Pharmacokinetics of Darolutamide in Mouse - Assessment of the Disposition of the Diastereomers, Key Active Metabolite and Interconversion Phenomenon: Implications to Cancer Patients.

BACKGROUND: Darolutamide is recently approved for the treatment of non-metastatic castrate resistance prostate cancer. Hitherto, no stereoselective pharmacokinetic data have been published pertaining to darolutamide and its diastereomers in animals or humans. The key aims of the experiment were to examine darolutamide, S,S-darolutamide and S,R-darolutamide with respect to (a) assessment of in vitro metabolic stability and protein binding and (b) characterization of in vivo oral and intravenous pharmacokinetics in mice. METHODS: In vitro (liver microsomes stability and protein binding) and in vivo experiments (oral/intravenous dosing to mice) were carried out using darolutamide, S,S-darolutamide and S,Rdarolutamide. Besides, tissue levels of darolutamide, S,S-darolutamide and S,R-darolutamide were measured following oral and intravenous dosing. Appropriate plasma/tissue samples served to determine the pharmacokinetics of various analytes in mice. Liquid chromatography in tandem with mass spectrometry procedures enabled the delineation of the plasma pharmacokinetics, in vitro and tissue uptake data of the various analytes. RESULTS: Chiral inversion was absent in the metabolic stability study. However, darolutamide showed profound stereoselectivity (S,S-darolutamide greater than S,R-darolutamide) after either intravenous or oral dosing. S,R-darolutamide but not S,S-darolutamide showed conversion to its antipode post oral and intravenous dosing to mice. Regardless of oral or intravenous dosing, active keto darolutamide formation was evident after administration of darolutamide, S,S-darolutamide or S,R- darolutamide. Tissue data supported the observations in plasma; however, tissue exposure of darolutamide, S,Sdarolutamide and S,R-darolutamide was much lower as compared to plasma. CONCLUSION: In lieu of the human pharmacokinetic data, although the administration of diastereomeric darolutamide was justified, it is proposed to delineate the clinical pharmacokinetics of S,Rdarolutamide and S,S-darolutamide relative to darolutamide in future clinical pharmacology studies.

Preparation and optimization of nilotinib self-micro-emulsifying drug delivery systems to enhance oral bioavailability.

Objective: The objective of the current research was to prepare self-micro-emulsifying drug delivery systems (SMEDDS) for BCS class II drug, nilotinib to enhance its oral bioavailability.Methodology: Different types of excipients like oil, surfactant, and co-surfactant were evaluated for drug solubility. Among the screened excipients, Capryol 90, Transcutol HP, and Tween 80 were selected as oil, co-surfactant, and surfactant, respectively, to construct a ternary phase diagram to identify a homogenous mixture. Characterization performed for the prepared SMEDDS for its particle size/droplet size, emulsification time, phase separation, droplet morphology, in vitro drug release, and oral bioavailability.Results: Prepared SMEDDS showed the highest of 87% drug release in in vitro drug release experiment. SMEDDS drug release was superior over suspension formulation, which could be attributed to oil/surfactant ratios and particle size of the SMEDDS. The acquired pharmacokinetic parameters indicate that twofold increase in systemic exposure of SMEDDS compared with nilotinib suspension formulation. A similar twofold increase in relative oral bioavailability was also observed when compared SMEDDS formulation with suspension formulation. Delayed Tmax (time to reach peak plasma concentrations) was observed with SMEDDS over suspension formulation, which was evident by slow rate of absorption of nilotinib from SMEDDS.Conclusion: This research demonstrated that SMEDDS could be an effective approach to improve solubility and oral bioavailability for the BCS class II poorly soluble nilotinib.

Stereoselective pharmacokinetics and tissue distribution of levodropropizine after administration of pure levodropropizine and the rac-dropropizine to Sprague-Dawley rats.

