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The USFDA recently approved mirabegron, a novel once-daily β-3 adrenoceptor agonist for oral administration, as a transformative treatment for overactive bladder. Despite the existence of numerous analytical methods for the assay and bioanalysis of mirabegron, it's perplexing that none have explored the domain of microwave-assisted sensitive spectrofluorimetric method for mirabegron estimation, even after extensive literature review. Adding to the enigma is the insistence of current analytical methods on using expensive and harmful organic solvents, posing a threat to marine life and the broader environment. Recently, the white analytical chemistry approach has been introduced to develop analytical methods that are cost-effective, environmentally friendly, and user-friendly. Consequently, a white analytical chemistry-based, sensitive, and eco-friendly spectrofluorimetric estimation of mirabegron has been initiated, using 4-Chloro-7-nitrobenzofurazan as a fluorescent biosensing probe. The development of this robust method involved a series of experiments designed to minimize solvent and time wastage. Through a combination of fractional factorial and Box-Behnken designs, researchers identified the critical variables and optimized the method to perfection. This method was validated according to the stringent ICH Q2 (R2) and USFDA guidelines, ensuring its reliability and accuracy. Once approved, this sensitive spectrofluorimetric method was tested, accurately estimating mirabegron levels in commercial formulations and rat plasma samples. To further enrich the study, a comprehensive evaluation of existing analytical methods was conducted alongside the proposed spectrofluorimetric method, using advanced tools like the AGREE calculator, GAPI software, and RGB model to assess their eco-friendliness and effectiveness in mirabegron estimation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mirabegron (MRB) is a β 3 -adrenoceptor agonist used for managing overactive bladder syndrome. A cost-effective, environmentally friendly, and highly sensitive spectrofluorimetric method was suggested to serve the purpose of quantifying MRB in its pure state, pharmaceutical tablets, spiked human plasma and urine, and testing content uniformity. In the present study, ninhydrin and phenylacetaldehyde react with the amino group moiety of MRB in Teorell-Stenhagen buffer (pH 7.5) to generate a strongly fluorescent diaryl pyrrolone compound that emits fluorescence at a wavelength of 477 nm upon excitation at 385 nm. The obtained calibration curve showed a linear relationship with a high correlation coefficient (r = 0.9997) in the concentration range of 0.25 to 5.0 µg mL -1 . Limits of detection (LOD) and quantitation (LOQ) were 0.082 and 0.248 µg mL -1 respectively. The procedure was verified in accordance with the ICH guidelines. The suggested approach could be utilized for the selective analysis of MRB in its pharmaceuticals, either containing a single drug or co-formulated with solifenacin succinate. The greenness of the suggested method was confirmed using different green analytical metrics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Here, a novel fluorescence strategy was established for the detection of mirabegron (MBG) sensitively on the basis of hantzsch dihydropyridine synthesis. The developed method adopts turn-on fluorescence of MBG for the first time, permitting its selective determination in spiked human plasma at 486 nm after excitation at 410 nm. The developed method exhibited a good linear range from 0.5 μgmL -1 to 2.0 μgmL -1 with detection and quantification limits of 0.05 and 0.2 (μgmL -1 ), respectively. The profitable applicability of the developed method in spiked human plasma samples was demonstrated, achieving limit of detection below the previously levels reported by spectroscopic methods, allowing application of the developed method for selective determination of MBG in its tablets and spiked human plasma samples with good recovery., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

LW6, an (aryloxyacetylamino)benzoic acid derivative, was recently identified to be an inhibitor of hypoxia-inducible factor-1α (HIF-1α), which is an attractive target for cancer therapeutics. Although LW6 is known to act by inhibiting the accumulation of HIF-1α, pharmacokinetics needs to be evaluated to assess its potential as an anti-tumor agent. Here, we investigated the plasma pharmacokinetics and metabolism of LW6 in mice. LW6 exhibited a small volume of distribution (0.5 ± 0.1 L/kg), and a short terminal half-life (0.6 ± 0.1 h). Following intravenous or oral administration, LW6 was rapidly converted to its active metabolite, (4-adamantan-1-yl-phenoxy)acetic acid (APA). Although LW6 was rapidly absorbed, its oral bioavailability, estimated using AUC last values, was low (1.7 ± 1.8%). It was slowly degraded in mouse liver microsomes ( t 1/2 > 1 h) and serum ( t 1/2 > 6 h). About 54% or 44.8% of LW6 was available systemically as APA in the mouse after a single intravenous or oral administration, respectively. Thus, our results indicated the need to simultaneously consider the active metabolite as well as the parent compound for successful evaluation during lead optimization.

A novel HIF (hypoxia-inducible factor)-1α inhibitor, the (aryloxyacetylamino)benzoic acid derivative LW6, is an anticancer agent that inhibits the accumulation of HIF-1α. The aim of this study was to characterize and determine the structures of the metabolites of LW6 in ICR mice. Metabolite identification was performed using a predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (pMRM-IDA-EPI) method in negative ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (QTRAP). A total of 12 metabolites were characterized based on their MS/MS spectra, and the retention times were compared with those of the parent compound. The metabolites were divided into five structural classes based on biotransformation reactions: amide hydrolysis, ester hydrolysis, mono-oxidation, glucuronidation, and a combination of these reactions. From this study, 2-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)acetic acid (APA, M7), the metabolite produced via amide hydrolysis, was found to be a major circulating metabolite of LW6 in mice. The results of this study can be used to improve the pharmacokinetic profile by lowering the clearance and increasing the exposure relative to LW6.

5-Hydroxymethyl tolterodine (5-HMT; the active fesoterodine metabolite) is metabolized via the cytochrome P450 (CYP) 2D6 and CYP3A pathways. Mirabegron is a moderate CYP2D6 inhibitor and weak CYP3A inhibitor. Potential drug-drug interactions (DDIs) following coadministration of these 2 overactive bladder treatments were estimated using physiologically based pharmacokinetic models, developed and verified by comparing predicted and observed pharmacokinetic profiles from clinical studies. Models predicted and verified mirabegron and desipramine (CYP2D6 substrate) and 5-HMT and ketoconazole (strong CYP3A inhibitor) DDIs. Mirabegron model-predicted mean steady-state AUC and C max were within 11% of clinical observations. The predicted versus observed geometric mean ratio (GMR) of AUC inf for CYP2D6 substrates desipramine and metoprolol coadministered with mirabegron 100 or 160 mg once daily were 3.47 versus 3.41 and 2.97 versus 3.29, respectively, indicating that the mirabegron model can be used to predict clinical CYP2D6 inhibition. 5-HMT fractional clearance by CYP3A and CYP2D6 was verified from clinical DDI studies with a potent CYP3A4 inhibitor (ketoconazole) and inducer (rifampicin) in CYP2D6 extensive and poor metabolizers and with a moderate CYP3A inhibitor (fluconazole) in healthy volunteers. 5-HMT AUC inf and C max GMRs for fesoterodine DDIs were all predicted within 1.26-fold of clinical observation, providing verification for the fesoterodine substrate model. The predicted changes in 5-HMT AUC inf and C max ratios for 8 mg fesoterodine when coadministered with 50 mg mirabegron were 1.22-fold and 1.17-fold, respectively, relative to 8 mg fesoterodine given alone. This modest increase in 5-HMT exposures by approximately 20% is considered clinically insignificant and would not require fesoterodine dose adjustment when coadministered with mirabegron within approved daily-dose ranges., (© 2019, The American College of Clinical Pharmacology.)

