Development of a high-performance liquid chromatography-tandem mass spectrometric method for the determination of Methimazole in human blood matrices.

Methimazole (MMI, 1-methyl-2-mercaptoimidazole) is widely used for the treatment of hyperthyroidisms. There are methods available for the measurement of MMI concentration in human serum or plasma from the past, but none meet the current regulatory standards for bioanalytical method validations. In this paper, we developed and validated a total MMI measurement method using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a technique that conforms to current bioanalytical method validation. To form a free sulfhydryl group on MMI, sodium bisulfite was added to 50 µl of plasma or serum samples containing MMI before the derivatization step. The internal standard (MMI-D3) was spiked into samples, then these samples were derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole. After derivatization, these samples were extracted by supported liquid extraction. Then, the organic solvent was evaporated and the residue was dissolved in 50% methanol and injected into the LC-MS/MS system. A calibration curve was plotted over the concentration range 1-1000 ng/mL (r2 = 0.999). The intra-day and inter-day precisions were less than 10.2% and 9.8%, respectively. The intra-day and inter-day accuracies were between 89.5% and 101.1%, and 96.0% and 99.7%, respectively. The long-term stability of samples showed good precision and accuracy. The validated method was successfully applied to determine serum total MMI concentration in Graves' disease patients after oral administration of 5, 10 or 15 mg MMI. The range of circulating total MMI concentrations was found to be between 2.69 and 304.27 ng/mL in this study. It was shown that the measured serum total MMI concentrations changed in a dose-dependent manner.

Unconjugated bile acids in rat brain: Analytical method based on LC/ESI-MS/MS with chemical derivatization and estimation of their origin by comparison to serum levels.

Although some studies have revealed the implication of bile acids (BAs) and neurological diseases, the levels and origin of the BAs in the brain are not fully understood. In this study, we first developed and validated a sensitive and specific method for the determination of three unconjugated BAs [cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA)] in the rat brain by liquid chromatography/electrospray ionization-tandem mass spectrometry combined with chemical derivatization. The measured brain concentrations (mean±standard deviation, n=10) of normal rats were 58.7±48.8, 14.2±11.7 and 13.2±8.7ng/g tissue for CA, CDCA and DCA, respectively. For their origin, we developed the hypothesis that they might be mostly derived from the periphery. To test this hypothesis, the brain BA levels were compared with the serum levels. The brain levels had high correlations with the serum levels, and were always lower than the serum levels for the three unconjugated BAs. Furthermore, the higher brain-to-serum concentration ratios were found for the BAs with higher logD values (higher lipophilicity). Moreover, the brains of the rats intraperitoneally administered with deuterium-labeled CA and CDCA were also analyzed; the deuterium-labeled BAs were detected in the brain of the rats administered with these compounds. Based on all the results, we concluded that the BAs found in the brain are mostly derived from the periphery and the major mechanism for the transportation of the unconjugated BAs to the brain is by passive diffusion.

Development of analytical method for simultaneous determination of five rodent unique bile acids in rat plasma using ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry.

Bile acids (BAs) are crucial for the diagnosis, follow-up, and prognostics of liver injuries and other BA metabolism related diseases. In particular, rodent unique BAs, α-muricholic acid (α-MCA), ß-MCA, ω-MCA, tauro-α-MCA (α-TMCA), and ß-TMCA, are valuable biomarkers for preclinical drug development. To the best of our knowledge, however, a simple, selective, sensitive, and robust analytical method for ω-MCA and taurine-conjugated MCAs has never been reported. We have developed a simple, selective, and sensitive analytical method for measurement of 16 BAs including the five rodent unique BAs in rat plasma using an ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) method. Activated charcoal was utilized to prepare BA-free plasma, which served as the surrogate matrix for the preparation of calibration standards and quality control (QC) samples. Results of matrix effects evaluation suggested that the BA-free plasma could be adequate as a surrogate matrix for BAs determination. Three stable isotope labelled internal standards were separated by reverse phase UPLC using gradient elution and were detected by TOF-MS in negative ion mode. The calibration curve was linear for all BAs over a range of 10-25ng/mL to 1000-10,000ng/mL, with overall imprecision below 15% and 20% at lower limit of quantification (LLOQ), respectively. This analytical method was used to determine BA concentrations in more than 300 plasma samples from rats with liver injuries induced using α-naphthylisocyanate, carbon tetrachloride, or flutamide. The alteration of BA concentrations was most evident for necrosis, and cholestasis hepatotoxins, with more subtle effects by steatosis and idiosyncratic hepatotoxins. In conclusion, we have developed a simple, selective, and sensitive analytical method to measure plasma 16 BAs including 5 rodent unique BAs, α-MCA, ß-MCA, ω-MCA, α-TMCA, and ß-TMCA. Our data suggested that α-TMCA and ß-TMCA could be useful for identification or prediction of liver injuries, a currently unmet need in preclinical toxicity. Our method using TOF-MS is useful to determine BAs in rat plasma and of use in structural analyses of metabolites in early stage of drug development.

