Metabolism, excretion, and pharmacokinetics of [14C]mirogabalin, a novel α2δ ligand, in healthy volunteers following oral administration.

The metabolism, excretion, and pharmacokinetics of mirogabalin were investigated following a single oral administration of [14C]mirogabalin at 30 mg/5.55 MBq to six healthy male subjects.The mean recovery values of radioactivity in urine and faeces were 96.85 and 1.21%, respectively. The main component of radioactivity in the plasma, urine, and faeces was mirogabalin. A204-4455 (lactam form), mirogabalin N-glucuronide, and glucuronide of oxidized A204-4455 were detected as minor components in the specimens. Renal clearance of mirogabalin was higher than the glomerular filtration rate of the human kidneys, indicating renal secretion is involved in the clearance.In vitro studies revealed that UDP-glucuronosyltransferase produced two metabolites: A204-4455, formed via mirogabalin acylglucuronide, and a ring-opened form of mirogabalin N-glucuronide.Mirogabalin was absorbed almost completely, and was eliminated via urine. A part of the orally administered dose of mirogabalin was metabolized through glucuronidation at the amine and carboxylic acid moiety, which represents the primary metabolic pathway.

Stereoselective hydroxylation by CYP2C19 and oxidation by ADH4 in the in vitro metabolism of tivantinib.

1. In prior studies, it has been shown that tivantinib is extensively metabolized in humans to many oxidative metabolites and glucuronides. In order to identify the responsible enzymes, we investigated the in vitro metabolism of tivantinib and its four major circulating metabolites. 2. The primary isoforms involved in the elimination of tivantinib were CYP2C19 and CYP3A4/5. CYP2C19 showed catalytic activity for the formation of M5 (hydroxylated metabolite), but not for M4 (a stereoisomer of M5), whereas CYP3A4/5 catalyzed the formation of both metabolites. For the elimination of M4, M5 and M8 (keto-metabolite), CYP3A4/5 was the major cytochrome P450 isoform and UGT1A9 was mainly involved in the glucuronidation of M4 and M5. 3. ADH4 was identified as one of the major alcohol dehydrogenase isoforms contributing to the formation of M6 (sequential keto-metabolite of M4 and M5) and M8. The substrate preference of ADH for M4, and not M5, was observed in the formation of M6. 4. In conclusion, CYP2C19, CYP3A4/5, UGT1A9 and ADH4 were the primary drug metabolizing enzymes involved in the in vitro metabolism of tivantinib and its metabolites. The stereoselective hydroxylation by CYP2C19 and substrate stereoselectivity of ADH4-catalyzed oxidation in the in vitro metabolism of tivantinib was discovered.

Alpha-amylase inhibitor, CS-1036 binds to serum amylase in a concentration-dependent and saturable manner.

(2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-ß-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036), which is an α-amylase inhibitor, exhibited biphasic and sustained elimination with a long t1/2 (18.4-30.0 hours) in rats and monkeys, but exhibited a short t1/2 (3.7-7.9 hours) in humans. To clarify the species differences in the t1/2, the plasma protein binding of CS-1036 was evaluated by ultrafiltration. A concentration-dependent and saturable plasma protein binding of CS-1036 was observed in rats and monkeys with the dissociation rate constant (KD) of 8.95 and 27.2 nM, and maximal binding capacity (Bmax) of 52.8 and 22.1 nM, respectively. By the assessments of the recombinant amylase and immunoprecipitation, the major binding protein of CS-1036 in rats was identified as salivary amylase (KD 5.64 nM). CS-1036 also showed concentration-dependent and saturable binding to human salivary and pancreatic amylase, with similar binding affinity in rats. However, the protein binding of CS-1036 was constant in human plasma (≤10.2%) due to the lower serum amylase level compared with rats and monkeys. From the calculation of the unbound fraction (fu) in plasma based on in vitro KD and Bmax, the dose-dependent increase in fu after oral administration is speculated to lead to a dose-dependent increase in total body clearance and a high area under the curve/dose at lower doses, such as 0.3 mg/kg in rats.

Absorption, elimination, and metabolism of CS-1036, a novel α-amylase inhibitor in rats and monkeys, and the relationship between gastrointestinal distribution and suppression of glucose absorption.