This was the first study to construct a physiologically-based pharmacokinetic (PBPK) model for mirabegron which incorporates the overall elimination pathways of metabolism by cytochrome P450 (CYP) 3A4, uridine 5'-diphosphate-glucuronosyltransferase (UGT) 2B7, and butyrylcholinesterase (BChE) and renal excretion. The objective was to assess the risk of drug-drug interactions (DDIs) by estimating the contribution of each elimination pathway and simulating the magnitude of the DDIs with UGT2B7 inhibitors. A PBPK model for mirabegron was constructed to reproduce the plasma concentration-time curves from a phase 1 study and the magnitude of the DDI with ketoconazole taking into account the overall elimination pathways. The PBPK model was subsequently verified using data from other DDI studies. The constructed PBPK model estimated the contribution for each elimination pathway: 44% and 29% for CYP3A4 and UGT2B7 in the liver, 1.6% for UGT2B7 in the kidney, 3.2% for BChE in plasma, and 22% for renal excretion. Co-administration of probenecid (an UGT2B7 inhibitor) or fluconazole (an UGT2B7 and CYP3A4 inhibitor) was predicted to increase area under the curve for mirabegron to 115% or 174%, respectively. In conclusion, PBPK modeling and simulation revealed a low DDI risk for mirabegron following co-administration with BChE or UGT2B7 inhibitors.

Background: SKLB023, a novel 5-benzylidenethiazolidine-2,4-dione based-derivative, specifically inhibits inducible nitric oxide synthase and shows promise for treating non-alcoholic steatohepatitis (NASH). However, its poor water solubility and low bioavailability limits its clinical use. Here the drug was loaded into phosphatidylcholine-bile salt-mixed micelles (PBMM/SKLB023) to overcome these limitations. Methods: PBMM/SKLB023 was developed using a simple co-precipitation method, and formulation parameters were optimized. The pharmacokinetics of PBMM/SKLB023 were investigated in Wistar rats, and therapeutic efficacy was assessed in a mouse model of NASH induced by a diet deficient in methionine- and choline. Results: PBMM/SKLB023 particles were 11.36±2.08 nm based on dynamic light scattering, and loading the drug into micelles improved its water solubility 300-fold. PBMM/SKLB023 inhibited proliferation and activation of HSC-T6 cells more strongly than free SKLB023. PBMM/SKLB023 showed longer mean retention time and higher bioavailability than the free drug after intravenous injection in Wistar rats. In the mouse model of NASH, PBMM/SKLB023 alleviated hepatic lipid accumulation, inflammation, and fibrosis to a significantly greater extent than free SKLB023. Conclusion: PBMM/SKLB023 shows therapeutic potential for treating NASH and liver fibrosis., Competing Interests: The authors report no conflicts of interest in this work.

We previously verified a physiologically based pharmacokinetic (PBPK) model for mirabegron in healthy subjects using the Simcyp Simulator by incorporating data on the inhibitory effect on cytochrome P450 (CYP) 2D6 and a multi-elimination pathway mediated by CYP3A4, uridine 5'-diphosphate-glucuronosyltransferase (UGT) 2B7 and butyrylcholinesterase (BChE). The aim of this study was to use this PBPK model to assess the magnitude of drug-drug interactions (DDIs) in an elderly population with severe renal impairment (sRI), which has not been evaluated in clinical trials. We first determined the system parameters, and meta-analyses of literature data suggested that the abundance of UGT2B7 and the BChE activity in an elderly population with sRI was almost equivalent to and 20% lower than that in healthy young subjects, respectively. Other parameters, such as the CYP3A4 abundance, for an sRI population were used according to those built into the Simcyp Simulator. Second, we confirmed that the PBPK model reproduced the plasma concentration-time profile for mirabegron in an sRI population (simulated area under the plasma concentration-time curve (AUC) was within 1.5-times that of the observed value). Finally, we applied the PBPK model to simulate DDIs in an sRI population. The PBPK model predicted that the AUC for mirabegron with itraconazole (a CYP3A4 inhibitor) was 4.12-times that in healthy elderly subjects administered mirabegron alone, and predicted that the proportional change in AUC for desipramine (a CYP2D6 substrate) with mirabegron was greater than that in healthy subjects. In conclusion, the PBPK model was verified for the purpose of DDI assessment in an elderly population with sRI., (© 2019 John Wiley & Sons, Ltd.)

Mirabegron is the first registered β3-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., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Mirabegron is a kind of β3 adrenergic receptor agonist which is an effective drug for the treatment of overactive bladder. In this research, a UPLC-MS/MS method is developed and validated for the study of mirabegron pharmacokinetic in rats. A protein precipitation method is applied for sample preparation with acetonitrile. m/z 397.3→379.6, m/z 326.4→121.0 for mirabegron, tolterodine (IS), respectively in the positive ion mode was performed for quantitation. The method is reliable and reproducible in our study (intra-day precision≤11.06%, inter-day precision≤11.43%) with concentration curves linear from 5 to 2500 ng/mL(R2>0.999). Stability studies demonstrated that mirabegron was stable under a variety of storage conditions. This method was successfully applied for determining mirabegron in rats after oral and intravenous administration.

Purpose: Hematopoietic cell transplant patients are exposed to numerous classes of medications. Transplant practitioners must vigilantly monitor for drug interactions especially involving immunosuppressants. We report a hematopoietic cell transplant patient receiving sirolimus who developed supratherapeutic serum concentrations after initiating mirabegron., Summary: A 31-year-old, 98 kg female received a second umbilical cord blood transplant four years after the first transplant for relapsed acute myeloid leukemia. Mycophenolate mofetil and sirolimus were utilized for graft versus host disease prophylaxis. The patient was receiving sirolimus 2 mg daily and the serum concentration on day 26 post-transplant (day + 26) was within therapeutic range (6.7 μg/L, goal range 3-12 μg/L). Her post-transplant course was complicated by BK viruria-associated cystitis for which she was started on mirabegron. Six days after starting the new medication (day + 33), the sirolimus serum concentration increased to 19.2 μg/L. Thus mirabegron was discontinued and sirolimus was held. Sirolimus was restarted once the serum concentration was within goal and subsequently stabilized with a combination of 1 mg and 2 mg daily for a total weekly dose of 10 mg. The proposed mechanisms of interaction include: (1) sirolimus inhibition of organic anion transporting polypeptide leading to increased mirabegron in the intestinal lumen; (2) mirabegron inhibition of P-glycoprotein leading to increased absorption of sirolimus and; (3) increased sirolimus absorption leading to increased sirolimus serum concentrations., Conclusion: To our knowledge, this is the first report of a potential drug interaction between sirolimus and mirabegron. Transplant specialists should be aware of this potential interaction when considering the concurrent use of these medications.

