Defining reality: the potential role of pharmacists in assessing the impact of progesterone receptor modulators and misoprostol in reproductive health.

Medical abortion is increasingly heralded as an ideal method for decreasing maternal mortality in health-care resource-deprived areas and as an answer to the shrinking pool of physicians willing to perform abortions. The advent of progesterone receptor modulators (PRMs) and the recent approval by the Food and Drug Administration of ella (ulipristal) as an emergency contraceptive put pharmacists in the center of abortion controversy. Pharmacists, worldwide, need to be aware of the controversy surrounding the introduction of PRMs, particularly with regard to the effect on health policy, their mechanism of action, associated adverse events, and common off-label uses. Once understood, genuine opportunity exists for pharmacists to serve a fundamental role in positively shaping public health policy.

A multiyear comparison of flipped- vs. lecture-based teaching on student success in a pharmaceutical science class.

BACKGROUND AND PURPOSE: To gauge the potential effect of mode of content delivery on overall student success in a pharmaceutical sciences course in a doctor of pharmacy program. EDUCATIONAL ACTIVITY AND SETTING: Principles of Drug Action I (PDAI) is a first-year pharmaceutical science course typically taught by multiple faculty, and each utilizes their own approach to deliver course content. Over a seven year period, the course naturally separated into blocks. Block-1 was taught using a traditional lecture-based approach while Block-2 varied between either a lecture-based or a flipped-classroom format. Student success was evaluated by exam at the end of each block. FINDINGS: For the four years in which Block-2 was taught by lecture, the number of exam failures was similar to Block-1. For each of the three years Block-2 was taught via the flipped-classroom format, the number of exam failures was approximately half that of Block-1. While grades for the lecture-based Block-1 trended downward over the seven-year evaluation period, average exam grades overall were similar between Block-1 and Block-2 each year regardless of teaching modality. SUMMARY: Retrospective analysis of this novel blocked approach within PDAI provided a means of internally evaluating the potential effect of teaching format on overall student performance. The results described here support previous studies that indicate that the flipped-classroom approach can reduce course failures. These findings also show that flipped-classroom teaching may have a greater impact on improving learning in lower-performing students.

Comparative metabolism of 14C-labeled apixaban in mice, rats, rabbits, dogs, and humans.

The metabolism and disposition of [(14)C]apixaban, a potent, reversible, and direct inhibitor of coagulation factor Xa, were investigated in mice, rats, rabbits, dogs, and humans after a single oral administration and in incubations with hepatocytes. In plasma, the parent compound was the major circulating component in mice, rats, dogs, and humans. O-Demethyl apixaban sulfate (M1) represented approximately 25% of the parent area under the time curve in human plasma. This sulfate metabolite was present, but in lower amounts relative to the parent, in plasma from mice, rats, and dogs. Rabbits showed a plasma metabolite profile distinct from that of other species with apixaban as a minor component and M2 (O-demethyl apixaban) and M14 (O-demethyl apixaban glucuronide) as prominent components. The fecal route was a major elimination pathway, accounting for >54% of the dose in animals and >46% in humans. The urinary route accounted for <15% of the dose in animals and 25 to 28% in humans. Apixaban was the major component in feces of every species and in urine of all species except rabbit. M1 and M2 were common prominent metabolites in urine and feces of all species as well as in bile of rats and humans. In vivo metabolite profiles showed quantitative differences between species and from in vitro metabolite profiles, but all human metabolites were found in animal species. After intravenous administration of [(14)C]apixaban to bile duct-cannulated rats, the significant portion (approximately 22%) of the dose was recovered as parent drug in the feces, suggesting direct excretion of the drug from gastrointestinal tracts of rats. Overall, apixaban was effectively eliminated via multiple elimination pathways in animals and humans, including oxidative metabolism, and direct renal and intestinal excretion.

Subchronic urinary bladder effects of muraglitazar in male rats.