Levodropropizine (LDP) is a non-opioid anti-tussive. The stereoselective pharmacokinetics and tissue distribution (TD) of LDP vs. dextrodropropizine (DDP) have been characterized after oral and intravenous (IV) administration of LDP and rac-dropropozine in rats.Oral/IV doses of 50/5.0 mg/kg and 25/2.5 rac-dropropizine and LDP were employed. TD study focused on tissues such as liver, lung and kidney. Blood samples were collected for pharmacokinetic and TD evaluation. Validated methods were used to quantitate LDP, DDP and rac-dropropizine.No stereoselectivity in pharmacokinetics was observed between LDP vs. DDP following rac-dropropizine. However, LDP pharmacokinetics after LDP administration (oral/IV) appeared to be different compared to LDP derived from rac-dropropizine.TD data were similar between the two enantiomers regardless of oral/IV rac-dropropizine administration. When LDP alone was administered, levels were comparable to those derived for LDP from rac-dropropizine after oral/IV. However, in the lung and kidney tissues, the exposure after oral dosing was higher for LDP alone as compared to LDP from rac-dropropizine.In summary, complete characterization of stereoselective pharmacokinetics and TD of rac-dropropizine has been reported after oral/IV routes. It was evident that the presence of DDP, increased the plasma/tissue exposure of LDP which was evident after oral rac-dropropizine dosing.

Validated LC-ESI-MS/MS method for the determination of tunicamycin in rat plasma: Application to a pharmacokinetic study.

A liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of tunicamycin in rat plasma as per regulatory guideline. Chromatography of tunicamycin and the IS in the processed plasma samples was achieved on an X-Terra phenyl column using a binary gradient (mobile phase A, acetonitrile and mobile phase B, 5 mm ammonium formate) elution at a flow rate of 0.6 ml/min. LC-MS/MS was operated under the multiple reaction monitoring mode using the electrospray ionization technique in positive ion mode and the transitions of m/z 817.18 → 596.10, 831.43 → 610.10, 845.29 → 624.10, 859.23 → 638.10 and 309.24 → 163.20 were used to quantitate homologs A-D and the IS, respectively. The total chromatographic run time was 4.5 min. The correlation coefficient (r2 ) was >0.99 for all homologs with accuracy 90.7-107.4% and precision 0.74-15.1%. The recovery of homologs was 78.6-90.2%. No carryover was observed and the matrix effect was minimal. Tunicamycin four homologs were found to be stable on the bench-top for 6 h, for up to three freeze-thaw cycles, in the injector for 24 h and for 1 month at -80°C. The applicability of the validated method has been demonstrated in a rat pharmacokinetic study.

Prediction of Human Pharmacokinetics of Fomepizole from Preclinical Species Pharmacokinetics Based on Normalizing Time Course Profiles.

Fomepizole is used as an antidote to treat methanol poisoning due to its selectivity towards alcohol dehydrogenase. In the present study, the goal is to develop a method to predict the fomepizole human plasma concentration versus time profile based on the preclinical pharmacokinetics using the assumption of superimposability on simulated time course profiles of animals and humans. Standard allometric equations with/without correction factors were also assimilated in the prediction. The volume of distribution at steady state (Vss) predicted by simple allometry (57.55 L) was very close to the reported value (42.17 L). However, clearance (CL) prediction by simple allometry was at least 3-fold higher to the reported value (33.86 mL/min); hence, multiple correction factors were used to predict the clearance. Both brain weight and maximum life span potential could predict the CL with 1.22- and 1.01-fold difference. Specifically, the predicted Vss and CL values via interspecies scaling were used in the prediction of series of human intravenous pharmacokinetic parameters, while the simulation of human oral profile was done by the use of absorption rate constant (Ka) from dog following the applicability of human bioavailability value scaled from dog data. In summary, the findings indicate that the utility of diverse allometry approaches to derive the human pharmacokinetics of fomepizole after intravenous/oral dosing.

Enantioselective LC-ESI-MS/MS determination of dropropizine enantiomers in rat plasma and application to a pharmacokinetic study.

We developed and validated a simple, sensitive, selective and reliable LC-ESI-MS/MS method for direct quantitation of dropropizine enantiomers namely levodropropizine (LDP) and dextrodropropizine (DDP) in rat plasma without the need for derivatization as per regulatory guidelines. Dropropizine enantiomers and carbamazepine (internal standard) were extracted from 50 µL rat plasma using ethyl acetate. LDP and DDP resolved with good baseline separation (Rs = 4.45) on a Chiralpak IG-3 column. The mobile phase consisted of methanol with 0.05% diethylamine pumped at a flow rate of 0.5 mL/min. Detection and quantitation were done in multiple reaction monitoring mode following the transitions m/z 237 → 160 and 237 → 194 for dropropizine enantiomers and the internal standard, respectively, in the positive ionization mode. The proposed method provided accurate and reproducible results over the linearity range of 3.23-2022 ng/mL for each enantiomer. The intra- and inter-day precisions were in the ranges of 3.38-13.6 and 5.11-13.8 for LDP and 4.19-11.8 and 8.89-10.1 for DDP. Both LDP and DDP were found to be stable under different stability conditions. The method was successfully used in a stereoselective pharmacokinetic study of dropropizine enantiomers in rats following oral administration of racemate dropropizine at 100 mg/kg. The pharmacokinetic results indicate that the disposition of dropropizine enantiomers is not stereoselective and chiral inversion does not occur in rats.

