Cheng Huei Hsiong

Novel inhibition of cis/trans retinoic acid interconversion in biological fluids-an accurate method for determination of trans and 13-cis retinoic acid in biological fluids

All-trans retinoic acid (tRA, or tretinoin) can be metabolized through stereoisomerization to 13-cis retinoic acid (13-cRA) in vivo. We have developed a simple, sensitive and accurate method for analyzing tRA and 13-cRA in plasma with the addition of N-ethylmaleimide (NEM) and Vitamin C (Vit. C) to prevent the interconversion of cis/trans retinoic acid. All-trans RA, 9-cRA, and 13-cRA were well separated from each other in plasma by using a C18 precolumn and a column with a gradient solvent system of mobile phases A and B at a flow rate of 1.0 ml/min. In addition, thermal stability of tRA and cRA in plasma during the sample preparation under the temperature of 0 and 25 degrees C were studied. Our results showed that (1) the interconversion ratios (%) (cRA/tRA and tRA/cRA) were decreased with the addition of NEM and Vit. C and the minimum concentrations of NEM and Vit. C to inhibit the interconversion were 50 and 150 microM, respectively, (2) higher concentrations of NEM and Vit. C were required to prevent the interconversion at higher temperature, (3) the precision and accuracy of calibration curve with various concentration of tRA (1-1000 ng/ml) and 13-cRA (5-800 ng/ml) in plasma showed good linearity (r(2)=0.9992 and 0.9994), and between-day errors expressed by coefficient of variation (CV, %) for tRA and 13-cRA which were both less than 5.6%, (4) the mean recovery of the analytes were 78-94% for tRA and 80-92% for 13-cRA at concentration range from 1 to 1000 ng/ml and 5 to 800 ng/ml, respectively, and (5) the limit of quantitation of tRA and 13-cRA were 1 and 5 ng/ml, respectively. In addition, the HPLC method had been successfully applied to the tRA pharmacokinetic study in two hepatoma patients after receiving 45 mg/m(2) per day orally. Thus, our results suggest that the HPLC method for analyzing tRA and 13-cRA in plasma with the addition of NEM and Vit. C is a simple, sensitive and accurate method.

Pharmacokinetics of Oral Rosiglitazone in Taiwanese and Post Hoc Comparisons with Caucasian, Japanese, Korean, and Mainland Chinese Subjects

PURPOSEnRosiglitazone, an insulin-sensitizing thiazolidinedione, acts as a ligand for the y-subtype of the peroxisome proliferator-activated receptor in the regulation of glucose homeostasis and lipid metabolism. The aims of this study were to determine the pharmacokinetics of oral rosiglitazone in Taiwanese and to post hoc compare the ethnic differences among Caucasian, Japanese, Korean, and Mainland Chinese.nnnMETHODSnTwelve Taiwanese healthy male subjects received 4 and 8 mg of rosiglitazone. Similar protocols were used in the previously unpublished studies conducted in 25 Caucasian, 32 Japanese, 8 Korean, and 12 Mainland Chinese healthy male subjects. The 4 mg dose data were used for ethnicity comparisons.nnnRESULTSnThe respective pharmacokinetic properties of Taiwanese, Caucasian, Japanese, Korean and Mainland Chinese are: terminal half-life (hr): 4.18 +/- 0.43, 3.96 +/- 1.31, 3.83 +/- 0.78, 4.70 +/- 1.19 and 4.37 +/- 0.63; Cmax (ng/ml): 384.1 +/- 59.3, 260.2 +/- 75.7, 401.9 +/- 102.3, 345.3 +/- 60.6, and 406.2 +/- 52.0; AUC0-inf (h*ng/ml): 2078 +/- 433, 1249 +/- 566, 1901 +/- 397, 1938 +/- 534, and 2158 +/- 498. The Cmax and AUC0-inf of Caucasian were significantly (p = 0.002, 0.008) lower and CL/F and V/F were significantly (p = 0.000, 0.003) higher than those of other races. These differences of Cmax, AUC0-inf, CL/F and V/F between Caucasian and other races became insignificant after normalized by dose and weight.nnnCONCLUSIONSnIn a given dose by body weight, ethnicity had no significant impact on the pharmacokinetics of rosiglitazone in normal healthy volunteers.

