Insights into Praziquantel Metabolism and Potential Enantiomeric Cytochrome P450-Mediated Drug-Drug Interaction.

The active enantiomer R-Praziquantel (PZQ) shows a clinically lower relative exposure when administered enantiomerically pure compared with a racemic form. We investigated the hypothesis that enantiomer-enantiomer interactions on cytochrome P450 (P450) enzymes could explain this observation and aimed to further deepen the understanding of PZQ metabolism. First, in an in vitro metabolite profiling study, the formation of multiple metabolites per P450, together with an observed interconversion of cis-4'-OH-PZQ to trans-4'-OH-PZQ in human hepatocytes, pointed out the inadequacy of measuring metabolite formation in kinetic studies. Thus, a substrate depletion approach to study PZQ enantiomeric interactions was applied. Second, an abundant CYP3A4 metabolite found in previous studies was structurally characterized. Third, substrate depletion methodologies were applied to determine P450 enzyme kinetics of PZQ and to further estimate enantiomer-enantiomer inhibitory parameters. A competitive inhibition between PZQ enantiomers for CYP2C9, 2C19, 3A4, and 3A5 was revealed. Analyses considering the clearance of only one or both enantiomers provided comparable enantiomer-enantiomer inhibition estimates. To conclude, this paper provides new insights into PZQ metabolic profile to enable a better understanding of enantioselective pharmacokinetics using substrate depletion-based methods. SIGNIFICANCE STATEMENT: In this study, enantiomer-enantiomer interactions of praziquantel on cytochrome P450 metabolizing enzymes are investigated via substrate depletion measurement using modeling methods. Together with new insights into the praziquantel metabolism, this work provides a novel data set to understand its pharmacokinetics.

A Decade in the MIST: Learnings from Investigations of Drug Metabolites in Drug Development under the "Metabolites in Safety Testing" Regulatory Guidance.

Since the introduction of metabolites in safety testing (MIST) guidance by the Food and Drug Administration in 2008, major changes have occurred in the experimental methods for the identification and quantification of metabolites, ways to evaluate coverage of metabolites, and the timing of critical clinical and nonclinical studies to generate this information. In this cross-industry review, we discuss how the increased focus on human drug metabolites and their potential contribution to safety and drug-drug interactions has influenced the approaches taken by industry for the identification and quantitation of human drug metabolites. Before the MIST guidance was issued, the method of choice for generating comprehensive metabolite profile was radio chromatography. The MIST guidance increased the focus on human drug metabolites and their potential contribution to safety and drug-drug interactions and led to changes in the practices of drug metabolism scientists. In addition, the guidance suggested that human metabolism studies should also be accelerated, which has led to more frequent determination of human metabolite profiles from multiple ascending-dose clinical studies. Generating a comprehensive and quantitative profile of human metabolites has become a more urgent task. Together with technological advances, these events have led to a general shift of focus toward earlier human metabolism studies using high-resolution mass spectrometry and to a reduction in animal radiolabel absorption/distribution/metabolism/excretion studies. The changes induced by the MIST guidance are highlighted by six case studies included herein, reflecting different stages of implementation of the MIST guidance within the pharmaceutical industry.

The impact of early human data on clinical development: there is time to win.

Modern accelerator mass spectrometry (AMS) methods enable the routine application of this technology in drug development. By the administration of a (14)C-labelled microdose or microtrace, pharmacokinetic (PK) data, such as mass balance, metabolite profiling, and absolute bioavailability (AB) data, can be generated easier, faster, and at lower costs. Here, we emphasize the advances and impact of this technology for pharmaceutical companies. The availability of accurate intravenous (iv) PK and human absorption, distribution, metabolism, and excretion (ADME) information, even before or during Phase I trials, can improve the clinical development plan. Moreover, applying the microtrace approach during early clinical development might impact the number of clinical pharmacology and preclinical safety pharmacology studies required, and shorten the overall drug discovery program.

ADME studies of [5-(3)H]-2'-O-methyluridine nucleoside in mice: a building block in siRNA therapeutics.