Muraglitazar, a PPARalpha/gamma dual agonist, was dosed orally to rats once daily for 13 weeks to evaluate urinary and urothelial changes of potential relevance to urinary bladder tumorigenesis. Groups of 17 young or aged rats per sex were fed a normal or 1% NH4Cl-supplemented diet and were dosed with 0, 1, or 50 mg/kg muraglitazar. Lithogenic ions and sediment were profiled from freshly voided urine samples collected 24 h after dosing, and drug exposures were measured. Urinary citrate, oxalate, and epidermal growth factor (EGF) were assayed from 18-h urine collections. Urothelium was assessed by light microscopy, scanning electron microscopy, and BrdU and TUNEL immunohistochemistry. When fed a normal diet, urine pH was higher in males (above 6.5). Urine volume/body weight was greater in females. Urine soluble/total calcium and magnesium and phosphorus/creatinine ratios were lower in male rats fed a normal diet. Urine citrate levels were decreased and oxalate was increased in young male rats treated with 50 mg/kg muraglitazar compared to age/sex/diet-matched controls. No changes in urine sediment were detected 24 h after dosing. In young male rats treated with 50 mg/kg on normal diet, multifocal urothelial necrosis and proliferation were observed, whereas urothelial apoptosis and urine EGF levels were unchanged compared to age/sex/diet-matched controls. Urothelial necrosis and proliferation were not correlated to systemic or urinary drug exposures and were prevented by dietary acidification. These data suggest that muraglitazar-associated changes in urine composition predispose to urothelial cytotoxicity and proliferation in the urinary bladder of young male rats and that urine sediment must be profiled at multiple daily timepoints to fully qualify drug-induced changes in urine composition.

Utility of imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization (MALDI) on an ion trap mass spectrometer in the analysis of drugs and metabolites in biological tissues.

INTRODUCTION: The properties and potential liabilities of drug candidate are investigated in detailed ADME assays and in toxicity studies, where findings are placed in context of exposure to dosed drug and metabolites. The complex nature of biological samples may necessitate work-up procedures prior to high performance liquid chromatography-mass spectrometric (HPLC-MS) analysis of endogenous or xenobiotic compounds. This concept can readily be applied to biological fluids such as blood or urine, but in localized samples such as organs and tissues potentially important spatial, thus anatomical, information is lost during sample preparation as the result of homogenization and extraction procedures. However, the localization of test article or spatial identification of metabolites may be critical to the understanding of the mechanism of target-organ toxicity and its relevance to clinical safety. METHODS: Tissue imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization (MALDI) and ion trap mass spectrometry (MS) with higher order mass spectrometric scanning functions was utilized for localization of dosed drug or metabolite in tissue. Laser capture microscopy (LCM) was used to obtain related samples from tissue for analyses by standard MALDI-MS and HPLC-MS. RESULTS: In a toxicology study, rats were administered with a high dosage of a prodrug for 2 weeks. Birefringent microcrystalline material (10-25 microm) was observed in histopathologic formalin-fixed tissue samples. Direct analysis by IMS provided the identity of material in the microcrystals as circulating active drug while maintaining spatial orientation. Complementary data from visual cross-polarized light microscopy as well as standard MALDI-MS and HPLC-MS experiments on LCM samples validated the qualitative results obtained by IMS. Furthermore, the HPLC-MS analysis on the LCM samples afforded a semi-quantitative assessment of the crystalline material in the tissue samples. DISCUSSION: IMS by MALDI ion trap MS proved sensitive, specific, and highly amenable to the image analysis of traditional small molecule drug candidates directly in tissue.

In vitro and in vivo metabolism of a gamma-secretase inhibitor BMS-299897 and generation of active metabolites in milligram quantities with a microbial bioreactor.