The NAT2 tag SNP rs1495741 correlates with the susceptibility of antituberculosis drug-induced hepatotoxicity.

Background Earlier studies have demonstrated an association between N-acetyltransferase 2 (NAT2) catalytic activity and the genotype of a recently published tag single nucleotide polymorphism (SNP), rs1495741. There have been no reports on the relationship between the rs1495741 genotype and antituberculosis drug-induced hepatotoxicity (ATDIH) to date. Objective The aim of the present study was to determine the frequency of the NAT2 tag SNP (rs1495741) in the Taiwanese and its relation to the incidence of ATDIH. Materials and methods A total of 348 tuberculosis patients were enrolled to determine the frequency of NAT2 tag SNP rs1495741 and its relation to the incidence of ATDIH. The conventional NAT2 variants alleles have also been investigated. Furthermore, to evaluate the correlation of NAT2 activity and rs1495741 genotypes, a pharmacokinetic study of isoniazid was also conducted in healthy volunteers. Results Among the 348 tuberculosis patients, 20 (5.7%) were diagnosed with ATDIH. The frequencies of the three rs1495741 genotypes, viz., AA, AG, and GG, were 24.7, 52.3, and 23.0%, respectively. Significant differences among rs1495741 genotypes and susceptibility to hepatotoxicity were noted (odds ratio=14.068, P<0.05). Moreover, the rs1495741 genotypes showed an association with the isoniazid dosage required for induction of hepatotoxicity. In the pharmacokinetic study, NAT2 activity was strongly associated with genotype categories (P<0.001). Conclusion The present study demonstrated that the three genotypes according to rs1495741 were in good accordance with conventional NAT2 alleles-inferred phenotypes and the tag SNP could be used as a proxy to determine the susceptibility to ATDIH.

Quantitative rat liver function test by galactose single point method

Summary The purpose of this study was to investigate the galactose single point (GSP) method, a residual liver function test recently recommended by the US Food and Drug Administration, which can be a useful tool for rat liver function measurement. Rats were treated either with carbon tetrachloride (CCl4) alone (1 mL/kg, intraperitoneally [i.p.]) for one day or with isoniazid (INH) alone (150 mg/kg, i.p.) or (in order to ameliorate the effects of INH) with a combination of INH and bis-p-nitrophenyl phosphate (BNPP) (25 mg/kg, i.p.) for 21 days. Hepatotoxicity was assayed by plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and scores of histological activity index-necroinflammation (HAI-NI) of the respective liver specimens. The GSP method in rats was defined by the galactose blood level after 60 min. Significant differences in GSP values were observed between controls and the CCl4-treated rats. After 21 days of treatment, no significant changes in AST and ALT values were observed among the control, INH and INH-BNPP groups. There were significant differences in average GSP values for controls (P < 0.001) and INH-BNPP (P < 0.001) compared with INH alone. Highly significant correlations (P < 0.001) were obtained between GSP and scores of HAI-NI for all the groups. GSP was concluded to be a more sensitive biomarker of INH-induced hepatotoxicity than AST or ALT in the rats. The GSP method has been proved to be a simple and useful tool for the quantitative determination of liver function in rats, which can possibly be extended to other animals.

Association of Galactose Single-Point Test Levels and Phenytoin Metabolic Polymorphisms with Gingival Hyperplasia in Patients Receiving Long-Term Phenytoin Therapy

Study Objective. To evaluate whether the occurrence or severity of gingival hyperplasia is associated with liver function test results or phenytoin metabolism.

