[Show abstract][Hide abstract] ABSTRACT: Triptolide (TP) is the major active principle of Tripterygium wilfordii Hook f. and very effective in treatment of autoimmune diseases. However, TP induced hepatotoxicity limited its clinical applications. Our previous study found that TP was a substrate of P-glycoprotein and its hepatobiliary clearance was markedly affected by P-gp modulation in sandwich-cultured rat hepatocytes. In this study, small interfering RNA (siRNA) and specific inhibitor tariquidar were used to investigate the impact of P-gp down regulation on TP-induced hepatotoxicity. The results showed that when the function of P-gp was inhibited by mdr1a-1 siRNA or tariquidar, the systemic and hepatic exposures of TP were significantly increased. The aggravated hepatotoxicity was evidenced with the remarkably lifted levels of serum biomarkers (ALT and AST) and pathological changes in liver. The other toxicological indicators (MDA, SOD and Bcl-2/Bax) were also significantly changed by P-gp inhibition. The data analysis showed that the increase of TP exposure in mice was quantitatively correlated to the enhanced hepatotoxicity, and the hepatic exposure was more relevant to the toxicity. P-gp mediated clearance played a significant role in TP detoxification. The risk of herb-drug interaction likely occurs when TP is concomitant with P-gp inhibitors or substrates in clinic.
[Show abstract][Hide abstract] ABSTRACT: Organophosphorus pesticides are the most widely used pesticides in modern agricultural systems to ensure good harvests. Isocarbophos (ICP), with a potent acetylcholinesterase inhibitory effect is widely utilized to control a variety of leaf-eating and soil insects. However, the characteristics of the bioactivation and detoxification of ICP in humans remain unclear. In this study, the oxidative metabolism, esterase hydrolysis, and chiral inversion of ICP in human liver microsomes (HLMs) were investigated with the aid of a stereoselective LC/MS/MS method. The depletion of ICP in HLMs was faster in the absence of carboxylesterase inhibitor (BNPP) than in the presence of NADPH and BNPP, with t1/2 of 5.2 and 90 min, respectively. Carboxylesterase was found to be responsible for the hydrolysis of ICP, the major metabolic pathway. CYP3A4, CYP1A2, CYP2D6, CYP2C9 and CYP2C19 were all involved in the secondary metabolism pathway of desulfuration of ICP. FMO didn't contribute to the clearance of ICP. The hydrolysis and desulfuration of (±) ICP, (+) ICP, and (-) ICP in HLMs follow Michaelis-Menten kinetics. Individual enantiomers of ICP and its oxidative desulfuration metabolite ICPO were found to be inhibitors of acetylcholinesterases at different extents. For example, (±) ICPO is more potent than ICP (IC50 0.031 v.s.192 μM), whereas (+) ICPO is more potent than (-) ICPO (IC50 0.017 v.s.1.55 μM). Given the finding of rapid hydrolysis of ICP and low abundance of oxidative metabolites presence in human liver, the current study highlights that human liver has a greater capacity for detoxification of ICP.
[Show abstract][Hide abstract] ABSTRACT: The metabolic characteristics of ligustrazin (TMPz) in liver microsomes were investigated in the present study. The reaction phenotyping of TMPz metabolism was also identified by in vitro assessment using recombinant human cytochrome P450 enzymes (CYP) and UDP glucuronosyltransferases (UGT). TMPz was incubated at 37 degrees C with human (HLM) and rat liver microsomes (RLM) in the presence of different co-factors. The metabolic stability and enzyme kinetics of TMPz were studied by determining its remaining concentrations with a LC-MS/MS method. TMPz was only metabolically eliminated in the microsomes with NADPH or NADPH+UDPGA. In the HLM and RLM with NADPH+UDPGA, t1/2, K(m) and V(max) of TMPz were 94.24 +/- 4.53 and 105.07 +/- 9.44 min, 22.74 +/- 1.89 and 33.09 +/- 2.74 micromol x L(-1), 253.50 +/- 10.06 and 190.40 +/- 8.35 nmol x min(-1) x mg(-1) (protein), respectively. TMPz showed a slightly higher metabolic rate in HLM than that in RLM. Its primary oxidative metabolites, 2-hydroxymethyl-3, 5, 6-trimethylpyrazine (HTMP), could undergo glucuronide conjugation. The CYP reaction phenotyping of TMPz metabolism was identified using a panel of recombinant CYP isoforms (rCYP) and specific CYP inhibitors in HLM. CYP1A2, 2C9 and 3A4 were found to be the major CYP isoforms involved in TMPz metabolism. Their individual contributions were assessed b) using the method of the total normalized rate to be 19.32%, 27.79% and 52.90%, respectively. It was observed that these CYP isoforms mediated the formation of HTMP in rCYP incubation. The UGT reaction phenotyping of HTMP glucuronidation was also investigated preliminarily by using a panel of 6 UGT isoforms (rUGT). UGT1A1, 1A4 and 1A6 were the predominant isoforms mediated the HTMP glucuronidation. The results above indicate that the metabolism of TMPz involves multiple enzymes mediated phase I and phase II reactions.