The absorption, metabolism, and excretion of (2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-ß-d-glucopyranosyl)-α-d-glucopyranoside (CS-1036), a novel and potent pancreatic and salivary α-amylase inhibitor, were evaluated in F344/DuCrlCrlj rats and cynomolgus monkeys. The total body clearance and volume of distribution of CS-1036 were low (2.67-3.44 ml/min/kg and 0.218-0.237 l/kg for rats and 2.25-2.84 ml/min/kg and 0.217-0.271 l/kg for monkeys). After intravenous administration of [(14)C]CS-1036 to rats and monkeys, radioactivity was mainly excreted into urine (77.2% for rats and 81.1% for monkeys). After oral administration, most of the radioactivity was recovered from feces (80.28% for rats and 88.13% for monkeys) with a low oral bioavailability (1.73-2.44% for rats and 0.983-1.20% for monkeys). In rats, intestinal secretion is suggested to be involved in the fecal excretion as a minor component because fecal excretion after intravenous administration was observed (15.66%) and biliary excretion was almost negligible. Although intestinal flora was involved in CS-1036 metabolism, CS-1036 was the main component in feces (70.3% for rats and 48.7% for monkeys) and in the intestinal contents (33-68% for rats up to 2 hours after the dose) after oral administration. In Zucker diabetic fatty rats, CS-1036 showed a suppressive effect on plasma glucose elevation after starch loading with a 50% effective dose at 0.015 mg/kg. In summary, CS-1036 showed optimal pharmacokinetic profiles: low oral absorption and favorable stability in gastrointestinal lumen, resulting in suppression of postprandial hyperglycemia by α-amylase inhibition.

Imaging Mass Spectrometry (IMS) for drug discovery and development survey: Results on methods, applications and regulatory compliance.

Imaging mass spectrometry (IMS) is increasingly used for drug discovery and development to understand target enagement, tissue distribution, drug toxicity, and disease mechanisms, etc. However, this is still a relatively new technique that requires further development validation before it will be an acceptable technique to support regulated development of new drugs. Thus, best practices will need to be established to build more confidence and gain wider acceptance by the scientific community, pharmaceutical industry, and regulatory authorities. The Imaging Mass Spectrometry Society (IMSS) and the Japan Association for Imaging Mass Spectrometry (JAIMS) have conducted a thorough survey to gather information on the current state of IMS and to identify key issues. The survey was sent to researchers or managers in the position who are currently using IMS techniques in support of their drug discovery and development efforts and/or who plan to use such tools as best practices are established. The survey probes questions related to details regarding technical aspects of IMS, which includes data acquisition, data analysis and quantitation, data integrity, reporting, applications, and regulatory concerns. This international survey was conducted online through the Survey Monkey (https://www.surveymonkey.com) in both English and Japanese from September 14 through September 30, 2020.

Enantioselective determination of azelnidipine in human plasma using liquid chromatography-tandem mass spectrometry.

A sensitive and simple method was developed for determination of the enantiomers of azelnidipine, (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, in human plasma using chiral liquid chromatography with positive ion atmospheric pressure chemical ionization tandem mass spectrometry. Plasma samples spiked with stable isotope-labeled azelnidipine, [(2)H(6)]-azelnidipine, as an internal standard, were processed for analysis using a solid-phase extraction in a 96-well plate format. The azelnidipine enantiomers were separated on a chiral column containing alpha(1)-acid glycoprotein as a chiral selector under isocratic mobile phase conditions. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, monitoring the transitions from m/z 583-->167 for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, and from m/z 589-->167 for [(2)H(6)]-azelnidipine. The standard curve was linear over the studied range (0.05-20 ng/mL), with r(2)>0.997 using weighted (1/x(2)) quadratic regression, and the chromatographic run time was 5.0 min/injection. The intra- and inter-assay precision (coefficient of variation), calculated from the assay data of the quality control samples, was 1.2-8.2% and 2.4-5.8% for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, respectively. The accuracy was 101.2-117.0% for (R)-(-)-azelnidipine and 100.0-107.0% for (S)-(+)-azelnidipine. The overall recoveries for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine were 71.4-79.7% and 71.7-84.2%, respectively. The lower limit of quantification for both enantiomers was 0.05 ng/mL using 1.0 mL of plasma. All the analytes showed acceptable short-term, long-term, auto-sampler and stock solution stability. Furthermore, the method described above was used to separately measure the concentrations of the azelnidipine enantiomers in plasma samples collected from healthy subjects who had received a single oral dose of 16 mg of azelnidipine.

Simultaneous determination of azelnidipine and two metabolites in human plasma using liquid chromatography-tandem mass spectrometry.

