations of biomarkers in the urine samples were corrected for creatinine concentrations. The creatinine levels in urine were determined at Swedish University of Agricultural Sciences, using the enzymatic assay (cat no. 8L24-01) on the Abbott system Architect c4000.
Statistical analysis
In the nonclinical studies, two-way ANOVA was used to assess the effects of MIV-711 on plasma CTX-I. Pearson’s correlation was used when comparing plasma exposure of MIV-711 with inhibitory effects on CTX-I. Student’s unpaired t test was used when assessing the effect of MIV-711 on urine biomarkers. In the clinical study, PK parameters were summarized by dose level using descriptive statistics. The dose proportionality for AUC and Cmax was assessed by using a power model with determination of the linear regression slope with a 95% confidence interval. Change in CTX-I from baseline at 24 h after dose was calculated by dose level and summarized using descriptive statistics. In addition, the 95% confidence interval for the difference between each MIV-711 group and the placebo group for these variables was calculated by dose level. Pearson’s correlation was used when comparing plasma exposure of MIV-711 with inhibitory effects on CTX-I. A time-matched and placebo-corrected approach was used to evaluate the effects of MIV-711 on the QTcF interval from the Holter ECG recordings. The change-from-baseline QTcF at post-dose time points was analyzed using an ANCOVA model including the treatment, time (categorical), and treatment-by-time interaction as fixed effects, baseline QTcF as a covariate, and subject as random effect. A p value < 0.05 was considered statistically significant. Data are expressed as mean ± SEM.