The chemical modification 2'-O-methyl of nucleosides is often used to increase siRNA stability towards nuclease activities. However, the metabolic fate of modified nucleosides remains unclear. Therefore, the aim of this study was to determine the mass balance, pharmacokinetic, and absorption, distribution, metabolism, and excretion (ADME)-properties of tritium-labeled 2'-O-methyluridine, following a single intravenous dose to male CD-1 mice. The single intravenous administration of [5-(3)H]-2'-O-methyluridine was well tolerated in mice. Radioactivity was rapidly and widely distributed throughout the body and remained detectable in all tissues investigated throughout the observation period of 48 h. After an initial rapid decline, blood concentrations of total radiolabeled components declined at a much slower rate. [(3)H]-2'-O-Methyluridine represented a minor component of the radioactivity in plasma (5.89% of [(3)H]-AUC 0-48 h). Three [(3)H]-2'-O-methyluridine metabolites namely uridine (M1), cytidine (M2), and uracil (M3) were the major circulating components representing 32.8%, 8.11%, and 23.6% of radioactivity area under the curve, respectively. The highest concentrations of total radiolabeled components and exposures were observed in kidney, spleen, pineal body, and lymph nodes. The mass balance, which is the sum of external recovery of radioactivity in excreta and remaining radioactivity in carcass and cage wash, was complete. Renal excretion accounted for about 52.7% of the dose with direct renal excretion of the parent in combination with metabolism to the endogenous compounds cytidine, uracil, cytosine, and cytidine.

Absorption, distribution, metabolism, and excretion of [(14)C]BYL719 (alpelisib) in healthy male volunteers.

PURPOSE: To determine the pharmacokinetics of the p110α-selective inhibitor alpelisib (BYL719) in humans, to identify metabolites in plasma and excreta, and to characterize pathways of biotransformation. METHODS: Four healthy male volunteers received a single oral dose of [(14)C]-labeled alpelisib (400 mg, 2.78 MBq). Blood, urine, and feces samples were collected throughout the study. Total radioactivity was measured by liquid scintillation counting, and metabolites were quantified and identified by radiometry and mass spectrometry. Complementary in vitro experiments characterized the hydrolytic, oxidative, and conjugative enzymes involved in metabolite formation. RESULTS: Over 50 % of [(14)C] alpelisib was absorbed, with a T(max) of 2 h and an elimination half-life from plasma of 13.7 h. Over the first 12 h, exposure to alpelisib and the primary metabolite M4 was 67.9 and 26.7 % of total drug-related material in circulation, respectively. Mass balance was achieved, with 94.5 % of administered radioactivity recovered in excreta. In total, 38.2 % of alpelisib was excreted unchanged, while 39.5 % was excreted as M4. Based on the excreta pools analyzed, excretion occurred mainly via feces (79.8 % of administered dose); 13.1 % was excreted via urine. In vitro experiments showed that spontaneous and enzymatic hydrolysis contributed to M4 formation, while CYP3A4-mediated oxidation and UGT1A9-mediated glucuronidation formed minor metabolites. Alpelisib was well tolerated, and no new safety concerns were raised during this study. CONCLUSIONS: Alpelisib was rapidly absorbed and cleared by multiple metabolic pathways; the primary metabolite M4 is pharmacologically inactive. Alpelisib has limited potential for drug-drug interactions and is therefore a promising candidate for combination therapy.

Phenotypic and metabolic investigation of a CSF-1R kinase receptor inhibitor (BLZ945) and its pharmacologically active metabolite.

1. 4-[2((1R,2R)-2-Hydroxycyclohexylamino)-benzothiazol-6-yloxyl]-pyridine-2-carboxylic acid methylamide (BLZ945) is a small molecule inhibitor of CSF-1R kinase activity within osteoclasts designed to prevent skeletal related events in metastatic disease. Key metabolites were enzymatically and structurally characterized to understand the metabolic fate of BLZ945 and pharmacological implications. The relative intrinsic clearances for metabolites were derived from in vitro studies using human hepatocytes, microsomes and phenotyped with recombinant P450 enzymes. 2. Formation of a pharmacologically active metabolite (M9) was observed in human hepatocytes. The M9 metabolite is a structural isomer (diastereomer) of BLZ945 and is about 4-fold less potent. This isomer was enzymatically formed via P450 oxidation of the BLZ945 hydroxyl group, followed by aldo-keto reduction to the alcohol (M9). 3. Two reaction phenotyping approaches based on fractional clearances were applied to BLZ945 using hepatocytes and liver microsomes. The fraction metabolized (fm) or contribution ratio was determined for each metabolic reaction type (oxidation, glucuronidation or isomerization) as well as for each metabolite. The results quantitatively illustrate contribution ratios of the involved enzymes and pathways, e.g. the isomerization to metabolite M9 accounted for 24% intrinsic clearance in human hepatocytes. In summary, contribution ratios for the Phase I and Phase II pathways can be determined in hepatocytes.