RP-HPLC-UV Method for Simultaneous Quantification of Second Generation Non-Steroidal Antiandrogens Along with their Active Metabolites in Mice Plasma: Application to a Pharmacokinetic Study.

A simple, specific and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the quantitation of second generation antiandrogens and their active metabolites namely apalutamide, enzalutamide, N-desmethylenzalutamide (active metabolite of enzalutamide), darolutamide and ORM-15341 (active metabolite of darolutamide) in mice plasma. The method involves extraction of apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 along with internal standard (IS) from 100 µL mice plasma through a simple protein precipitation process. The chromatographic analysis was performed on a Waters Alliance HPLC system using a gradient mobile phase (comprising 10 mM ammonium acetate and acetonitrile in a flow-gradient) and X-Terra Phenyl column. The UV detection wave length was set at λmax 250 nm. Apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 and the IS eluted at 13.6, 11.4, 9.68, 6.11, 6.93 and 4.69 min, respectively with a total run time of 15 min. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 209 - 5215 ng/mL (r 2=0.998). The intra- and inter-day precisions were in the range of 0.56-13.5 and 1.04-13.9%, respectively. The validated HPLC method was successfully applied to a pharmacokinetic study in mice.

Validated LC-MS-MS Method for Determination of SF0034 in Mice Plasma: Application to a Pharmacokinetic Study in Mice.

A rapid and sensitive assay method has been developed and validated using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode for the estimation of SF0034 in mice plasma. The assay procedure involves a simple protein precipitation of SF0034 and tolbutamide (internal standard, IS) from mice plasma. Chromatographic separation was performed on an Atlantis dC18 column using an isocratic mobile phase comprising 0.2% formic acid:acetonitrile (10:90, v/v) at a flow rate of 0.60 mL/min. The total run time was 2.5 min. For mass spectrometric detection, the multiple reaction monitoring was used and ion transitions monitored were m/z 322 → 248 for SF0034 and 271 → 155 for IS. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. A calibration curve was constructed in the range of 2.08-2,078 ng/mL. The intra- and inter-day precision was in the range of 1.06-14.4% and 7.16-11.7%, respectively. This novel method has been applied to a pharmacokinetic study in mice.

Effect of bromocriptine on cardiovascular complications associated with metabolic syndrome in fructose fed rats.

OBJECTIVE: The objective of the present study was to evaluate the effect of bromocriptine on cardiovascular complications associated with type-2 diabetes mellitus (DM). MATERIALS AND METHODS: Metabolic syndrome or type 2 DM was induced by administration of fructose (66% solution, p.o.) in rats. Bromocriptine mesylate (10 mg/kg, i.p.) was given in fructose-treated rats for a period of 6 weeks after induction of diabetes. After drug treatment, the parameters such as body weight, food and water intake, serum glucose, triglycerides, cholesterol, insulin, and blood pressure (BP) were measured weekly and at the end of study. At the end of treatment, BP was determined by invasive method and vascular reactivity was tested with adrenaline (Adr), noradrenaline (NA), and phenylephrine (PE). Acetylcholine-induced vasorelaxation was tested on isolated rat aorta and histopathology of hearts was also done. RESULTS: Fructose-fed rats showed significant weight gain, hyperglycemia, hyperlipidemia, hyperinsulinemia, and rise of BP. Administration of bromocriptine at a dose 10 mg/kg, i.p. significantly decreased weight gain, serum glucose, triglyceride, cholesterol and insulin levels in rats fed on fructose. Bromocriptine also significantly reduced elevated BP in fructose-fed hypertensive rats. Chronic treatment with bromocriptine significantly improved the relaxant response to acetylcholine on fructose-fed hyperinsulinemic rat aorta and also reduced the pressor response to Adr, NA, and PE. Bromocriptine also showed a protection from hypertrophy and degenerative changes in myocardium. CONCLUSION: Bromocriptine has beneficial effect in reduction of cardiovascular complications associated with metabolic syndrome.