BMS-299897 is a gamma-secretase inhibitor that has the potential for treatment of Alzheimer's disease. The metabolism of [(14)C]BMS-299897 was investigated in human liver microsomes, in rat, dog, monkey and human hepatocytes and in bile duct cannulated rats. Seven metabolites (M1-M7) were identified from in vitro and in vivo studies. LC-MS/MS analysis showed that M1 and M2 were regioisomeric acylglucuronide conjugates of BMS-299897. Metabolites M3, M4 and M6 were identified as monohydroxylated metabolites of BMS-299897 and M5 was identified as the dehydrogenated product of monooxygenated BMS-299897. In vivo, 52% of the radioactive dose was excreted in bile within 0-6 h from bile duct cannulated rats following a single oral dose of 15 mg/kg of [(14)C]BMS-299897. Glucuronide conjugates, M1 and M2 accounted for 80% of the total radioactivity in rat bile. In addition to M1 and M2, M7 was observed in rat bile which was identified as a glucuronide conjugate of an oxidative metabolite M5. For structure elucidation and pharmacological activity testing of the metabolites, ten microbial cultures were screened for their ability to metabolize BMS-299897 to form these metabolites. Among them, the fungus Cunninghamella elegans produced two major oxidative metabolites M3 and M4 that had the same HPLC retention time and mass spectral properties as those found in in vitro incubations. NMR analysis indicated that M3 and M4 were stereoisomers, with the hydroxyl group on the benzylic position. However, M3 and M4 were unstable and converted to their corresponding lactones readily. Based on x-ray analysis of the synthetically prepared lactone of M3, the stereochemistry of benzylic hydroxyl group was assigned as the R configuration. Both the hydroxy metabolites (M3 and M4) and the lactone of M3 showed gamma-secretase inhibition with IC(50) values similar to that of the parent compound. This study demonstrates the usefulness of microbial systems as bioreactors to generate metabolites of BMS-299897 in large quantities for structure elucidation and activity testing. This study also demonstrates the biotransformation profile of BMS-299897 is qualitatively similar across the species including rat, dog, monkey and human which provides a basis to support rat, dog and monkey as preclinical models for toxicological testing.

Analysis of low level radioactive metabolites in biological fluids using high-performance liquid chromatography with microplate scintillation counting: method validation and application.

TopCount, a microplate scintillation counter (MSC), has been recently employed as an off-line liquid radiochromatographic detector for radioactive metabolite profile analysis. The present study was undertaken to validate TopCount for metabolite profiling with respect to sensitivity, accuracy, precision and radioactivity recovery. Matrix effects of various human samples on TopCount performance and capability of MSC for volatile metabolite analysis were also investigated. TopCount had a limit of detection (LOD) of 5 DPM and a limit of quantification (LOQ) of 15 DPM for [(14)C]-labeled compounds at a 10min counting time. It was two-fold more sensitive than a liquid scintillation counter (LSC), and 50-100-fold more sensitive than a radioactivity flow detector (RFD). TopCount had comparable accuracy and precision to RFD, and comparable precision to LSC for determining relative abundance of metabolites. Human liver microsome incubation (up to 1 mL), plasma (up to 1 mL), urine (up to 2 mL) and feces (up to 50mg) had no significant quenching effects on TopCount performance. Benzoic acid, a volatile metabolite, was detected by TopCount, but not by Microbeta counter after microplates were dried under vacuum. Radioactivity recovery in HPLC-MSC analysis was reliably determined using an LSC-based method. Examples of using HPLC-MSC for analysis of low levels of radioactive metabolites are presented, including determination of plasma metabolite profile, in vitro reactive metabolites trapped by [(3)H]glutathione, and metabolite concentrations in an enzyme kinetic experiment. The data from this study strongly suggest that HPLC in combination with TopCount is a viable alternative analytical tool for detection and quantification of low levels of radioactive metabolites in biological fluids.

Comparative metabolism of radiolabeled muraglitazar in animals and humans by quantitative and qualitative metabolite profiling.