A highly sensitive, specific and enantioselective assay has been validated for the quantitation of OTX015 enantiomers [(+)-OTX015 and (-)-OTX015] in mice plasma on LC-MS/MS-electrospray ionization as per regulatory guidelines. Protein precipitation was used to extract (±)-OTX015 enantiomers and internal standard (IS) from mice plasma. The active [(-)-OTX015] and inactive [(+)-OTX015] enantiomers were resolved on a Chiralpak-IA column using an isocratic mobile phase (0.2% ammonia/acetonitrile 20 : 80, v/v) at a flow rate of 1.2 mL/min. The total run time was 6.0 min. (+)-OTX015, (-)-OTX015 and IS eluted at 3.34, 4.08 and 4.77 min, respectively. The MS/MS ion transitions monitored were m/z 492 → 383 for OTX015 and m/z 457 → 401 for IS. The standard curves for OTX015 enantiomers were linear (r 2  > 0.998) in the concentration range 1.03-1030 ng/mL. The inter- and intraday precisions were in the range 2.20-13.3 and 8.03-12.1% and 3.80-14.4 and 8.97-13.6% for (+)-OTX015 and (-)-OTX015, respectively. Both the enantiomers were found to be stable in a battery of stability studies. This novel method has been applied to the study of stereoselective oral pharmacokinetics of (-)-OTX015 and unequivocally demonstrated that (-)-OTX015 does not undergo chiral inversion to its antipode in vivo in mice., (Copyright © 2016 John Wiley & Sons, Ltd.)

A new concept of using thin-layer chromatography to sample preparation for the quantitative determination of solute/s followed by instrumental techniques is presented Thin-layer chromatography (TLC) is used to completely separate acetaminophen and its internal standard from other components (matrix) and to form a single spot/zone containing them at the solvent front position (after the final stage of the thin-layer chromatogram development). The location of the analytes and internal standard in the solvent front zone allows their easy extraction followed by quantitation by HPLC. The exctraction procedure of the solute/s and internal standard can proceed from whole solute frontal zone or its part without lowering in accuracy of quantitative analysis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background and Objectives: OTX015 (MK-8628) is a novel inhibitor of the bromodomain and extraterminal (BET)-bromodomain (BRD) protein family, binding specifically to bromodomains BRD2/3/4 and impacting the epigenetic regulation of several oncogenes. We characterized the pharmacokinetics of this first-in-class BET-BRD inhibitor administered as a single agent, including population pharmacokinetic modelling., Methods: A dose-escalation, phase Ib study was performed with oral OTX015 in patients with haematologic malignancies, at doses starting from 10 mg once daily (QD) with continuous or discontinuous schedules. Five or eight blood samples were collected per patient for pharmacokinetic analysis. OTX015 plasma concentrations were determined using validated ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and analysed using a nonlinear mixed-effects modelling software program. A population pharmacokinetic model was fitted to the data, and patient demographics and clinical chemistry parameters were tested as predictive covariates on the model parameters., Results: Blood samples were analysed from 81 patients treated with OTX015 at doses ranging from 10 to 160 mg QD or 40 mg twice daily (BID), and 633 time-plasma concentrations were available for analysis. A one-compartment open model with linear elimination adequately described OTX015 pharmacokinetics. The most significant covariate was lean body mass (LBM), which decreased the between-subject variability in apparent total body clearance (CL) and the volume of distribution (V). The estimated pharmacokinetic parameters were the absorption rate constant (k a) = 0.731 h(-1), V = 71.4 L and CL = 8.47 L·h(-1)., Conclusion: The pharmacokinetics of oral OTX015 in patients with haematologic malignancies can be described with a one-compartment model. Population pharmacokinetic modelling of OTX015 plasma concentrations showed that LBM influences V and CL. These findings do not suggest the need for dose adjustment.

The bioavailability of whole-grain rye-derived phytochemicals has not yet been comprehensively characterized, and different baking and manufacturing processes can modulate the phytochemical composition of breads and other rye products. The aim of our study was to find key differences in the phytochemical profile of plasma after the consumption of 3 breads containing rye bran when compared with a plain white wheat bread control. Plasma metabolite profiles of 12 healthy middle-aged men and women were analyzed using LC quadrupole time-of-flight mass spectrometry metabolomics analysis while fasting and at 60 min, 120 min, 240 min, and 24 h after consuming a meal that contained either 100% whole-grain sourdough rye bread or white wheat bread enriched with native unprocessed rye bran or bioprocessed rye bran. White wheat bread was used as the control. The meals were served in random order after a 12-h overnight fast, with at least 3 d between each occasion. Two sulfonated phenylacetamides, hydroxy-N-(2-hydroxyphenyl) acetamide and N-(2-hydroxyphenyl) acetamide, potentially derived from the benzoxazinoid metabolites, were among the most discriminant postprandial plasma biomarkers distinguishing intake of breads containing whole-meal rye or rye bran from the control white wheat bread. Furthermore, subsequent metabolite profiling analysis of the consumed breads indicated that different bioprocessing/baking techniques involving exposure to microbial metabolism (e.g., sourdough fermentation) have a central role in modulating the phytochemical content of the whole-grain and bran-rich breads., (© 2014 American Society for Nutrition.)

Mirabegron is a novel β3-adrenoceptor agonist developed for the treatment of overactive bladder. To clarify the relationship between the pharmacological effects of mirabegron in monkeys and the clinical efficacy in patients with overactive bladder, the effect of mirabegron on bladder function was evaluated using cynomolgus monkeys. Quantitative PCR revealed that mRNA expression of β3-adrenoceptors was most abundant (98 %) among β-adrenoceptor subtypes in the bladder of cynomolgus monkeys. Mirabegron, which showed selective and potent agonistic activity on monkey β3-adrenoceptors expressed in Chinese hamster ovary cells with EC50 value of 32 nmol/L and intrinsic activity of 0.8, induced concentration-dependent relaxation of bladder smooth muscle strips isolated from cynomolgus monkeys with EC50 values of 120 nmol/L in 20 mmol/L KCl stimulation and 43 nmol/L under 9.81 mN resting tension. In conscious cynomolgus monkeys, mirabegron decreased micturition frequency at oral doses of 1 and 3 mg/kg and increased mean volume voided per micturition at an oral dose of 3 mg/kg. Plasma concentration at which bladder function improved in the cynomolgus monkeys was similar to that at the clinically effective dose in patients with overactive bladder. These data suggest that the relaxant function in monkey bladder is mainly mediated by β3-adrenoceptors similar to that in the human bladder and mirabegron showed efficacy on the bladder functions of the same parameters in clinical evaluation endpoints.