Commonly Used Excipients Modulate UDP-Glucuronosyltransferase 2B7 Activity to Improve Nalbuphine Oral Bioavailability in Humans

PurposeNalbuphine (NAL) is a potent opioid analgesic, but can only be administered by injection. The major aim of this study was to develop an oral NAL formulation employing known excipients as UDP-glucuronosyltransferase 2B7 (UGT2B7) inhibitors to improve its oral bioavailability.MethodsTwenty commonly used pharmaceutical excipients were screened in vitro by using liver microsomes to identify inhibitors of UGT2B7, the major NAL metabolic enzyme. Tween 20 and PEG 400 were potent UGT2B7 inhibitors and both were co-administered (Tween-PEG) with NAL to rats and humans for pharmacokinetic and/or pharmacodynamic analyses.ResultsIn animal studies, oral Tween-PEG (4xa0mg/kg of each) significantly increased the area under the plasma NAL concentration-time curve (AUC) and the maximal plasma concentration (Cmax) by 4- and 5-fold, respectively. The results of the pharmacodynamic analysis were in agreement with those of the pharmacokinetic analysis, and showed that Tween-PEG significantly enhanced the analgesic effects of orally administered NAL. In humans, oral Tween-PEG (240xa0mg of each) also increased NAL Cmax 2.5-fold, and AUC by 1.6-fold.ConclusionsTween-PEG successfully improved oral NAL bioavailability and could formulate a useful oral dosage form for patient’s convenience.

Simultaneous determination of nalbuphine and its prodrug sebacoly dinalbuphine ester in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic study in humans

A rapid, simple, sensitive and selective ultraperformance liquid chromatography-tandem spectrometry (UPLC-MS/MS) method for the determination of nalbuphine and its prodrug sebacoly dinalbuphine ester (SDE) was developed and validated in human plasma. The sample pretreatment involves basification and iterative liquid-liquid extraction with ethyl-ether-dichloromethane (7:3, v/v) solution, followed by LC separation and positive electrospray ionization (ESI) API-3000 mass spectrometry detection. The chromatography was on a Waters Acquity UPLC BEH HILIC column (2.1 × 100 mm, 1.7 µm). The mobile phase was composed of acetonitrile and water (83:17, v/v) that contained 0.2% formic acid and 4 mm ammonium formate at a flow rate of 0.25 mL/min. Ethylmorphine and naloxine were selected as the SDE and nalbuphine internal standard (IS), respectively. The calibration curve for both was linear over the range from 0.05 to 20 ng/mL, with correlation coefficients ≥0.995. The lower limit of quantification was set at 0.05 ng/mL. The intra- and inter-day precision values for nalbuphine and SDE were acceptable as per FDA guidelines. The method was applied successfully to determine nalbuphine concentration in human plasma samples obtained from four Taiwanese volunteers receiving intramuscularly administration of sebacoyl dinalbuphine ester. The method is sensitive, selective and directly applicable to human pharmacokinetic studies involving nalbuphine.

Selected Pharmaceutical Excipient Prevent Isoniazid and Rifampicin Induced Hepatotoxicity

BACKGROUND & AIMSnThe incidence of isoniazid (INH)- and rifampicin (RIF)-induced abnormal liver enzyme activity is 27% but only 19% with INH alone. Cytochrome P450 2E1 (CYP2E1) is thought to contribute to the synergistic effects of RIF and INH. Pharmaceutical excipients are inactive ingredients that are added to a pharmaceutical compound. The purpose of this study was to screen excipients for CYP2E1 inhibition and identify whether the screened excipients prevented INH/RIF-induced hepatotoxicity.nnnMETHODSnFifty-five known pharmaceutical excipients were screened for CYP2E1 inhibition. The hepatotoxic doses of INH and RIF were 50 and 100 mg/kg/day, respectively. Hepatotoxicity was assessed by the galactose single point (GSP) method (a US Food and Drug Administration (FDA) recommended quantitative liver function test), liver histopathology, malondialdehyde (MDA) assay, and measurement of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity. We chose the CYP2E1-specific substrate chlorzoxazone to assess CYP2E1 activity in animal and human.nnnRESULTSnMannitol inhibited CYP2E1 activity by 54% in mice with INH/RIF-induced hepatotoxicity (p < 0.005). Serum AST, ALT and GSP levels were significantly increased 3.8- to 7.8-fold in these mice (p < 0.005), and these levels could be lowered by mannitol. Mannitol significantly alleviated the depletion of hepatic glutathione (GSH) and partially reversed the increase in MDA formation in mice treated with INH/RIF (p < 0.005). Mannitol also decreased CYP2E1 activity by 58% in humans (p < 0.005). Furthermore, an antituberculosis (TB) efficacy assay revealed that mannitol did not affect the anti-TB effects of INH/RIF.nnnCONCLUSIONSnMannitol, an FDA-approved excipient, was found to be a CYP2E1 inhibitor. Mannitol may be a useful adjuvant for drugs that induce hepatotoxicity through CYP2E1, such as INH and RIF.