Analysis of the disposition of a novel p38 MAPK inhibitor, AKP-001, and its metabolites in rats with a simple physiologically based pharmacokinetic model.

5-[(2-Chloro-6-fluorophenyl)acetylamino]-3-(4-fluorophenyl)-4-(4-pyrimidinyl)isoxazole (AKP-001) is a potent p38 mitogen-activated protein kinase inhibitor that is being developed to specifically target the intestines for the treatment of inflammatory bowel disease. According to the ante-drug concept, AKP-001 was designed to be metabolized to inactive forms via the first-pass metabolism to avoid undesirable systemic exposure. The purpose of this study is to investigate the pharmacokinetic characteristics of AKP-001 and its metabolites (M1 and M2) in rats, utilizing a simple physiologically based pharmacokinetic (PBPK) model. In vitro metabolic activity of AKP-001 in the S9 fraction of rat liver was examined, and plasma concentration-time profiles were developed following intravenous and/or oral administration of AKP-001 and its metabolites. AKP-001 was primarily metabolized to M1; however, M2 was not detected in liver S9 fractions. In accordance with this observation in vitro, M2 was detected in plasma after oral dosing of AKP-001 with a lag time of 1.5 hours, but not after intravenous dosing. To analyze pharmacokinetics in rats in vivo, a simple PBPK model was developed by simultaneous fitting of the plasma concentrations after treatment with AKP-001 and its metabolites. The observed plasma concentration-time profiles of AKP-001 and metabolites were described by the model adequately. Intestinal and systemic exposures of AKP-001 were simulated using the model to assess the relationship between pharmacokinetics and efficacy/safety. Model analysis suggested that oral bioavailability of intestine-targeting ante-drugs should be low to avoid systemic side effects. The pharmacokinetic properties of AKP-001 meet this criterion owing to extensive first-pass metabolism.

Effects of triiodothyronine on turnover rate and metabolizing enzymes for thyroxine in thyroidectomized rats.

AIM: Previous studies in rats have indicated that surgical thyroidectomy represses turnover of serum thyroxine (T4). However, the mechanism of this process has not been identified. To clarify the mechanism, we studied adaptive variation of metabolic enzymes involved in T4 turnover. MAIN METHODS: We compared serum T4 turnover rates in thyroidectomized (Tx) rats with or without infusion of active thyroid hormone, triiodothyronine (T3). Furthermore, the levels of mRNA expression and activity of the metabolizing enzymes, deiodinase type 1 (D1), type 2 (D2), uridine diphosphate-glucuronosyltransferase (UGT), and sulfotransferase were also compared in several tissues with or without T3 infusion. KEY FINDINGS: After the T3 infusion, the turnover rate of serum T4 in Tx rats returned to normal. Although mRNA expression and activity of D1 decreased significantly in both liver and kidneys without T3 infusion, D2 expression and activity increased markedly in the brain, brown adipose tissue, and skeletal muscle. Surprisingly, hepatic UGT mRNA expression and activity in Tx rats increased significantly in comparison with normal rats, and returned to normal after T3 infusion. SIGNIFICANCE: This study suggests that repression of the disappearance of serum T4 in rats after Tx is a homeostatic response to decreased serum T3 concentrations. Additionally, T4 glucuronide is a storage form of T4, but may also have biological significance. These results suggest strongly that repression of deiodination of T4 by D1 in the liver and kidneys plays a major role in thyroid hormone homeostasis in Tx rats, and that hepatic UGT also plays a key role in this mechanism.

Perspectives on non-clinical safety evaluation of drug metabolites through the JSOT workshop.

The prompt and appropriate safety assessment of drug metabolite(s) was mentioned in regulatory guidances such as an International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidance, entitled "Guidance on Non-clinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals" (ICH M3(R2)) implemented in January 1 of 2011 in Japan, and has become a significant issue in the drug development. Upon release of ICH M3(R2) Step 4, a survey was conducted between March and April 2010 on the safety assessment of drug metabolites in 63 member companies of the Japan Pharmaceutical Manufacturers Association (JPMA). The Pharmacokinetics Team in the Non-Clinical Evaluation Expert Committee in JPMA conducted a questionnaire survey and compiled the results to comprehend how safety of drug metabolites are currently assessed at research-based pharmaceutical companies in Japan. The assessment of "Metabolites in Safety Testing" (MIST) can be divided into three stages based on the research purpose as follows: MIST 1 is a stage of estimating human drug metabolites and predicting their potential risks, MIST 2 is a stage of deciding the necessity for non-clinical safety studies, and MIST 3 is a stage of conducting non-clinical safety studies. In this paper, we propose typical approaches on safety assessment of metabolites that meet the purpose of each stage, considering the current level of scientific technology. Our proposals are based on the results from our survey and a symposium about the safety assessment of drug metabolites at the 37th annual meeting of the Japanese Society of Toxicology held in June 2010.