The aim of our study was to enhance the bioavailability of ranolazine by using herbal-bioenhancer quercetin in rats and to study the role of P-glycoprotein (P-gp) in vitro models. In single dose study (SDS), rats were divided into four groups, Group I was treated with 0.5% sodium carboxy methyl cellulose (SCMC), Group II was treated with ranolazine (14 mg/kg), Group III was treated with quercetin (20 mg/kg) and Group IV was treated with both ranolazine and quercetin. The blood samples were collected at 0.5, 1, 2, 3, 4, 6, 8 and 12 h, and the concentration of ranolazine in the plasma was estimated by reverse phase high performance liquid chromatography (RP-HPLC) method. In multiple dose study (MDS), rats were treated with same drugs for 7 days. On 8th day, the concentration of ranolazine in plasma was estimated. In vitro study performed on the rat and chick intestinal sacs to study the intestinal transport of ranolazine in the presence and absence of quercetin and verapamil (P-gp-inhibitor). Quercetin increased the peak concentration (Cmax) of ranolazine from 254 ± 8.45 to 324 ± 10.21 and 331 ± 9.65 ng/mL in SDS and MDS, respectively. Quercetin also increased area under the curve (AUC) of ranolazine from 1565.12 ± 52.24 to 2016.98 ± 142.65 and 2070.85 ± 271.60 ng/mL/h in SDS and MDS, respectively. The transport of ranolazine from mucosal side to serosal side was increased in presence of quercetin. Quercetin is an inhibitor of CYP3A4 and P-gp. So it increased the AUC and Cmax of ranolazine.

Background: Mirabegron is a β3-adrenoceptor agonist used for the treatment of overactive bladder. Mirabegron is formulated as an extended-release tablet using oral controlled-absorption system (OCAS) technology., Objective: This study was designed to assess the effects of food on the pharmacokinetic properties of mirabegron OCAS in accordance with regulatory requirements to support dosing recommendations., Methods: In this single-dose, randomized, open-label, 3-period, parallel-dose-group, crossover study, mirabegron OCAS 50 or 100 mg was administered orally to healthy adult subjects in the fasted state or after a high- or low-fat breakfast. Dose administrations were separated by a washout period of at least 10 days. Blood samples were drawn up to 96 hours after dosing, and plasma concentrations of mirabegron were analyzed by LC/MS-MS. PK properties were determined using noncompartmental methods. Primary end points for the assessment of food effects were Cmax and AUC0-∞. For tolerability assessment, adverse events (AEs) were monitored using investigators' questionnaires and subjects' spontaneous reports, vital sign measurements, hematology, clinical chemistry, and ECG., Results: Thirty-eight subjects (male, 50%; mean age, 32.1 years; mean weight, 77.3 kg; race, 76.3% white) were enrolled in the 50-mg dose group and 38 subjects (male, 52.6%; mean age, 30.9 years; mean weight, 74.5 kg; race, 63.2% white) in the 100-mg dose group. With either fed condition or dose, the 90% CIs for the fed/fasted ratios of both Cmax and AUC0-∞ of mirabegron fell below the predetermined range for bioequivalence (80.0%-125.0%), suggesting that food had no effect on exposure to mirabegron OCAS. With the 50-mg dose, mirabegron Cmax was reduced by 45% with a high-fat breakfast compared with fasted conditions (geometric mean ratio [GMR], 54.8% [90% CI, 43.7%-68.6%]) and AUC0-∞, by 17% (GMR, 83.2% [90% CI, 74.2%-93.4%]). With the 100-mg dose, mirabegron Cmax and AUC0-∞ were reduced by 39% (GMR, 61.3% [90% CI, 47.8%-78.7%]) and 18% (82.4% [72.6%-93.5%]), respectively, after a high-fat breakfast. With the 50-mg dose, mirabegron Cmax was decreased by 75% (GMR, 25.0% [90% CI, 19.9%-31.3%]) and AUC0-∞ by 51% (48.7% [43.3%-54.7%]) after a low-fat breakfast. Corresponding reductions with the 100-mg dose were 64% (GMR, 36.3% [90% CI, 28.2%-46.8%]) for Cmax and 47% (GMR, 53.2% [90% CI, 46.8%-60.5%]) for AUC0-∞. The fed/fasted ratios for mirabegron Cmax and AUC0-∞ were in general independent of dose or sex. Food delayed Tmax compared with the fasted state, with similar increases with the high- and low-fat meals (0.9 hours with 50 mg and 1.5-2.0 hours with 100 mg). Mirabegron was generally well tolerated, with no apparent difference in AE frequency between the fasted and fed states., Conclusions: Mirabegron OCAS tablets exhibited a decrease in mirabegron plasma exposure with food that was independent of dose (50 or 100 mg) or gender but dependent on meal composition. A greater reduction in mirabegron exposure was observed after a low-fat breakfast compared with after a high-fat breakfast. Based on findings from previous studies, the effects of food observed in this study do not warrant dose adjustment in clinical practice. ClinicalTrials.gov identifier: NCT00939757., (Copyright © 2013 Elsevier HS Journals, Inc. All rights reserved.)

Background and Objectives: Mirabegron, a selective β3-adrenoceptor agonist for the treatment of overactive bladder (OAB), is eliminated by renal and metabolic routes. The potential influence of renal or hepatic impairment on the pharmacokinetics of mirabegron was evaluated., Methods: Two separate open-label, single-dose, parallel-group studies were conducted. Male and female subjects (n = 8 per group) were categorized according to their baseline renal function (mild, moderate, severe or no impairment as determined by estimated glomerular filtration rate [eGFR] using the abbreviated modification of diet in renal disease formula) or hepatic function (mild, moderate or no impairment as determined by the Child-Pugh classification). All subjects received a single oral 100 mg dose of mirabegron. Non-compartmental pharmacokinetic parameters were determined from plasma and urine concentration-time data of mirabegron and metabolites., Results: Compared with healthy subjects who were similar overall in terms of age, sex and body mass index (BMI), the geometric mean area under the plasma concentration-time curve from time zero extrapolated to infinity (AUC(∞)) for mirabegron was 31, 66 and 118 % higher in subjects with mild, moderate and severe renal impairment, respectively. Peak plasma concentrations (C(max)) increased 6, 23 and 92 %, respectively, in subjects with mild, moderate and severe renal impairment. Renal clearance but not apparent total body clearance of mirabegron correlated well with renal function. Compared with healthy subjects matched for age, sex and BMI, mirabegron AUC(∞) values were 19 and 65 % higher in subjects with mild and moderate hepatic impairment, respectively. Mirabegron C(max) was 9 and 175 % higher, respectively, compared with matched healthy subjects. No clear relationship was evident between pharmacokinetic parameters and Child-Pugh scores. Protein binding was approximately 71 % in healthy subjects and was not altered to a clinically significant extent in subjects with renal or hepatic impairment. No consistent changes in mirabegron elimination half-life were observed in subjects with renal or hepatic impairment. There was high pharmacokinetic variability and significant overlap in exposures between subjects with renal or hepatic impairment and healthy subjects., Conclusion: Mirabegron AUC(∞) and C(max) increased 118 and 92 %, respectively, in subjects with severe renal impairment, and 65 and 175 %, respectively, in subjects with moderate hepatic impairment. Pharmacokinetic changes observed in subjects with mild or moderate renal impairment or mild hepatic impairment are of small magnitude and likely to be without clinical importance.