Muraglitazar (Pargluva), a dual alpha/gamma peroxisome proliferator-activated receptor (PPAR) activator, has both glucose- and lipid-lowering effects in animal models and in patients with diabetes. This study describes the in vivo and in vitro comparative metabolism of [(14)C]muraglitazar in rats, dogs, monkeys, and humans by quantitative and qualitative metabolite profiling. Metabolite identification and quantification methods used in these studies included liquid chromatography/mass spectrometry (LC/MS), LC/tandem MS, LC/radiodetection, LC/UV, and a newly described mass defect filtering technique in conjunction with high resolution MS. After oral administration of [(14)C]muraglitazar, absorption was rapid in all species, reaching a concentration peak for parent and total radioactivity in plasma within 1 h. The most abundant component in plasma at all times in all species was the parent drug, and no metabolite was present in greater than 2.5% of the muraglitazar concentrations at 1 h postdose in rats, dogs, and humans. All metabolites observed in human plasma were also present in rats, dogs, or monkeys. Urinary excretion of radioactivity was low (<5% of the dose) in all intact species, and the primary route of elimination was via biliary excretion in rats, monkeys, and humans. Based on recovered doses in urine and bile, muraglitazar showed a very good absorption in rats, monkeys, and humans. The major drug-related components in bile of rats, monkeys, and humans were glucuronides of muraglitazar and its oxidative metabolites. The parent compound was a minor component in bile, suggesting extensive metabolism of the drug. In contrast, the parent drug and oxidative metabolites were the major components in feces, and no glucuronide conjugates were found, suggesting that glucuronide metabolites were excreted in bile and hydrolyzed in the gastrointestinal tract. The metabolites of muraglitazar resulted from both glucuronidation and oxidation. The metabolites in general had greatly reduced activity as PPARalpha/gamma activators relative to muraglitazar. In conclusion, muraglitazar was rapidly absorbed, extensively metabolized through glucuronidation and oxidation, and mainly eliminated in the feces via biliary excretion of glucuronide metabolites in all species studied. Disposition and metabolic pathways were qualitatively similar in rats, dogs, monkeys, and humans.

Metabolism of [14C]gemopatrilat after oral administration to rats, dogs, and humans.

This study describes the pharmacokinetic parameters of gemopatrilat, a potent vasopeptidase inhibitor, in humans and the comparative biotransformation of the compound in rats, dogs, and humans after administration of a single oral dose of [14C]gemopatrilat. Gemopatrilat was rapidly absorbed in humans with an oral bioavailability of 49%. Within 5 h after dose, the mean concentrations of gemopatrilat were less than 1% of the mean Cmax values. The total area under the first-moment time curve extrapolated to infinity [AUC(INF)] value for gemopatrilat was only 2% of the AUC(INF) of radioactivity in plasma. Gemopatrilat showed a large apparent steady-state volume of distribution (2500 liters) and a prolonged terminal-phase decline in plasma concentration. These results are consistent with the idea that the free sulfhydryl group of gemopatrilat forms reversible disulfide linkages with plasma and tissue proteins and is thus eliminated from the body at a very slow rate. Approximately half of the drug-related radioactivity in 1-h plasma samples from rat, dog, and human was reduced chemically with dithiothreitol to gemopatrilat, suggesting that disulfide linkage occurred in all species. In addition, metabolites formed through S-methylation and amide hydrolysis were also detected in rat, dog, and human plasma. No gemopatrilat was detected in urine and fecal samples from all three species, indicating that the compound is extensively metabolized in vivo. The major metabolites identified in human urine and feces were also present in rat and dog. These data suggest that the metabolism of gemopatrilat in all three species were qualitatively very similar.

Cytochrome P450 3A-mediated metabolism of buspirone in human liver microsomes.

This study was carried out to determine the metabolic pathways of buspirone and cytochrome P450 (P450) isoform(s) responsible for buspirone metabolism in human liver microsomes (HLMs). Buspirone mainly underwent N-dealkylation to 1-pyrimidinylpiperazine (1-PP), N-oxidation on the piperazine ring to buspirone N-oxide (Bu N-oxide), and hydroxylation to 3'-hydroxybuspirone (3'-OH-Bu), 5-hydroxybuspirone (5-OH-Bu), and 6'-hydroxybuspirone (6'-OH-Bu) in HLMs. The apparent K(m) values for buspirone metabolite formation in pooled HLMs were 8.7 (1-PP), 34.0 (Bu N-oxide), 4.3 (3'-OH-Bu), 11.4/514 (5-OH-Bu), and 8.8 microM (6'-OH-Bu). CYP3A inhibitor ketoconazole (1 microM) completely inhibited the formation of all major metabolites in HLMs (0-16% of control), whereas the chemical inhibitor selective to other P450 isoforms had little or no inhibitory effect. Recombinant CYP3A4, CYP3A5, and CYP2D6 exhibited buspirone oxidation activities among nine P450 isoforms tested. The overall metabolism rate of 5 microM buspirone by CYP3A4 was 18-fold greater than that by CYP2D6 and 35-fold greater than that by CYP3A5. In a panel of HLMs from 16 donors, buspirone metabolism correlated well CYP3A activity (r2 = 0.85-0.96, rho < 0.0005), but not the activities of other P450 isoforms. The metabolism rates of buspirone in CYP2D6 poor-metabolizer genotype HLMs were comparable to those in pooled HLMs. Taken together, these data suggest that CYP3A, mostly likely CYP3A4, is primarily responsible for the metabolism of buspirone in HLMs.