A quantitative assay method by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) for the simultaneous determination of azelnidipine and its two metabolites, M-1 (aromatized form) and M-2 (hydroxylated form), in human plasma was developed and validated. Plasma samples, each of 1.0mL, were extracted by a single step liquid-liquid extraction using a mixture of ethyl acetate and hexane (1:1, v/v), and analyzed by the LC/ESI-MS/MS method. Three analytes were separated by isocratic elution on a C(18) column, and ionized using a positive ion electrospray ionization source. The ion transitions were monitored in selected reaction monitoring (SRM) mode. The chromatographic run time was 11min per injection, with retention time of 3.6, 10.2 and 6.8min for azelnidipine, M-1 and M-2, respectively. The calibration curves for azelnidipine, M-1 and M-2 well fitted to equations by a weighted (1/X(2)) quadratic regression over the range of 0.5-40.0ng/mL (r(2)>0.9979). The intra- and inter-assay precisions (coefficient of variation: C.V.), calculated from quality control (QC) samples, were less than 8.7 and 8.4%, 3.8 and 4.7%, and 11.9 and 13.9%, respectively, for azelnidipine, M-1 and M-2. The accuracy was within +/-9% for azelnidipine, within +/-7% for M-1 and within +/-16% for M-2. The overall recoveries for azelnidipine, M-1 and M-2 were 68.8-78.6%, 54.3-62.9% and 80.4-89.7%, respectively. All analytes evaluated demonstrated acceptable short-term, long-term, auto-sampler and stock solution stabilities. Furthermore, the method developed was successfully applied to pharmacokinetic studies on azelnidipine, M-1 and M-2 after an oral dose of 16mg CALBLOCK tablets (2mgx8mg tablets) to healthy volunteers.

Quantitative determination of pravastatin and R-416, its main metabolite in human plasma, by liquid chromatography-tandem mass spectrometry.

A quantitative method was developed and validated for rapid and sensitive analysis of pravastatin and R-416, the main metabolite of pravastatin, in human plasma. The analytes were extracted from plasma samples by a solid phase extraction method using a Bond Elut C(8). The method involved the use of liquid chromatography coupled with atmospheric pressure chemical ionization (APCI) and selected reaction monitoring (SRM) mass spectrometry. A pravastatin analog, R-122798, was used as the internal standard (I.S.). Separation of pravastatin, R-416 and the I.S. was accomplished using a reverse-phase column (C(18)). The components eluted were ionized by the APCI source (negative ion) and subsequently detected by a highly selective triple quadrupole mass spectrometer in the SRM mode. Linear standard curves were obtained from 0.1 ng/mL (lower limit of quantification, LLOQ) to 100 ng/mL. The intra-assay precisions (coefficient of variation) for the samples at the LLOQ were 1.8% for pravastatin and 1.6% for R-416. The intra-assay accuracy values were 95.8-107.6% for pravastatin, and 92.6-109.0% for R-416, respectively. Precision and accuracy of quality control (QC) samples were determined at concentrations of 0.5, 10 and 80 ng/mL for all analytes. The intra- and inter-assay precision calculated from QC samples were within 10% for pravastatin and within 11% for R-416. The overall recoveries for pravastatin and R-416 were 75.7-82.1% and 68.6-74.3%, respectively. Pravastatin and R-416 were stable in human plasma for 3 weeks at -20 degrees C in a freezer, up to 6h at room temperature, and up to 48 h at 6 degrees C. This assay method was successfully used to evaluate the pravastatin and R-416 levels in healthy volunteers following oral administration of Mevalotin.

Polymorphisms of OATP-C (SLC21A6) and OAT3 (SLC22A8) genes: consequences for pravastatin pharmacokinetics.

OBJECTIVE: Our objective was to quantitate the contribution of the genetic polymorphisms of the genes for 2 human organic anion transporters-organic anion transporting polypeptide C (OATP-C) and organic anion transporter 3 (OAT3)-to the pharmacokinetics of pravastatin. METHODS: Genetic polymorphisms were screened by polymerase chain reaction-single-strand conformation polymorphism analysis, after sequencing with deoxyribonucleic acid obtained from 120 healthy volunteers. To examine whether polymorphisms in these 2 genes of interest alter transport activity, we conducted a clinical study (n = 23) with pravastatin as a selective probe drug. RESULTS: Among 120 healthy individuals, 5 nonsynonymous variants and 1 nonsynonymous variant were observed in the OATP-C and OAT3 genes, respectively. The polymorphisms in the OAT3 gene did not appear to be associated with changes in renal and tubular secretory clearance. In contrast, the OATP-C variants were associated with differences in the disposition kinetics of pravastatin. Subjects with the OATP-C*15 allele (Asp130Ala174) had a reduced total and nonrenal clearance, as compared with those with the OATP-C*1b allele (Asp130Val174); nonrenal clearance values in *1b/*1b (n = 4), *1b/*15 (n = 9), and *15/*15 (n = 1) subjects were 2.01 +/- 0.42 L. kg(-1). h(-1), 1.11 +/- 0.34 L. kg(-1). h(-1), and 0.29 L. kg(-1). h(-1), respectively, and the difference between *1b/*1b and *1b/*15 subjects was significant (P <.05). CONCLUSION: Certain commonly occurring single-nucleotide polymorphisms in OATP-C, such as T521C (Val174Ala), are likely to be associated with altered pharmacokinetics of pravastatin. Large clinical studies are needed to confirm these observations.