Background and Objectives: Mirabegron is a potent and selective β3-adrenoceptor agonist in development for treatment of overactive bladder., Methods: Mirabegron pharmacokinetics after single intravenous (i.v.) and oral doses, absolute bioavailability (F), dose proportionality, sex differences and tolerability were assessed in 2 single-dose, open-label, randomized, parallel-group, cross-over studies in healthy men (exploratory Study 1, n = 12) and men and women (Study 2, n = 91)., Results: After oral dosing (25 - 150 mg), peak plasma concentrations were attained after ~ 4 h. Mean half-life was around 40 h for both routes of administration. Volume of distribution at steady state was 1,670 l and total clearance was around 57 l/h for i.v. dosing. Mirabegron pharmacokinetics were linear after i.v. dosing (7.5 - 50 mg), but exposure increased more than proportionally after oral dosing due to increased F (29% for 25 mg to 45% at 150 mg). About 20% of the (absorbed) dose was excreted unchanged into urine. Area under the curve (AUC) was 27% and 64% higher in females than males after i.v. and oral dosing respectively; differences were mostly attributed to body weight, and for oral dosing, also to F., Conclusions: Mirabegron pharmacokinetics were linear after i.v. dosing (7.5 - 50 mg), but increased more than proportionally after oral dosing (25 - 150 mg) as a result of increased F. Sex differences in exposure could be explained by body weight and for oral dosing, also by F. Mirabegron was in general well tolerated up to the highest doses studied, 50 mg i.v. and 150 mg oral.

1. Human cytochrome P450 (CYP) enzymes and esterases involved in the metabolism of mirabegron, a potent and selective human β(3)-adrenoceptor agonist intended for the treatment of overactive bladder, were identified in in vitro studies. 2. Incubations of mirabegron with recombinant human CYP enzymes showed significant metabolism of mirabegron by CYP2D6 and CYP3A4 only. Correlation analyses showed a significant correlation between mirabegron metabolism and testosterone 6β-hydroxylation (CYP3A4/5 marker activity). In inhibition studies using antiserum against CYP3A4, a strong inhibition (at maximum 80% inhibition) of the metabolism of mirabegron was observed, whereas the inhibitory effects of monoclonal antibodies against CYP2D6 were small (at maximum 10% inhibition). These findings suggest that CYP3A4 is the primary CYP enzyme responsible for in vitro oxidative metabolism of mirabegron, with a minor role of CYP2D6. 3. Mirabegron hydrolysis was catalyzed in human blood, plasma and butyrylcholinesterase (BChE) solution, but not in human liver microsomes, intestinal microsomes, liver S9, intestinal S9 and recombinant acetylcholinesterase solution. K(m) values of mirabegron hydrolysis in human blood, plasma and BChE solution were all similar (13.4-15.2 μM). The inhibition profiles in human blood and plasma were also similar to those in BChE solution, suggesting that mirabegron hydrolysis is catalyzed by BChE.

A simple, sensitive and high-throughput ultra high-performance liquid chromatography electrospray ionization mass spectrometry (U-HPLC-ESI-MS/MS) method has been developed and validated for the determination of ranolazine in human plasma. Propafenone was employed as the internal standard (I.S.). The analytes were chromatographically separated on a BEH C(18) column (50 mm × 2.1 mm, 1.7 μm) with a mobile phase consisting of acetonitrile and aqueous ammonium acetate solution (0.06% formic acid, 7.5 mmol L(-1) ammonium acetate, 40:60, v/v). Detection of the analytes was achieved using positive ion electrospray ionization via multiple reactions monitoring mode. The mass transitions were m/z 428.3→279.3 for ranolazine and m/z 342.4→115.9 for propafenone. The assay was linear over the concentration range 1-3000 ng mL(-1), with correlation coefficients ≥0.997. The intra- and inter-day coefficients of variation were less than 8.9% in terms of relative standard deviation and accuracy ranged from 93.0 to 108.9% at all quality control levels. The validated method was a simple sample preparation procedure and short run-time (<2.0 min) method, which was successfully applied to a phase I pharmacokinetic study of ranolazine in Chinese healthy volunteers., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The mass balance and metabolite profiles of 2-(2-amino-1,3-thiazol-4-yl)-N-[4-(2-{[(2R)-2-hydroxy-2-phenylethyl]amino}ethyl)[U-(14)C]phenyl]acetamide ([(14)C]mirabegron, YM178), a β(3)-adrenoceptor agonist for the treatment of overactive bladder, were characterized in four young, healthy, fasted male subjects after a single oral dose of [(14)C]mirabegron (160 mg, 1.85 MBq) in a solution. [(14)C]Mirabegron was rapidly absorbed with a plasma t(max) for mirabegron and total radioactivity of 1.0 and 2.3 h postdose, respectively. Unchanged mirabegron was the most abundant component of radioactivity, accounting for approximately 22% of circulating radioactivity in plasma. Mean recovery in urine and feces amounted to 55 and 34%, respectively. No radioactivity was detected in expired air. The main component of radioactivity in urine was unchanged mirabegron, which accounted for 45% of the excreted radioactivity. A total of 10 metabolites were found in urine. On the basis of the metabolites found in urine, major primary metabolic reactions of mirabegron were estimated to be amide hydrolysis (M5, M16, and M17), accounting for 48% of the identified metabolites in urine, followed by glucuronidation (M11, M12, M13, and M14) and N-dealkylation or oxidation of the secondary amine (M8, M9, and M15), accounting for 34 and 18% of the identified metabolites, respectively. In feces, the radioactivity was recovered almost entirely as the unchanged form. Eight of the metabolites characterized in urine were also observed in plasma. These findings indicate that mirabegron, administered as a solution, is rapidly absorbed after oral administration, circulates in plasma as the unchanged form and metabolites, and is recovered in urine and feces mainly as the unchanged form.

Rationale: Previously, we demonstrated that a deoxycorticosterone acetate (DOCA)-salt hypertensive mouse model produces cardiac oxidative stress and diastolic dysfunction with preserved systolic function. Oxidative stress has been shown to increase late inward sodium current (I(Na)), reducing the net cytosolic Ca(2+) efflux., Objective: Oxidative stress in the DOCA-salt model may increase late I(Na), resulting in diastolic dysfunction amenable to treatment with ranolazine., Methods and Results: Echocardiography detected evidence of diastolic dysfunction in hypertensive mice that improved after treatment with ranolazine (E/E':sham, 31.9 ± 2.8, sham+ranolazine, 30.2 ± 1.9, DOCA-salt, 41.8 ± 2.6, and DOCA-salt+ranolazine, 31.9 ± 2.6; P=0.018). The end-diastolic pressure-volume relationship slope was elevated in DOCA-salt mice, improving to sham levels with treatment (sham, 0.16 ± 0.01 versus sham+ranolazine, 0.18 ± 0.01 versus DOCA-salt, 0.23 ± 0.2 versus DOCA-salt+ranolazine, 0.17 ± 0.0 1 mm Hg/L; P<0.005). DOCA-salt myocytes demonstrated impaired relaxation, τ, improving with ranolazine (DOCA-salt, 0.18 ± 0.02, DOCA-salt+ranolazine, 0.13 ± 0.01, sham, 0.11 ± 0.01, sham+ranolazine, 0.09 ± 0.02 seconds; P=0.0004). Neither late I(Na) nor the Ca(2+) transients were different from sham myocytes. Detergent extracted fiber bundles from DOCA-salt hearts demonstrated increased myofilament response to Ca(2+) with glutathionylation of myosin binding protein C. Treatment with ranolazine ameliorated the Ca(2+) response and cross-bridge kinetics., Conclusions: Diastolic dysfunction could be reversed by ranolazine, probably resulting from a direct effect on myofilaments, indicating that cardiac oxidative stress may mediate diastolic dysfunction through altering the contractile apparatus.

