Pilocarpine-Induced Effects on Salivary Secretion as a Pharmacological Biomarker for Cholinergic Parasympathetic Activation.

Pilocarpine-induced salivary secretion could serve as a nontherapeutic target engagement biomarker in a clinical setting to test the activity of an M3 positive allosteric modulator (PAM). The potentiating effect on the reactivity of the M3 receptor to the agonistic effect of pilocarpine would support the mechanism of action of an M3 PAM in a variety of therapeutic areas. The aim of this study was to determine the optimal pilocarpine dose needed for evaluation of this potentiating effect. Therefore, the effects of pilocarpine on salivary secretion rate and its pharmacokinetics were explored at single doses of 2.5, 5, and 10 mg of pilocarpine or placebo. The study also explored the test-retest variability of the pilocarpine-induced effects on salivary secretion. Pilocarpine caused a reproducible, dose-related increase in overall and maximum salivary secretion rate, in line with pilocarpine exposure. Oral doses of pilocarpine from 2.5 to 10 mg were safe and well tolerated, consistent with the published safety profile. These results support the use of pilocarpine in single-dose pharmacological challenge studies. The recommended dose for evaluating M3 PAM activity would be between 2.5 and 5 mg, showing a small increase in salivary secretion rate with room for further increase due to PAM activation.

Quantitation of Tacrolimus in Human Whole Blood Samples Using the MITRA Microsampling Device.

BACKGROUND: The calcineurin inhibitor tacrolimus is a narrow therapeutic index medication, which requires therapeutic drug monitoring to optimize dose on the basis of systemic exposure. MITRA microsampling offers a minimally invasive approach for the collection of capillary blood samples from a fingerprick as an alternative to conventional venous blood sampling for quantitation of tacrolimus concentrations. METHODS: A bioanalytical method for the quantitation of tacrolimus in human whole blood samples collected on MITRA tips was developed, using liquid-liquid extraction followed by liquid chromatography with tandem mass spectrometry detection. Validation experiments were performed according to the current Food and Drug Administration and European Medicines Agency guidelines on validation of bioanalytical methods. Validation criteria included assay specificity and sensitivity, interference, carryover, accuracy, precision, dilution integrity, matrix effect, extraction recovery, effect of hematocrit and hyperlipidemia, and stability. RESULTS: All assay validation results were within the required acceptance criteria, indicating a precise and accurate tacrolimus quantitation method. The validated assay range was 1.00-50.0 ng/mL. No interference, carryover or matrix effect was observed. Extraction recovery was acceptable across the assay range. Samples were stable for up to 96 days at -20°C and 20°C, and 28 days at 40°C. Hematocrit, hyperlipidemia, and lot-to-lot differences in the nominal absorption volume of the 10-µL MITRA tips were shown not to influence tacrolimus quantitation by this assay method. CONCLUSIONS: The bioanalytical method validated in this study is appropriate and practical for the quantitation of tacrolimus in human whole blood samples collected using the MITRA microsampling device.

Pharmacokinetics of solifenacin in pediatric populations with overactive bladder or neurogenic detrusor overactivity.

The aim of this investigation was to characterize and compare the pharmacokinetics (PK) of the antimuscarinic drug solifenacin in pediatric patients with overactive bladder (OAB) or neurogenic detrusor overactivity (NDO) utilizing data from three phase III trials. LION was a placebo-controlled, 12-week trial in children (5-<12 years) and adolescents (12-<18 years) with OAB. MONKEY and MARMOSET were open-label, 52-week trials in children and adolescents or younger children (6 months-<5 years), respectively, with NDO. During the trials, solifenacin doses could be titrated to weight-adjusted pediatric equivalent doses (PEDs) of 2.5, 5, 7.5, or 10 mg day-1 . Nonlinear mixed effects modeling was used to develop population PK models to characterize the PK in patients with either OAB or NDO. Overall, 194 children and adolescents received solifenacin. At the time of PK sampling, the majority (119/164 [72.6%] patients) were receiving PED10 once daily. All population models included first-order oral absorption, a lag time, and interindividual variability. PK analysis showed that apparent clearance was similar in both patient populations. Mean apparent oral plasma clearance (CL/F), apparent volume of distribution during the terminal phase (Vz /F), and terminal half-life (t1/2 ) were higher in adolescents than in children, but median time to maximum plasma concentration (tmax ) was similar. Dose-normalized exposure results were similar for both younger and older patients with OAB or NDO. In conclusion, population PK modeling was used to successfully characterize solifenacin PK in pediatric patients with OAB or NDO. Similar solifenacin PK characteristics were observed in both populations.

The Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of ASP3652 in First-in-Human and Ascending Multiple Oral Dose Studies in Healthy Subjects.