New finding of nalbuphine metabolites in men: NMR spectroscopy and UPLC–MS/MS spectrometry assays in a pilot human study

A safe and efficient semi-synthetic narcotic nalbuphine (NAL) which was broadly applied in analgesic therapy has long been considered to eliminate from human body via phase II conjugation. However, up to the present, neither the complete metabolic pathways nor the identified metabolites of NAL have been clarified in documented reports. In this study, four novel metabolites were discovered by incubating NAL with human liver microsomes. These metabolites were later quantified in blood samples from human volunteers treated with NAL. An accurate and precise new method for simultaneously determining NAL and its metabolites was also established. Their chemical structures were elucidated on the basis of one- and two-dimensional NMR spectroscopic analyses including 1H–1H correlation spectroscopy, nuclear overhauser enhancement spectroscopy, heteronuclear single-quantum correlation, and heteronuclear multiple bond correlation, and further confirmed by mass spectrometry. The analytical method was validated and applied successfully to a pilot human study with ultra-high performance liquid chromatography–tandem mass spectrometry employed with positive ion electrospray ionization via multiple reaction monitoring mode. This is the first report on the qualitative and quantitative analysis of NAL coupled with its two hydroxylated (3′-hydroxynalbuphine and 4′-hydroxynalbuphine) and two conjugated metabolites (nalbuphine-3-β-d-glucuronide and nalbuphine-6-β-d-glucuronide). The present method offers a rapid and simple way of performing pharmacokinetic studies of NAL, and assists in elucidating its metabolic pathway in humans.

Genetic Polymorphisms of Metabolic Enzymes and the Pharmacokinetics of Indapamide in Taiwanese Subjects

To understand the genetic makeup and impact on pharmacokinetics (PK) in the Taiwanese population, we analyzed the pharmacogenetic (PG) profile and demonstrated its effects on enzyme metabolism using indapamide as an example. A multiplex mass spectrometry method was used to examine the single nucleotide polymorphism (SNP) profile of eight major phases I and II metabolic enzymes in 1,038 Taiwanese subjects. A PG/PK study was conducted in 24 healthy subjects to investigate the possible effects of 28 SNPs on drug biotransformation. Among the genetic profile analyzed, eight SNPs from CYP2A6, CYP2C19, CYP2D6, CYP2E1, CYP3A5, and UGT2B7 showed higher variant frequencies than those previously reported in Caucasians or Africans. For instance, we observed 14.7% frequency of the SNP rs5031016 (I471T) from CYP2A6 in Taiwanese, whereas 0% variation was reported in Caucasians and Africans. The PG/PK study of indapamide demonstrated that the polymorphic SNPs CYP2C9 rs4918758 and CYP2C19 rs4244285 appeared to confer lowered enzyme activity, as indicated by increased Cmax (25%u2009∼u200964%), increased area under the plasma level-time curves (30∼76%), increased area under the time infinity (43%u2009∼u200980%), and lower apparent clearance values than PK for wild-type indapamide. Our results reinforce the biochemical support of CYP2C19 in indapamide metabolism and identify a possible new participating enzyme CYP2C9. The PG/PK approach contributed toward understanding the genetic makeup of different ethnic groups and associations of enzymes in drug metabolism. It could be used to identify two genetic markers that enable to differentiate subjects with varied PK outcomes of indapamide.