A rapid, sensitive and reliable LC-MS/MS method was developed and validated for the simultaneous determination of ranolazine and its three metabolites, CVT-2514, CVT-2738, and CVT-4786, in human plasma. The plasma samples were prepared by protein precipitation. Chromatographic separation was achieved on a Gemini C(18) column (50 mm × 2.0 mm, 5 μm) using methanol: 5 mM ammonium acetate as the mobile phase with gradient elution. Mass detection was carried out by electrospray ionization in both positive and negative ion multiple reaction monitoring (MRM) modes. The calibration curves were linear over a concentration range of 4-2000 ng/mL for ranolazine, 4-1000 ng/mL for CVT-2514 and CVT-2738 and 8-1000 ng/mL for CVT-4786. The intra-day and inter-day accuracy and precision were within the acceptable limits of ±15% at all concentrations. The method was successfully applied for the simultaneous estimation of ranolazine and its three metabolites in human plasma from a clinical pharmacokinetics study., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Mirabegron is being developed for the treatment of overactive bladder. To support the development of mirabegron, including pharmacokinetic studies, liquid chromatography/tandem mass spectrometry methods for mirabegron and eight metabolites (M5, M8, M11-M16) were developed and validated for heparinized human plasma containing sodium fluoride. Four separate bioanalytical methods were developed for the analysis of: (1) mirabegron; (2) M5 and M16; (3) M8; and (4) M11-M15. Either solid-phase extraction or liquid-liquid extraction was used to extract the analytes of interest from matrix constituents. For mirabegron, an Inertsil C₈-3 analytical column was used and detection was performed using a triple-quad mass spectrometer equipped with an atmospheric pressure chemical ionization interface. For the metabolite assays, chromatographic separation was performed through a Phenomenex Synergi Fusion-RP C₁₈ analytical column and detection was performed using a triple-quad mass spectrometer equipped with a Heated Electrospray Ionization interface. The validation results demonstrated that the developed liquid chromatography/tandem mass spectrometry methods were precise, accurate, and selective for the determination of mirabegron and its metabolites in human plasma. All methods were successfully applied in evaluating the pharmacokinetic parameters of mirabegron and metabolites in human plasma., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This study aimed to develop a joint population pharmacokinetic model for an antipsychotic agent in development (S33138) and its active metabolite (S35424) produced by reversible metabolism. Because such a model leads to identifiability problems and numerical difficulties, the model building was performed using the FOCE-I and the Stochastic Approximation Expectation Maximization (SAEM) estimation algorithms in NONMEM and MONOLIX, respectively. Four different structural models were compared based on Bayesian information criteria. Models were first written as ordinary differential equations systems and then in closed form (CF) to facilitate further analyses. The impact of polymorphisms on genes coding for the CYP2C19 and CYP2D6 enzymes, respectively involved in the parent drug and the metabolite elimination were investigated using permutation Wald test. The parent drug and metabolite plasma concentrations of 101 patients were analyzed on two occasions after 4 and 8 weeks of treatment at 1, 3, 6, and 24 h following daily oral administration. All configurations led to a two compartment model with back-transformation of the metabolite into the parent drug and a first-pass effect. The elimination clearance of the metabolite through other processes than back-transformation was decreased by 35% [9-53%] in CYP2D6 poor metabolizer. Permutation tests were performed to ensure the robustness of the analysis, using SAEM and CF. In conclusion, we developed a complex joint pharmacokinetic model adequately predicting the impact of CYP2D6 polymorphisms on the parent drug and its metabolite concentrations through the back-transformation mechanism.

Effects of ranolazine alone and in the presence of phenylephrine (PE) or isoproterenol (ISO) on hemodynamics, coronary blood flow and heart rate (HR) in the absence and presence of hexamethonium (a ganglionic blocker) were studied in conscious dogs. Ranolazine (0.4, 1.2, 3.6, and 6 mg/kg, intravenous) alone caused transient (<1 minute) and reversible hemodynamic changes. PE (0.3-10 μg/kg) caused a dose-dependent increase in blood pressure and decrease in HR. ISO (0.01-0.3 μg/kg) caused a dose-dependent decrease in blood pressure and an increase in HR. Ranolazine at high (11-13 mM), but not at moderate (4-5 mM) concentrations partially attenuated changes in mean arterial blood pressure and HR caused by either PE or ISO in normal conscious dogs. However, in dogs treated with hexamethonium (20 mg/kg) to cause autonomic blockade, ranolazine (both 4-5 and 11-13 μM) significantly attenuated both the PE- and ISO-induced changes in mean arterial blood pressure. The results suggest that a potential antiadrenergic effect of ranolazine was masked by autonomic control mechanisms in conscious dogs but could be observed when these mechanisms were inhibited (eg, in the hexamethonium-treated dog). Ranolazine, at plasma concentrations <10 μM and in conscious dogs with intact autonomic regulation, had minimal antiadrenergic (α and β) effects.

Purpose: Translocator protein (TSPO) is a promising biomarker for neuroinflammation. We developed two new PET ligands, (18)F-PBR06 and (11)C-PBR28, to image TSPOs. Although our prior studies suggest that either of the two ligands could be used to quantify TSPOs in human brain, the studies were done in different sets of subjects. In this study, we directly compared (18)F-PBR06 and (11)C-PBR28 in eight human subjects to determine (1) whether either ligand provides more precise measurements of TSPOs and (2) whether the higher in vitro affinity of PBR06 compared to PBR28 led to higher in vivo binding of (18)F-PBR06 compared to (11)C-PBR28., Methods: In vivo binding was calculated as total distribution volume (V(T)), using an unconstrained two-tissue compartment model. V(T) was corrected for plasma free fraction (f (P)) to measure ligand binding based on free ligand concentration in brain., Results: Both ligands measured V(T) with similar precision, as evidenced by similarly good identifiability. However, V(T) for both radioligands increased with increasing lengths of data acquisition, consistent with the accumulation of radiometabolites in brain. Despite its higher lipophilicity and higher in vitro affinity, V(T)/f(P) of (18)F-PBR06 was similar to that of (11)C-PBR28., Conclusion: Both (18)F-PBR06 and (11)C-PBR28 are similar in terms of precision, sensitivity to accumulation of radiometabolites, and magnitude of in vivo binding. Thus, selection between the two radioligands will be primarily determined by the logistical impact of the different half-lives of the two radionuclides (110 vs 20 min).