INTRODUCTION: Inhibitors of fatty acid amide hydrolase (FAAH) increase the levels of endocannabinoids and have shown analgesic and anti-inflammatory activity in animal models. ASP3652 is a peripherally acting FAAH inhibitor in development for the treatment of chronic bladder and pelvic pain disorders. Here we describe the safety, pharmacokinetics, and pharmacodynamics of single and multiple oral doses of ASP3652 administered in healthy non-elderly and elderly male and female volunteers. METHODS: Study 1 was a combined single-ascending dose and food-effect study in which ASP3652 was given as single doses (1-600 mg) or matching placebo in healthy subjects. Study 2 was a multiple ascending dose study in which ASP3652 or matching placebo was administered in multiple oral doses (10-300 mg bid and 600 mg qd for 14 days) to healthy subjects. In both studies, the levels of ASP3652, FAAH, endocannabinoids (eCBs) and safety were evaluated. RESULTS: ASP3652 was readily absorbed to reach Cmax at 1 h after a single dose. Steady state was reached within 3 days after the start of multiple dosing. The Cmax and AUC of ASP3652 increased in a slightly more than dose-proportional manner after a single dose of ASP3652 at 30-600 mg. There was some accumulation (15-38%) based on Cmax and AUC12h upon multiple doses. Cmax was 47% lower in combination with food. There was no significant effect of gender or age on the pharmacokinetics of ASP3652. FAAH activity was inhibited in a dose-dependent manner in all dose groups after single and multiple doses of ASP3652, paralleled by an increase in plasma levels of anandamide (AEA). The incidence of adverse events following multiple doses was similar across all treatment groups including the placebo group. CONCLUSIONS: Single and multiple doses of ASP3652 were safe and well tolerated and increased endogenous cannabinoid plasma levels.

Investigation of Safety and Tolerability of ASP3652 Based on Clinical Studies of Cerebrospinal Fluid Transfer After Multiple Doses and Exposure After Single Doses at High Dose Levels.

INTRODUCTION: The studies described here were conducted to investigate the central nervous system (CNS) transfer of ASP3652, a peripherally acting inhibitor of fatty acid amide hydrolase, after multiple doses at around the anticipated therapeutic dose and the safety, tolerability, and pharmacokinetics after single doses at corresponding supratherapeutic doses in healthy subjects. METHODS: Study 1 was an open-label multiple dose study in which ASP3652 (300 mg bid) or matching placebo was administered in multiple doses to healthy subjects. Study 2 was a placebo-controlled, randomized 4 × 4 crossover study in which ASP3652 was given as three single ascending doses of ASP3652 (600-1800 mg) or matching placebo to healthy subjects. Levels of ASP3652 and endocannabinoids (eCBs) in plasma, cerebrospinal fluid (CSF) (study 1 only), and safety were evaluated. RESULTS: In study 1, ASP3652 was readily absorbed to reach Cmax at 1 h after dosing. AUCtau and Cmax of ASP3652 in CSF were approximately 0.2% and 0.06% of the AUCtau and Cmax in plasma after multiple doses of ASP3652 300 mg bid. At steady state the area under the response-time curve (AURC) from 0 to 12 h and the maximum response for anandamide in plasma were approximately 550-fold and 230-fold higher than those in CSF. In study 2, the Cmax and AUC of ASP3652 increased higher than dose proportionally in subjects receiving 600-1800 mg ASP3652. For eCBs, although the AURC increased less than dose proportionally, maximum plasma levels were comparable across all treatment groups. The incidence of adverse events (AEs) was similar across all treatment groups including the placebo group. There was no evidence of CNS-related side effects. CONCLUSIONS: ASP3652 showed low CNS penetration at the anticipated therapeutic dose and was well tolerable without any CNS-related AEs at supratherapeutic doses, supporting that the drug can be safely tested at the anticipated therapeutic dose. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT02034734 for study 1, NCT01815684 for study 2.

Validation of LC-MS/MS methods for the determination of mirabegron and eight metabolites in human plasma in a paediatric population.

Mirabegron is the first registered ß3-adrenoceptor agonist for treatment of overactive bladder as an alternative to antimuscarinics. Previously four liquid chromatography-tandem mass spectrometry assays were published to determine mirabegron and eight metabolites (M5, M8, M11-M16) in human plasma. In order to support paediatric development, the assays were further optimized to reduce the required blood volume and increase the sensitivity. The assays were miniaturized by using 96-well supported liquid extraction plates (mirabegron, M5, M16) or 96-well mixed-mode cation exchange solid phase extraction plates (M8, M11-M15) and a more sensitive MS-system was used. For the analytes, up to fivefold increase in assay sensitivity was achieved. The required blood sample volume was reduced from 10 to 2 mL for each timepoint. Validation demonstrated that the assays were accurate, precise and selective in the determination of mirabegron and metabolites. The assays were successfully applied to evaluate the pharmacokinetics of mirabegron in a paediatric population.