Phase II metabolism in human skin: skin explants show full coverage for glucuronidation, sulfation, N-acetylation, catechol methylation, and glutathione conjugation.

Although skin is the largest organ of the human body, cutaneous drug metabolism is often overlooked, and existing experimental models are insufficiently validated. This proof-of-concept study investigated phase II biotransformation of 11 test substrates in fresh full-thickness human skin explants, a model containing all skin cell types. Results show that skin explants have significant capacity for glucuronidation, sulfation, N-acetylation, catechol methylation, and glutathione conjugation. Novel skin metabolites were identified, including acyl glucuronides of indomethacin and diclofenac, glucuronides of 17ß-estradiol, N-acetylprocainamide, and methoxy derivatives of 4-nitrocatechol and 2,3-dihydroxynaphthalene. Measured activities for 10 µM substrate incubations spanned a 1000-fold: from the highest 4.758 pmol·mg skin(-1)·h(-1) for p-toluidine N-acetylation to the lowest 0.006 pmol·mg skin(-1)·h(-1) for 17ß-estradiol 17-glucuronidation. Interindividual variability was 1.4- to 13.0-fold, the highest being 4-methylumbelliferone and diclofenac glucuronidation. Reaction rates were generally linear up to 4 hours, although 24-hour incubations enabled detection of metabolites in trace amounts. All reactions were unaffected by the inclusion of cosubstrates, and freezing of the fresh skin led to loss of glucuronidation activity. The predicted whole-skin intrinsic metabolic clearances were significantly lower compared with corresponding whole-liver intrinsic clearances, suggesting a relatively limited contribution of the skin to the body's total systemic phase II enzyme-mediated metabolic clearance. Nevertheless, the fresh full-thickness skin explants represent a suitable model to study cutaneous phase II metabolism not only in drug elimination but also in toxicity, as formation of acyl glucuronides and sulfate conjugates could play a role in skin adverse reactions.

Aldehyde oxidase activity in fresh human skin.

Human aldehyde oxidase (AO) is a molybdoflavoenzyme that commonly oxidizes azaheterocycles in therapeutic drugs. Although high metabolic clearance by AO resulted in several drug failures, existing in vitro-in vivo correlations are often poor and the extrahepatic role of AO practically unknown. This study investigated enzymatic activity of AO in fresh human skin, the largest organ of the body, frequently exposed to therapeutic drugs and xenobiotics. Fresh, full-thickness human skin was obtained from 13 individual donors and assayed with two specific AO substrates: carbazeran and zoniporide. Human skin explants from all donors metabolized carbazeran to 4-hydroxycarbazeran and zoniporide to 2-oxo-zoniporide. Average rates of carbazeran and zoniporide hydroxylations were 1.301 and 0.164 pmol⋅mg skin(-1)⋅h(-1), resulting in 13 and 2% substrate turnover, respectively, after 24 hours of incubation with 10 µM substrate. Hydroxylation activities for the two substrates were significantly correlated (r(2) = 0.769), with interindividual variability ranging from 3-fold (zoniporide) to 6-fold (carbazeran). Inclusion of hydralazine, an irreversible inhibitor of AO, resulted in concentration-dependent decrease of hydroxylation activities, exceeding 90% inhibition of carbazeran 4-hydroxylation at 100 µM inhibitor. Reaction rates were linear up to 4 hours and well described by Michaelis-Menten enzyme kinetics. Comparison of carbazeran and zoniporide hydroxylation with rates of triclosan glucuronidation and sulfation and p-toluidine N-acetylation showed that cutaneous AO activity is comparable to tested phase II metabolic reactions, indicating a significant role of AO in cutaneous drug metabolism. To our best knowledge, this is the first report of AO enzymatic activity in human skin.