Metabolism, pharmacokinetics, and protein covalent binding of radiolabeled MaxiPost (BMS-204352) in humans.

MaxiPost [(3S)-(+)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one); BMS-204352] is an investigational maxi-K channel opener to treat ischemic stroke. This study reports the disposition, metabolism, pharmacokinetics, and protein covalent binding of (14)C-labeled MaxiPost in healthy male volunteers as well as in dogs and rats. After each human subject received a single dose of 10 mg (14)C-labeled BMS-204352 (50 microCi) as a 5-ml intravenous infusion lasting 5 min, the plasma radioactivity concentrations showed a unique profile, wherein the concentration appeared to increase initially, followed by a terminal decline. The mean terminal t(1/2) of plasma radioactivity (259 h) was prolonged compared with that of unchanged parent (37 h). Furthermore, the extractability of radioactivity in plasma decreased over time, reaching approximately 20% at 4 h after dosing. The unextractable radioactivity was covalently bound to plasma proteins through a des-fluoro-des-methyl BMS-204352 lysine adduct. Unchanged BMS-204352 and minor metabolites were identified in plasma extract following protein precipitation. The recovery of the radioactive dose in urine and feces was nearly complete in 14-day collections (approximately 37% in urine and 60% in feces). The N-glucuronide of the parent was the prominent metabolite in urine (16.5% of dose), whereas the parent was a major drug-related component in feces (11% of dose). Similar disposition, metabolism, pharmacokinetic, and protein covalent binding properties of (14)C-labeled BMS-204352 were observed in humans, dogs, and rats.

Amide N-glucuronidation of MaxiPost catalyzed by UDP-glucuronosyltransferase 2B7 in humans.

MaxiPost [(3S)-(+)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one), or BMS-204352)] is a potent and specific maxi-K channel opener for potential use to treat stroke. This article describes structural characterization of a major human N-glucuronide metabolite of BMS-204352 and identification of the enzyme responsible for the N-glucuronidation reaction. After intravenous administrations of [(14)C]BMS-204352 (10 mg, 50 microCi) to eight healthy human subjects, one major metabolite M representing an average of 17% of the radioactive dose was excreted in pooled urine collected over 0 to 336 h after dosing. A major biliary metabolite of dogs dosed with [(14)C]BMS-204352 (5 mg/kg), which represented about 33% of the dose, has the same retention time and the same tandem mass spectrometry fragmentation pattern as the human urinary metabolite M. Four hundred fifty micrograms of the metabolite was isolated from the dog bile and analyzed by NMR. Long-range (1)H-(13)C NMR experimentation indicated that the glucuronic acid moiety was at the nitrogen site. The N-glucuronide of BMS-204352 was stable up to 24 h at 37 degrees C in the incubations at different pH values (3.0, 7.4, and 9.0) and with glucuronidases from Escherichia coli and Helix pomatia. Of the seven human UDP-glucuronosyltransferases (UGT) isozymes (1A1, 1A3, 1A4, 1A6, 1A7, 1A10, and 2B7) tested, only UGT2B7 produced metabolite M. UGT2B7-catalyzed N-glucuronidation of BMS-204352 exhibited Michaelis-Menten kinetics with a K(m) of 14.2 microM and V(max) of 0.29 nmol/min. mg of protein. Collectively, these results suggest that amide N-glucuronidation, a major elimination pathway of MaxiPost, is catalyzed by UGT2B7 in humans. This N-glucuronide represents a fully characterized amide N-glucuronide, and glucuronidation at the nitrogen represents a newly identified conjugation reaction for UGT2B7.