Determination of methionine-enkephalin and leucine-enkephalin by LC-MS in human plasma: Study of pre-analytical stability.

The aim of this work was to assess the influence of preanalytical variables on the stability of two endogenous opioid peptides (Methionine-Enkephalin and Leucine-Enkephalin) in human plasma. For this purpose, first a sensitive LC-MS/MS analytical method was developed and validated for the simultaneous quantitative analysis of these two peptides. The methodology consisted of a simple protein precipitation step followed by UPLC separation and MRM quantitative analysis using a stable isotope labelled Methionine-Enkephalin as internal standard. The method with a limit of quantitation of 10 pg/mL showed good reproducibility with excellent accuracy and precision, and was linear up to 2000 pg/mL. An extensive evaluation of the pre-analytical stability of these peptides in human blood was carried out to ensure an adequate sample collection procedure to obtain reliable results in the analysis of clinical samples.

The application of control charts in regulated bioanalysis for monitoring long-term reproducibility.

In regulated bioanalysis, the acceptance of results is batch-wise. When during clinical development derived pharmacokinetic or pharmacodynamic results from different studies will be combined or compared, it is recommendable to monitor the long-term reproducibility of bioanalytical assays. Long-term reproducibility can be evaluated by control charts generated from control samples included in each batch. We present a methodology for the implementation, construction and evaluation of control charts next to the regular batch acceptance of bioanalytical results. Decision rules can be set up for a statistical evaluation of the results. Violation of a decision rule may lead to a root-cause investigation and corrective actions to improve assay robustness. Three examples of control charts, for pharmacokinetic and pharmacodynamic analytes are presented.

An Exploratory Study in Healthy Male Subjects of the Mechanism of Mirabegron-Induced Cardiovascular Effects.

To explore the role of ß1 -adrenoceptors (ARs) in the heart rate response to the selective ß3 -adrenoceptor agonist mirabegron, 12 young male volunteers received single oral doses of the nonselective ß1/2 -AR antagonist propranolol (160 mg), the selective ß1 -AR antagonist bisoprolol (10 mg), or placebo on days 1 and 5 of each period in a 3-period crossover study. On day 5, dosing was followed by a supratherapeutic dose of mirabegron (200 mg). Vital signs, impedance cardiography, and plasma renin activity were collected. Mirabegron increased heart rate and systolic blood pressure and reduced stroke volume, whereas cardiac output and diastolic blood pressure were unaffected. Mirabegron-induced changes were attenuated by propranolol and bisoprolol. The data indicate that mirabegron has a positive chronotropic effect at supratherapeutic concentrations, which is at least partly mediated by stimulation of ß1 -AR.

Pharmacokinetic Interactions Between Mirabegron and Metformin, Warfarin, Digoxin or Combined Oral Contraceptives.

BACKGROUND AND OBJECTIVES: Mirabeg ron is a selective ß3-adrenoceptor agonist approved for the treatment of overactive bladder (OAB). Four phase 1 studies were conducted in healthy subjects to evaluate the potential for pharmacokinetic interactions between mirabegron and metformin, warfarin, digoxin, or a combination oral contraceptive (COC). METHODS: Thirty-two male subjects received metformin (500 mg twice daily) or mirabegron (160 mg once daily) alone, in combination or with placebo. Twenty-four male and female subjects received single doses of warfarin (25 mg) alone and in combination with mirabegron (100 mg once daily). Twenty-five male and female subjects were administered digoxin (0.25 mg) alone and in combination with mirabegron (100 mg once daily). Thirty female subjects received low-dose COC containing ethinylestradiol (EE)/levonorgestrel (LNG) (30/150 µg once daily) in combination with mirabegron (100 mg once daily) or placebo. Pharmacokinetic parameters were determined by non-compartmental methods. Absence of a Pharmacokinetic interaction was concluded if the 90 % confidence intervals (CI) of geometric least-squares means ratio of area under the curve (AUC) and maximum concentration (C max) were contained within the standard 80-125 % no-effect boundaries. The effect of mirabegron on warfarin International Normalized Ratio (INR) was also assessed. RESULTS: Mirabegron increased digoxin AUC and Cmax by 27 and 29 %, respectively, indicating that mirabegron is a weak inhibitor of P-glycoprotein (P-gp) in vivo. Co-administration of mirabegron did not affect the pharmacokinetics of metformin, warfarin, EE and LNG, or warfarin INR, except for a slight extension of the 90 % CI for the C max ratio for metformin (lower limit 79 %). Metformin decreased mirabegron AUC and C max by 21 %. Most treatment-emergent adverse events were mild, and all resolved by study end. CONCLUSIONS: No dose adjustment of either drug is required when mirabegron is administered concomitantly with metformin, warfarin or COC. Patients receiving mirabegron with digoxin may require additional monitoring of digoxin concentrations with dose adjustments where necessary, due to its narrow therapeutic index.