An iron stable isotope comparison between human erythrocytes and plasma.

We present precise iron stable isotope ratios measured by multicollector-ICP mass spectrometry (MC-ICP-MS) of human red blood cells (erythrocytes) and blood plasma from 12 healthy male adults taken during a clinical study. The accurate determination of stable isotope ratios in plasma first required substantial method development work, as minor iron amounts in plasma had to be separated from a large organic matrix prior to mass-spectrometric analysis to avoid spectroscopic interferences and shifts in the mass spectrometer's mass-bias. The (56)Fe/(54)Fe ratio in erythrocytes, expressed as permil difference from the "IRMM-014" iron reference standard (δ(56/54)Fe), ranges from -3.1‰ to -2.2‰, a range typical for male Caucasian adults. The individual subject erythrocyte iron isotope composition can be regarded as uniform over the 21 days investigated, as variations (±0.059 to ±0.15‰) are mostly within the analytical precision of reference materials. In plasma, δ(56/54)Fe values measured in two different laboratories range from -3.0‰ to -2.0‰, and are on average 0.24‰ higher than those in erythrocytes. However, this difference is barely resolvable within one standard deviation of the differences (0.22‰). Taking into account the possible contamination due to hemolysis (iron concentrations are only 0.4 to 2 ppm in plasma compared to approx. 480 ppm in erythrocytes), we model the pure plasma δ(56/54)Fe to be on average 0.4‰ higher than that in erythrocytes. Hence, the plasma iron isotope signature lies between that of the liver and that of erythrocytes. This difference can be explained by redox processes involved during cycling of iron between transferrin and ferritin.

Iron uptake and ferrokinetics in healthy male subjects of an iron-based oral phosphate binder (SBR759) labeled with the stable isotope (58)Fe.

SBR759 is a novel polynuclear iron(III) oxide-hydroxide starch·sucrose·carbonate complex being developed for oral use in chronic kidney disease (CKD) patients with hyperphosphatemia on hemodialysis. SBR759 binds inorganic phosphate released by food uptake and digestion in the gastro-intestinal tract increasing the fecal excretion of phosphate with concomitant reduction of serum phosphate concentrations. Considering the high content of ∼20% w/w covalently bound iron in SBR759 and expected chronic administration to patients, absorption of small amounts of iron released from the drug substance could result in potential iron overload and toxicity. In a mechanistic iron uptake study, 12 healthy male subjects (receiving comparable low phosphorus-containing meal typical for CKD patients: ≤1000 mg phosphate per day) were treated with 12 g (divided in 3 × 4 g) of stable (58)Fe isotope-labeled SBR759. The ferrokinetics of [(58)Fe]SBR759-related total iron was followed in blood (over 3 weeks) and in plasma (over 26 hours) by analyzing with high precision the isotope ratios of the natural iron isotopes (58)Fe, (57)Fe, (56)Fe and (54)Fe by multi-collector inductively coupled mass spectrometry (MC-ICP-MS). Three weeks following dosing, the subjects cumulatively absorbed on average 7.8 ± 3.2 mg (3.8-13.9 mg) iron corresponding to 0.30 ± 0.12% (0.15-0.54%) SBR759-related iron which amounts to approx. 5-fold the basal daily iron absorption of 1-2 mg in humans. SBR759 was well-tolerated and there was no serious adverse event and no clinically significant changes in the iron indices hemoglobin, hematocrit, ferritin concentration and transferrin saturation.

Absorption, distribution, metabolism, and excretion (ADME) of ¹4C-sonidegib (LDE225) in healthy volunteers.