We tested the effect of the antianginal agent ranolazine on ventricular arrhythmias in an ischemic model using two protocols. In protocol 1, anesthetized rats received either vehicle or ranolazine (10 mg/kg, iv bolus) and were subjected to 5 min of left coronary artery (LCA) occlusion and 5 min of reperfusion with electrocardiogram and blood pressure monitoring. In protocol 2, rats received either vehicle or three doses of ranolazine (iv bolus followed by infusion) and 20 min of LCA occlusion. With protocol 1, ventricular tachycardia (VT) occurred in 9/12 (75%) vehicle-treated rats and 1/11 (9%) ranolazine-treated rats during reperfusion (P = 0.003). Sustained VT occurred in 5/12 (42%) vehicle-treated but 0/11 in ranolazine-treated rats (P = 0.037). The median number of episodes of VT during reperfusion in vehicle and ranolazine groups was 5.5 and 0, respectively (P = 0.0006); median duration of VT was 22.2 and 0 s in vehicle and ranolazine rats, respectively (P = 0.0006). With protocol 2, mortality in the vehicle group was 42 vs. 17% (P = 0.371), 10% (P = 0.162) and 0% (P = 0.0373) with ranolazine at plasma concentrations of 2, 4, and 8 microM, respectively. Ranolazine significantly reduced the incidence of ventricular fibrillation [67% in controls vs. 42% (P = 0.414), 30% (P = 0.198) and 8% (P = 0.0094) in ranolazine at 2, 4, and 8 microM, respectively]. Median number (2.5 vs. 0; P = 0.0431) of sustained VT episodes, incidence of sustained VT (83 vs. 33%, P = 0.0361), and the duration of VT per animal (159 vs. 19 s; P = 0.0410) were also significantly reduced by ranolazine at 8 microM. Ranolazine markedly reduced ischemia-reperfusion induced ventricular arrhythmias. Ranolazine demonstrated promising anti-arrhythmic properties that warrant further investigation.

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantification of ranolazine in human plasma. The analytical method consists in the precipitation of plasma sample with methanol, followed by the determination of ranolazine by an LC-MS/MS. The analyte was separated on a Peerless Cyano column (33 mm x 4.6 mm, 3 microm) an isocratic mobile phase of methanol-water containing formic acid (1.0%, v/v) (65:35, v/v) at a flow rate of 1.0 ml/min. Protonated ions formed by a turbo ionspray in positive mode were used to detect analyte and internal standard (IS). The MS/MS detection was made by monitoring the fragmentation of m/z 428.20-->279.50 for ranolazine and m/z 448.30-->285.20 for internal standard on a triple quadrupole mass spectrometer. The method was validated over the concentration range of 5-2000 ng/ml for ranolazine in human plasma with correlation coefficient of 0.9937 (S.D.: +/-0.00367, range: 0.9895-0.9963). The accuracy and precision values obtained from six different sets of quality control samples analyzed in separate occasions ranged from 94.53 to 117.86 and 0.14% to 4.56%, respectively. Mean extraction recovery was 82.36-94.25% for three quality control (QC) samples and 88.37% for IS. Plasma samples were stable for three freeze-thaw cycles, or 24h ambient storage, or 1 and 3 months storage at -20 degrees C. Processed samples (ready for injection) were stable up to 72 h at autosampler (4 degrees C). The developed method was successfully applied for analyzing ranolazine in plasma samples for a bioequivalence study with 12 healthy volunteers.

Agina pectoris is a discomfort in the chest or adjacent areas caused by myocardial ischemia. It is most commonly caused by the inability of narrowed atherosclerotic coronary arteries to supply adequate oxygen to the heart under conditions of increase demand. This review article will focus in the medical treatment of chronic stable angina, with a focus in new strategies or medications. Treatment by revascularization techniques will not be discussed in this article. The goal of treatment is to improve quality of life, decrease cardiovascular events and mortality. All patients should be evaluated for reversible causes of their angina, such as anemia, hyperthyroidism, sympathomimetic drugs and hypertension. Sublingual nitroglycerin should be used for immediate relief of symptoms. In general, all patients should be on aspirin (ASA) unless they are allergic or other contraindications, if so; clopidogrel should be added to the therapy. In addition to the antiplatelet therapy, which decreases mortality, patients should be started on beta blockers and nitrates. If there is no improvement in symptoms then a calcium channel blockers of the dihydropyridine family should be added. Patients with Diabetes Mellitus and/or left ventricular systolic dysfunction should be also started on angiotensin converting enzyme inhibitors. If the patient continues with limiting angina, ranolazine should be started and finally enhanced external counterpulsation should be considered in those patients who have not responded to maximal drug therapy.

A highly sensitive liquid chromatographic-tandem mass spectrometric method (LC-MS-MS) is developed to quantitate ranolazine in human plasma. The analyte and internal standard tramadol are extracted from plasma by liquid-liquid extraction using diethyl ether-dichloromethane (60:40 v/v), and separated on a Zorbax extend C18 column using methanol-10mM ammonium acetate (60:40 v/v, pH 4.0) at a flow of 1.0 mL/min. Detection is carried out by multiple reaction monitoring on a QtrapTM LC-MS-MS system with an electrospray ionization interface. The assay is linear over the range 10-5000 ng/mL with a limit of quantitation of 10 ng/mL and a lower limit of detection (S/N > 3) of 1 ng/mL. Intra- and inter-day precision are < 3.1% and < 2.8%, respectively, and the accuracy is in the range 96.7-101.6%. The validated method is successfully used to analyze the drug in samples of human plasma for pharmacokinetic studies.

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method with positive electrospray ionization (ESI) was developed for the quantification of ranolazine in human plasma. After liquid-liquid extraction of ranolazine and internal standard (ISTD) phenoprolamine from a 100 microl specimen of plasma, HPLC separation was achieved on a Nova-Pak C(18) column, using acetonitrile-water-formic acid-10% n-butylamine (70:30:0.5:0.08, v/v/v/v) as the mobile phase. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using the transition m/z 428.5-->m/z 279.1 for ranolazine and m/z 344.3-->m/z 165.1 for the internal standard, respectively. Linear calibration curves were obtained in the concentration range of 5-4000 ng/ml, with a lower limit of quantitation (LLOQ) of 5 ng/ml. The intra- and inter-day precision values were below 3.7% and accuracy was within +/-3.2% at all three quality control (QC) levels. This method was found suitable for the analysis of plasma samples collected during the phase I pharmacokinetic studies of ranolazine performed in 28 healthy volunteers after single oral doses from 200 mg to 800 mg.

A liquid chromatography with an electrochemical detector method has been developed for the quantitative measurement for three diamine derivatives (p-phenylenediamine, N,N(')-p-phenylenebisacetamide, and 4-aminoacetanilide) in human urine and rabbit blood, urine, and feces. The detection cell consisted of a glassy carbon electrochemical signal obtained with a supporting electrolyte containing 20% methanol-5mM octylammonium orthophosphate (pH 6.30) as the mobile phase. A comparison of the results obtained from HPLC-UV shows agreement.