Influence of iodine-deficiency on thyroid hormones homeostasis in rats.

Little is known about the kinetics and metabolism of thyroid hormones in the hypothyroid state. In order to optimize hormone replacement therapy, it is important to understand variations in the kinetics and metabolism of thyroid hormones. To investigate these factors, we monitored serum thyroxine (T4) and triiodothyronine (T3) levels in iodine-deficient diet (ID) rats using online solid-phase extraction liquid chromatography-mass spectrometry/mass spectrometry (Online SPE LC-MS/MS). Furthermore, we evaluated supply and turnover rates of T4 in ID rats using a stable isotope-labeled T4 ([¹³C9]T4). Although serum T4 levels gradually declined after beginning ID treatment, T3 levels were unchanged throughout the experimental period. After intravenous administration of [¹³C9]T4 to ID rats, [¹³C9]T4 levels were monitored. We previously reported that significant differences of supply and turnover rates for T4 were observed in surgically thyroidectomized (Tx) rats. Surprisingly, there were no differences of supply and turnover rates for T4 between ID rats and intact rats. In conclusion, there were significant differences of supply and turnover rates for T4 between the hypothyroid states of ID and Tx rats. In ID rats, T3 might be preferentially biosynthesized in the thyroid, and ID treatment might not affect T4 kinetics. Our method, online SPE LC-MS/MS monitoring using a stable isotope tracer, has the potential to be used as a diagnostic tool to investigate the pathogenesis of thyroid disease and is valuable for optimizing the dosage in thyroid hormone replacement therapy.

Influence of thyroidectomy on thyroxine metabolism and turnover rate in rats.

Little is known about the kinetics and metabolism of thyroid hormones in the hypothyroid state. To investigate these factors, we developed a reliable method for measurement of serum thyroxine (T(4)), triiodothyronine (T(3)), reverse-T(3) (rT(3)) and stable isotope-labeled T(4) ([(13)C(9)]T(4)), using online solid-phase extraction liquid chromatography-mass spectrometry/mass spectrometry (online SPE LC-MS/MS). We measured supply and turnover rates of T(4) in thyroidectomized (Tx) rats using [(13)C(9)]T(4) as a tracer. In rats, serum T(4), T(3) and rT(3) were decreased but not completely ablated after surgical Tx. Endogenous T(4) and T(3) levels in Tx rats were maintained at a constant low level throughout the experimental period. [(13)C(9)]T(4) levels declined with a half-life of ∼1.2 days after it was administered to Tx rats intravenously. These findings strongly suggest that serum T(4) levels in Tx rats are maintained by T(4) supplied by extra-thyroidal tissues (e.g. secretion of extra-thyroidal storage, enhancement of enterohepatic recirculation, and production in extra-thyroidal tissues). Moreover, the turnover rate of T(4) in Tx rats was approximately twofold lower than in controls. This finding suggests that degradation of serum T(4) is repressed by Tx. In conclusion, serum T(4) is maintained at a constant low level by T(4) supply from extra-thyroidal tissues and repression of T(4) degradation in Tx rats. The powerful online SPE LC-MS/MS tool can be used to investigate thyroid hormones kinetics and metabolism, and thus has the potential to be used as a diagnostic tool and to investigate the pathogenesis of thyroid disease.

Discovery of a novel 5-HT(3) antagonist/5-HT(1A) agonist 3-amino-5,6,7,8-tetrahydro-2-{4-[4-(quinolin-2-yl)piperazin-1-yl]butyl}quinazolin-4(3H)-one (TZB-30878) as an orally bioavailable agent for irritable bowel syndrome.