PURPOSE: The absorption, distribution, metabolism, and excretion of the hedgehog pathway inhibitor sonidegib (LDE225) were determined in healthy male subjects. METHODS: Six subjects received a single oral dose of 800 mg ¹4C-sonidegib (74 kBq, 2.0 µCi) under fasting conditions. Blood, plasma, urine, and fecal samples were collected predose, postdose in-house (days 1-22), and during 24-h visits (weekly, days 29-43; biweekly, days 57-99). Radioactivity was determined in all samples using accelerator mass spectrometry (AMS). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine concentrations of sonidegib and its main circulating metabolite in plasma. Metabolite profiles and structures were determined in pooled plasma, urine, and fecal samples using high-performance LC-AMS and LC-MS/MS, respectively. RESULTS: A single dose of ¹4C-sonidegib was well tolerated in healthy subjects. Unchanged sonidegib and total radioactivity reached peak concentration in plasma by 2 and 3 h, respectively, and demonstrated similarly long half-lives of 319 and 331 h, respectively. Absorbed sonidegib (estimated 6-7 %) was extensively distributed, and the approximate terminal volume of distribution was 2,500 L. Unchanged sonidegib and a metabolite resulting from amide hydrolysis were the major circulating components (36.4 and 15.4 % of radioactivity area under the curve, respectively). Absorbed sonidegib was eliminated predominantly through oxidative metabolism of the morpholine part and amide hydrolysis. Unabsorbed sonidegib was excreted through the feces. Metabolites in excreta accounted for 4.49 % of the dose (1.20 % in urine, 3.29 % in feces). The recovery of radioactivity in urine and feces was essentially complete (95.3 ± 1.93 % of the dose in five subjects; 56.9 % of the dose in one subject with incomplete feces collection suspected). CONCLUSIONS: Sonidegib exhibited low absorption, was extensively distributed, and was slowly metabolized. Elimination of absorbed sonidegib occurred largely by oxidative and hydrolytic metabolism.

Biodistribution and metabolism studies of lipid nanoparticle-formulated internally [3H]-labeled siRNA in mice.

Absorption, distribution, metabolism, and excretion properties of a small interfering RNA (siRNA) formulated in a lipid nanoparticle (LNP) vehicle were determined in male CD-1 mice following a single intravenous administration of LNP-formulated [(3)H]-SSB siRNA, at a target dose of 2.5 mg/kg. Tissue distribution of the [(3)H]-SSB siRNA was determined using quantitative whole-body autoradiography, and the biostability was determined by both liquid chromatography mass spectrometry (LC-MS) with radiodetection and reverse-transcriptase polymerase chain reaction techniques. Furthermore, the pharmacokinetics and distribution of the cationic lipid (one of the main excipients of the LNP vehicle) were investigated by LC-MS and matrix-assisted laser desorption ionization mass spectrometry imaging techniques, respectively. Following i.v. administration of [(3)H]-SSB siRNA in the LNP vehicle, the concentration of parent guide strand could be determined up to 168 hours p.d. (post dose), which was ascribed to the use of the vehicle. This was significantly longer than what was observed after i.v. administration of the unformulated [(3)H]-SSB siRNA, where no intact parent guide strand could be observed 5 minutes post dosing. The disposition of the siRNA was determined by the pharmacokinetics of the formulated LNP vehicle itself. In this study, the radioactivity was widely distributed throughout the body, and the total radioactivity concentration was determined in selected tissues. The highest concentrations of radioactivity were found in the spleen, liver, esophagus, stomach, adrenal, and seminal vesicle wall. In conclusion, the LNP vehicle was found to drive the kinetics and biodistribution of the SSB siRNA. The renal clearance was significantly reduced and its exposure in plasma significantly increased compared with the unformulated [(3)H]-SSB siRNA.

Metabolism and disposition of the metabotropic glutamate receptor 5 antagonist (mGluR5) mavoglurant (AFQ056) in healthy subjects.