Protein covalent binding of maxipost through a cytochrome P450-mediated ortho-quinone methide intermediate in rats.

(3S)-(+)-(5-Chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one) (MaxiPost, BMS-204352) is a potent and specific opener for maxi-K channels and has potential to prevent and treat ischemic stroke. Following single intravenous doses of [14C]BMS-204352 to rats, only 10 to 12% of radioactivity was extractable from plasma with organic solvents. The unextractable radioactivity remained associated with the proteins (mostly albumin) after SDS-polyacrylamide gel electrophoresis or dialysis. Following acid hydrolysis in 6 M HCl for 24 h at 110 degrees C from plasma proteins collected from nine rats dosed with [14C]BMS-204352, one major radioactive product was isolated and identified as a lysine-adduct of des-fluoro des-O-methyl BMS-204352 by liquid chromatography/mass spectrometry and NMR analyses as well as by comparison with the synthetic analog, lysine-adduct of des-fluoro BMS-204352 (BMS-349821). The covalent binding of BMS-204352 results from the displacement of the ring-fluorine atom of des-O-methyl BMS-204352 with the epsilon-amino group of a lysine residue. Microsomal incubations of [14C]BMS-204352 resulted in low levels of covalent binding of radioactivity to proteins. This in vitro covalent binding required cytochrome P450-reductase cofactor NADPH and was attenuated by glutathione. P4503A inhibitors ketoconazole and troleadomycin selectively prevented the covalent binding in vitro. Based on these observations, a two-step bioactivation process for the protein covalent binding of BMS-204352 was postulated: 1) P4503A-mediated O-demethylation leading to spontaneous release of HF and the formation of an ortho-quinone methide reactive metabolite and 2) nucleophilic addition of the epsilon-amino group of protein lysine residue(s) in protein to form des-fluoro des-O-methyl BMS-204352 lysine adduct.

Comparative biotransformation of radiolabeled [(14)C]omapatrilat and stable-labeled [(13)C(2)]omapatrilat after oral administration to rats, dogs, and humans.

Omapatrilat, a novel vasopeptidase inhibitor, is under development for the treatment of hypertension and congestive heart failure. This study describes the comparative biotransformation of radiolabeled [(14)C]- and stable-labeled [(13)C(2)]omapatrilat after administration of single oral doses to rats, dogs, and humans. The metabolites were identified by a combination of methods including reduction, hydrolysis, and comparison of high performance liquid chromatography retention times with those of the synthetic standards. Urinary metabolites were further characterized by liquid chromatography tandem mass spectrometry analysis. Prominent metabolites identified in human plasma, which were also present in rat and dog plasma, were S-methyl omapatrilat and S-2-thiomethyl-3-phenylpropionic acid. Omapatrilat accounted for only a small portion of the extractable radioactivity in plasma in all three species. A portion of the plasma radioactivity was unextractable in all three species (27-53%). The majority of unextractable radioactivity in plasma was characterized after dithiothreitol reduction to be omapatrilat and (S)-2-thio-3-phenylpropionic acid, both apparently bound to plasma proteins by reversible disulfide bonds. The major human urinary metabolites were the amine hydrolysis product, diasteromeric sulfoxide of (S)-2-thiomethyl-3-phenylpropionic acid, acyl glucuronide of S-methyl omapatrilat, and S-methyl omapatrilat. The minor metabolites were acyl glucuronide of (S)-2-thiomethyl-3-phenylpropionic acid, L-cysteine mixed disulfide of omapatrilat, diastereomers of S-methyl sulfoxide of omapatrilat, and S-methyl omapatrilat ring sulfoxide. The metabolic profiles of dog and human urine were qualitatively similar whereas rat urine showed only metabolites arising from hydrolysis of omapatrilat. Unchanged omapatrilat was not found in rat, dog, or human urine samples indicating extensive metabolism in vivo.