We have prepared a series of quinazolinone derivatives linked with piperazinylquinoline for the treatment of irritable bowel syndrome (IBS). Using pharmacophore analysis, we designed and synthesized compounds which bind to both serotonin receptor subtype 1A (5-HT(1A)) and subtype 3 (5-HT(3)). Quinazolinone derivatives with a sulfur atom in the linker showed high affinity in in vitro assays, but low in vivo activity. Focusing on the linker to improve the pharmacokinetic profile, the sulfur atom in the linker was replaced with a methylene group. Further optimization led to the discovery of compound 17m (TZB-30878) ( J. Pharmacol. Exp. Ther. 2007 , 322 , 1315 - 1323 , Patent WO2005082887 (A1), 2005 ), a novel 5-HT(1A) agonist/5-HT(3) antagonist in the 3-aminoquinazolinone series. In in vivo functional assays, 17m dose dependently inhibited the Bezold-Jarisch reflex and induced 5-HT(1A)-mediated behaviors, and in an IBS animal model, 17m significantly inhibited stress-induced defecation. Pretreatment by WAY-100635 (5-HT(1A) antagonist) significantly attenuated but did not abolish the inhibitory effects of 17m. These results suggested that 17m exerted inhibitory effects via both 5-HT(1A) agonistic and 5-HT(3) antagonistic activities and that 17m would be useful as a therapeutic agent for IBS.

Biotransformation of 3-amino-5,6,7,8-tetrahydro-2-{4-[4-(quinolin-2-yl)piperazin-1-yl]butyl}quinazolin-4(3H)-one (TZB-30878), a novel 5-hydroxytryptamine (5-HT)1A agonist/5-HT3 antagonist, in human hepatic cytochrome P450 enzymes.

3-Amino-5,6,7,8-tetrahydro-2-{4-[4-(quinolin-2-yl)piperazin-1-yl]-butyl}quinazolin-4(3H)-one (TZB-30878), a novel 5-hydroxytryptamine (5-HT)(1A) agonist/5-HT(3) antagonist, is currently under development for the treatment of irritable bowel syndrome. The objective of this investigation was to obtain information on the biotransformation of TZB-30878. This compound has quinazoline, piperazine, and quinoline rings. Metabolites of [quinazoline-2-(14)C]TZB-30878 were determined using radio high-performance liquid chromatography on samples obtained after incubation with human hepatic microsomes. Eight metabolites were detected in the microsomal incubation mixture, and their structures were proposed by mass spectrometry techniques using TZB-30878 and two stable labeled TZB-30878 analogs, [quinoline-deuterium (D)(6)]TZB-30878 and [piperazin-D(8)]TZB-30878. Liquid chromatography/tandem mass spectrometry analyses suggested that the eight metabolites consisted of a cyclic metabolite (M6), four hydroxylated metabolites (M1, M2, M3, and M4) (three on quinoline ring and one on quinazoline ring), a deaminated metabolite (M5), and two metabolites (M7 and M8) that were presumably intermediates leading to the formation of the cyclic metabolite M6. Hydroxylation sites in the quinoline and quinazoline rings were predicted by electron density calculations and confirmed by comparison with authentic standards. To the best of our knowledge, N-deamination by microsomes leading to the formation of M5 appears to be novel. In addition, in vitro experiments in human liver microsomes with cytochrome P450 (P450)-specific inhibitors revealed that CYP3A4 was the major enzyme responsible for the metabolism of TZB-30878. Other P450 enzymes, such as a CYP2D6, played a minor role in its metabolism.

Metabolism of osaterone acetate in dogs and humans.

Osaterone acetate (17 alpha-acetoxy-6-chloro-2-oxa-4,6-pregnadiene-3,20-dione; OA) is a steroidal antiandrogen. In order to clarify the species differences, metabolites of OA were examined in plasma, urine, and feces of dogs and humans after oral administration of OA. Eleven metabolites in plasma, urine, and feces were identified by their spectral properties and comparison to appropriate standards. The primary routes of OA metabolism involve 11 beta-, 15 beta- and 21-hydroxylation, 17 alpha-deacetylation, and dechlorination. Other metabolites arise from combinations of these pathways to form multiple oxidized metabolites. All metabolites observed in humans occurred in dogs. 11 beta-Hydroxylated metabolites (11 beta-OH OA and 11-oxo OA) were found in the plasma and urine of dogs, but there was no evidence of their presence in humans. 11 beta-Hydroxylation of exogenous steroids represents a distinctive biotransformation pathway.

Pharmacokinetics and biliary excretion of osaterone acetate, a new steroidal antiandrogen, in dogs.

The pharmacokinetics and biliary excretion of osaterone acetate (17alpha-acetoxy-6-chloro-2-oxa-4,6-pregnadiene-3,20-dione; OA), a new steroidal antiandrogen, were investigated in intact dogs and biliary fistula dogs after bolus intravenous administration of (14)C-labeled drug. In intact dogs, OA exhibited a biexponential disposition with a very long half-life of 197.9 +/- 109.9 h. OA accounted for almost all the plasma radioactivity. The major route of excretion was in feces via the bile. One-third of the radioactivity in the bile was due to OA. The major biliary metabolite was identified as a glucuronide of 17alpha-acetoxy-6-chloro-21-hydroxy-2-oxa-4,6-pregnadiene-3,20-dione. A significant amount of biliary recycling occurs in dogs.