The disposition and biotransformation of (14)C-radiolabeled mavoglurant were investigated in four healthy male subjects after a single oral dose of 200 mg. Blood, plasma, urine, and feces collected over 7 days were analyzed for total radioactivity, mavoglurant was quantified in plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and metabolite profiles were generated in plasma and excreta by high-performance liquid chromatography (HPLC) and radioactivity detection. The chemical structures of mavoglurant metabolites were characterized by LC-MS/MS, wet-chemical and enzymatic methods, NMR spectroscopy, and comparison with reference compounds. Mavoglurant was safe and well tolerated in this study population. Mavoglurant absorption was ≥50% of dose reaching mean plasma Cmax values of 140 ng/ml (mavoglurant) and 855 ng-eq/ml (total radioactivity) at 2.5 and 3.6 hours, respectively. Thereafter, mavoglurant and total radioactivity concentrations declined with mean apparent half-lives of 12 and 18 hours, respectively. The elimination of mavoglurant occurred predominantly by oxidative metabolism involving primarily 1) oxidation of the tolyl-methyl group to a benzyl-alcohol metabolite (M7) and subsequently to a benzoic acid metabolite (M6), and 2) oxidation of the phenyl-ring leading to a hydroxylated metabolite (M3). The subjects were mainly exposed to mavoglurant and seven main metabolites, which combined accounted for 60% of (14)C-AUC0-72 h (area under the concentration-time curve from time 0 to infinity). The primary steps of mavoglurant metabolism observed in vivo could partially be reproduced in vitro in incubations with human liver microsomes and recombinant cytochrome P450 enzymes. After 7 days, the mean balance of total radioactivity excretion was almost complete (95.3% of dose) with 36.7% recovered in urine and 58.6% in feces.

Metabolism studies of unformulated internally [3H]-labeled short interfering RNAs in mice.

Absorption, distribution, metabolism, and excretion properties of two unformulated model short interfering RNA (siRNAs) were determined using a single internal [(3)H]-radiolabeling procedure, in which the full-length oligonucleotides were radiolabeled by Br/(3)H -exchange. Tissue distribution, excretion, and mass balance of radioactivity were investigated in male CD-1 mice after a single intravenous administration of the [(3)H]siRNAs, at a target dose level of 5 mg/kg. Quantitative whole-body autoradiography and liquid scintillation counting techniques were used to determine tissue distribution. Radiochromatogram profiles were determined in plasma, tissue extracts, and urine. Metabolites were separated by liquid chromatography and identified by radiodetection and high-resolution accurate mass spectrometry. In general, there was little difference in the distribution of total radiolabeled components after administration of the two unformulated [(3)H]siRNAs. The radioactivity was rapidly and widely distributed throughout the body and remained detectable in all tissues investigated at later time points (24 and 48 hours for [(3)H]MRP4 (multidrug resistance protein isoform 4) and [(3)H]SSB (Sjögren Syndrome antigen B) siRNA, respectively). After an initial rapid decrease, concentrations of total radiolabeled components in dried blood decreased at a much slower rate. A nearly complete mass balance was obtained for the [(3)H]SSB siRNA, and renal excretion was the main route of elimination (38%). The metabolism of the two model siRNAs was rapid and extensive. Five minutes after administration, no parent compound could be detected in plasma. Instead, radiolabeled nucleosides resulting from nuclease hydrolysis were observed. In the metabolism profiles obtained from various tissues, only radiolabeled nucleosides were found, suggesting that siRNAs are rapidly metabolized and that the distribution pattern of total radiolabeled components can be ascribed to small molecular weight metabolites.

Metabolism of patupilone in patients with advanced solid tumor malignancies.

A phase 1, open-label, non-randomized, single center study was conducted to determine the pharmacokinetics, distribution, metabolism, elimination, and mass balance of patupilone in patients with advanced solid tumors. Five patients with advanced solid tumors received 10 mg/m(2) (1.1 MBq) of (14) C-radiolabeled patupilone at cycle 1 as a 20-minute intravenous infusion every 3 weeks until disease progression. Sequential samples of blood/plasma were taken for 3 weeks and urine and fecal samples were collected for seven days after the first dose of patupilone. Patupilone blood levels decreased rapidly after the infusion. The compound showed a large volume of distribution (Vss: 2242 L). The main radiolabeled component in blood was patupilone itself, accompanied by the lactone hydrolysis products that are unlikely to contribute to the pharmacological effect of patupilone. The blood clearance of patupilone was relatively low at 14 L/h. The administered radioactivity dose was excreted slowly (46 % of dose up to 168 h) but ultimately accounted for 91 % of the dose by extrapolation. The fecal excretion of radioactivity was 2-3 times higher than the urinary excretion consistent with hepato-biliary elimination. Three patients had progressive disease and two patients had stable disease as their best response. Patupilone was generally well tolerated in patients with advanced solid tumors with no newly occurring safety events compared to previous clinical studies. In adult solid tumor patients, intravenous radiolabeled patupilone undergoes extensive metabolism with fecal excretion of radioactive metabolites predominating over renal excretion.