A 47-year-old woman unwittingly ingested an unknown substance together with her breakfast coffee. She suffered effects such as strong headache, generalized cyanosis, and a burning sensation of the lips and collapsed some minutes later. After admission into hospital a methemoglobin level of 35% was determined in the blood. Treatment by administration of tolonium chloride (toluidine blue) resulted in complete recovery of the patient. The toxic agent was identified as aniline by GC with mass selective detection after organic solvent extraction and 11 h after ingestion the plasma aniline level was 0.13 mg/l. Acetanilide (0.79 mg/ml) and acetaminophen (2.3 mg/ml) were identified in plasma as metabolites of aniline. It was assumed that a high metabolic capacity for acetylation protected the victim from more severe reactions. Her husband confessed later that he had tried to poison her.

4,4'-Methylenediphenyl diisocyanate (MDI) is a very important component in the production of polyurethane. In a long-term experiment, designed to determine the carcinogenic and toxic effects of MDI, rats were exposed chronically for 3 and 12 months, to 0.0 (control), 0.26, 0.70 and 2.06 mg MDI/m3 as aerosols. Hemoglobin adducts and urine metabolites of MDI were determined at the different doses in order to develop methods to biomonitor workers exposed to MDI and to assess a risk resulting from such exposure. Hemoglobin adducts and urine metabolites of 4,4'-methylenedianiline (MDA) were found in all rats, including controls. MDA and N-acetyl-MDA (AcMDA) were quantified by GC-MS after derivatization with heptafluorobutyric anhydride. The dose-response relationships for hemoglobin adducts and urine metabolites were non-linear over this dose range. In urine, free AcMDA and MDA were found after base extraction. The amount of MDA present in urine and to a lesser extent the AcMDA found in urine correlate well with the corresponding amount determined as hemoglobin adducts for all dose groups. In order to release MDA from possible conjugates of MDA and AcMDA, urine was treated under strong acidic conditions. Following this procedure higher MDA levels were found than the sum of MDA and AcMDA from mild base hydrolysis. Similar results were obtained with the rats exposed for 3 and 12 months, indicating that a steady state had been reached by 3 months. In order to perform further investigations of the bronchoalveolar lavage fluid one group of animals was given a 1 week recovery period before sacrifice. Hemoglobin adducts from these animals showed a decrease of approximately 40% for all dose groups. According to the lifetime of rat erythrocytes the levels of hemoglobin adducts should have decreased by only 22%. This suggests that the erythrocytes with modified hemoglobin have a shorter lifespan. In order to exclude the possibility that hemoglobin adducts may have resulted from ingestion of hydrolyzed MDI via licking of the fur, a single dose experiment with rats exposed through the nose only or with the whole body was carried out. The only difference observed between these two exposure regimes was that the hemoglobin adduct levels of AcMDA after nose only exposure were significantly higher than after total body exposure. The presence of AcMDA in urine and as a hemoglobin adduct indicates that MDA was bioavailable after MDI exposure. The presence of MDA may contribute significantly to the carciongenic potential of MDI, since MDA has been shown to be carcinogenic in animals.

1. Propachlor-S-cysteine was the major metabolite found in systemic blood from rat, pig and calf given propachlor via the stomach. It was also the major metabolite found in the portal blood of pig; the portal blood of rat and calf was not examined. 2. Erythrocytes were the major transporter of propachlor metabolites in rat blood whereas plasma was the major transporter of these metabolites in pig and calf. 3. There was no evidence for metabolism of propachlor-S-cysteine by rat blood or by cytosol from rat, pig and calf erythrocytes.

The plasma elimination rates of phenacetin, acetanilide and theophylline have been determined in 32 healthy subjects in an effort to find drugs resembling in their metabolism the carcinogen benzo(a)pyrene. The plasma half-lives and metabolic clearance rates of the three drugs were correlated with the inducibilities of aryl hydrocarbon hydroxylase (AHH) in mitogen-stimulated lymphocytes and the plasma half-lives and metabolic clearance rates of antipyrine determined in previous studies. Statistically significant correlations were found between the half-lives and metabolic clearance rates of phenacetin, acetanilide and theophylline and the AHH ratios except for the metabolic clearance rates of phenacetin which did not correlate. The correlations of the three drugs with the half-lives and metabolic clearance rates of antipyrine were equally good. Of all the drugs tested thus far for similarity in metabolism to benzo(a)pyrene, antipyrine showed the best association followed closely by theophylline.

The plasma level of etidocaine was studied within the first two hours of axillary block in 7 patients with renal insufficiency and 7 healthy adults. After 15-20 min the maximum of plasma concentration was found in the group with renal failure and after 30-45 min in the healthy adults. 30 min after the block the plasma level was 1,26 +/- 0,62 mug/ml in the ill patients and only 0,73 +/- 0,31 in the normal group. Using a mathematical model the ratio of permeation into the blood is significantly higher in the group with renal failure than in the healthy adults. The main reason for these results seems to be the acidosis, which is often combined with renal insufficiency. The meaning of these results for regional anaesthesia in patients like these is discussed.

Acetanilide like phenylbutazone is paraoxidized by the liver endoplasmic reticulum as a primary biotransformation step. Both compounds were given at different times to each of 10 healthy volunteer subjects and the plasma disappearances measured. Correlation was shown between plasma clearance values of the two compounds (r = + 0.7067; P < 0.05).Eight patients with hypoplastic anaemia after phenylbutazone therapy were investigated. Plasma clearance values and half lives of acetanilide were measured in this group of patients and compared with those of a group of 30 healthy volunteer controls. There was a significant decrease in clearance (P < 0.01) and lengthening of half lives (P < 0.001 in the patients with phenylbutazone-associated hypoplasia. Five patients with idiopathic aplastic anaemia-that is, without history of antecedent phenylbutazone ingestion-were similarly investigated with acetanilide and there was no significant difference between the results in these patients and those in the control group.It is suggested that relatively poor paraoxidation of phenylbutazone producing high blood concentrations on a given dose may be a factor responsible for the drug-associated hypoplasia even though it does not explain the similar pattern of adverse reactions reported in association with oral administration of the metabolite oxyphenbutazone.

The proposed in vivo methodology for the investigation of hydroxylation rates consists of of the i.v. administration of tritiated substrates and the collection of tritiated water (HTO) from exhaled air as a measure of HTO accumulation in body water. Specifically, HTO was assessed in exhaled water after i.v. administration of 3H-acetanilide. Over a wide range the half lives of accumulation of HTO in exhaled water (T50) were almost identical with the half lives of elimination of 3H-acetanilide in blood, evaluated by an inverse isotope dilution method (r = 0.96, N = 18). Average T50 amounted to 29 min in controls, was reduced to 20 min after enzyme induction by phenobarbital or 3-methylcholanthrene, and prolonged to 45, 46 and 66 min after bile duct ligation, portacaval shunt and a single dose of ethanol, respectively. It is concluded that the chosen pharmacokinetic approach corrects for the NIH-shift and the results adequately reflect changes in acetanilide hydroxylation related to enzyme induction or inhibition and to liver pathology.