A unique automation platform for measuring low level radioactivity in metabolite identification studies.

Generation and interpretation of biotransformation data on drugs, i.e. identification of physiologically relevant metabolites, defining metabolic pathways and elucidation of metabolite structures, have become increasingly important to the drug development process. Profiling using (14)C or (3)H radiolabel is defined as the chromatographic separation and quantification of drug-related material in a given biological sample derived from an in vitro, preclinical in vivo or clinical study. Metabolite profiling is a very time intensive activity, particularly for preclinical in vivo or clinical studies which have defined limitations on radiation burden and exposure levels. A clear gap exists for certain studies which do not require specialized high volume automation technologies, yet these studies would still clearly benefit from automation. Use of radiolabeled compounds in preclinical and clinical ADME studies, specifically for metabolite profiling and identification are a very good example. The current lack of automation for measuring low level radioactivity in metabolite profiling requires substantial capacity, personal attention and resources from laboratory scientists. To help address these challenges and improve efficiency, we have innovated, developed and implemented a novel and flexible automation platform that integrates a robotic plate handling platform, HPLC or UPLC system, mass spectrometer and an automated fraction collector.

Pharmacokinetic interaction of solifenacin with an oral contraceptive containing ethinyl estradiol and levonorgestrel in healthy women: a double-blind, placebo-controlled study.

BACKGROUND: Solifenacin succinate (YM905; Vesicare, Astellas Pharma Inc., Tokyo, Japan) is a new once-daily, orally administered muscarinic receptor antagonist under investigation for the treatment of overactive bladder. OBJECTIVE: The aim of this study was to evaluate the effect of solifenacin on the pharmacokinetic (PK) parameters of an oral contraceptive (OC) containing ethinyl estradiol (EE) and levonorgestrel (LNG). METHODS: In a double-blind, placebo-controlled, 2-period, crossover study, 24 healthy, young, white women received a combined OC (EE 30 microg + LNG 150 microg) daily for two 21-day cycles, separated by a 7-day washout. On day 12 of each cycle, subjects began a 10-day regimen of solifenacin 10 mg QD, which is 2 times the suggested starting dose, or placebo. Subjects crossed over to the other treatment arm for the second cycle. Primary PK end points were C(max) and AUC from time 0 to 24 hours (AUC(0-24 h)) for EE and LNG. Women ranged in age from 20 to 37 years and had a mean body weight of 64 kg, mean height of 167.4 cm, and mean body mass index of 23 kg/m2. Seven women had never smoked, while 5 were former smokers and 12 were regular smokers. Safety assessments included the nature, frequency, and severity of spontaneously reported or observed adverse events, vital signs, electrocardiogram, laboratory values, and physical examination. RESULTS: Statistical analysis of AUC(0-24 h)/product of baseline concentration and total blood sampling time, and C(max)/baseline concentration ratios of solifenacin versus placebo for EE and LNG found the 90% CI to be within the predefined range of 0.8 to 1.25 (EE: 0.854-1.164 and 0.822-1.167; LNG: 0.920-1.125 and 0.910-1.139). The number of samples with non-quantifiable luteinizing hormone (LH) and folliclestimulating hormone (FSH) levels were comparable after administration of the OC with either solifenacin or placebo. The adverse event most frequently reported was dry mouth (solifenacin, n = 25 [9 mild, 13 moderate, and 3 severe] vs placebo, n = 1 [moderate]). There were no clinically relevant effects on vital signs, electrocardiogram, or laboratory parameters. CONCLUSIONS: A PK interaction between solifenacin and the OC containing EE and LNG was not found in this study. Solifenacin was not found to have altered suppression of LH or FSH. The drug was well tolerated in these healthy, young, white, adult female volunteers.