No preview · Article · Mar 2014 · Yao xue xue bao = Acta pharmaceutica Sinica
[Show abstract][Hide abstract] ABSTRACT: Triptolide (TP), a main bioactive component of Tripterygium wilfordii Hook F., is a promising agent for treatment of autoimmune diseases. However, a high incidence of dose-limiting hepatotoxicity was observed in the clinic. Sandwich-cultured rat heptocyte model was used in this study to identify the involvement of P-glycoprotein (P-gp) in TP disposition and to evaluate TP-induced hepatotocivity after modulation of P-gp by the known inhibitors, ritonavir and tariquidar, and known inducers, phenobarbital, quercetin, and H2O2. Our data showed that biliary clearance of TP reduced 73.7% and 84.2% upon treatment of ritonavir (25 μM) and tariquidar (5 μM), respectively. In contrast, increases of 346%, 280%, and 273% in biliary clearance of TP were observed with treatment of Phenobarbital (1.0 mM), quercetin (20 μM), and H2O2 (0.5 mM), respectively. The TP-induced heaptotoxicity increased by 2-fold when CYP activity was blocked by 1-Aminobenzotriazole, suggesting that CYP and P-gp may both contribute to the detoxification of TP in the SCRH model. In addition, hepatotoxicity and the expression of apoptosis proteins, Bax and Bcl-2, were correlated qualitatively with the TP exposure duration and its intracellular concentration which, in turn, can be modulated by P-gp inhibitors or inducers. Our results, for the first time, demonstrated that in addition to CYP-mediated metabolism, P-gp also plays an important role in the disposition of TP and TP induced hepatoxicity. Thus, the modulation of canalicular P-gp has a potential to cause drug-drug interaction between TP and the co-administered P-gp inhibitors or inducers in the clinic.
No preview · Article · Sep 2013 · Drug metabolism and disposition: the biological fate of chemicals
[Show abstract][Hide abstract] ABSTRACT: Psoralen (PRN) and isopsoralen (IPRN), naturally occurred furanocoumarin compounds, are the bioactive constituents found in herbaceous plants. They are widely used as active ingredients in several Chinese herb medicines. In this study, the CYP1A2 inhibitory potential of PRN and IPRN was investigated in rat in vitro and in vivo, and as well as in human liver microsomes. Both compounds exhibited reversible and time-dependent inhibition toward rat microsomal cyp1a2. The IC50, kinact, and KI were 10.4 ± 1.4 μM, 0.060 ± 0.002 min(-1), and 1.13 ± 0.12 μM for PRN, and 7.1 ± 0.6 μM, 0.10 ± 0.01 min(-1) and 1.95 ±0.31 μM for IPRN, respectively. In human liver microsomal incubations, much potent reversible CYP1A2 inhibition was observed for both compounds as the IC50 values were 0.26 ± 0.01 and 0.22 ± 0.03 μM for PRN and IPRN, respectively. However, time-dependent inhibition was only observed for IPRN with kinact and KI of 0.050 ± 0.002 min(-1) and 0.40 ± 0.06 μM, respectively. Co-administered with PRN or IPRN significantly inhibited cyp1a2 activity in rats, with AUC of phenacetin increasing more than 5-fold. Simcyp(®) simulation predicted that PRN would cause 1.71 and 2.12-fold increase in phenacetin AUC in healthy and smoker populations, respectively. IPRN, on the other hand, would result in 3.24 and 5.01-fold increase in phenacetin AUC in healthy and smoker populations, respectively. These findings represent the first detailed report on DDI potential comparison of PRN and IPRN, and provide useful information for balancing the safe and efficacious doses of PRN and IPRN.
Full-text · Article · Aug 2013 · Drug metabolism and disposition: the biological fate of chemicals
[Show abstract][Hide abstract] ABSTRACT: A quantitative method was established and validated for the simultaneous determination of six cytochrome P450 enzyme (CYP) probe metabolites using ultra. high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Human liver microsomal samples were processed with the mixed solvent of methanol and acetonitrile to precipitate proteins. The separation of the 6 probe metabolites was performed on a ZORBAX-C-18 column (100 mm x 4. 6 mm, 3. 5 mu m) by the gradient elution with the mobile phase consisting of 5mmol/L ammonium formate-0. 1% formic acid aqueous solution and acetonitrile. The mass spectrometric quantification was carried out in positive electrospray ionization and multiple reactions monitoring mode, with propranolol as the internal standard. The validation results indicated that the calibration curves of the 6 metabolites were linear (r(2) > 0. 994) over the range of 1. 0 similar to 1000 mu g/L, with the lower limits of quantification of 1 mu g/L. The intra. and inter. day precisions (RSD) were all below 12%. The recoveries for the spiked samples were in the range from 92. 8% to 104. 4%. The concentration variations (RSD) of the stability tests under different experimental conditions were all less than 10%. The results of human liver microsomes activity measurement showed that the CYP1A2 and CYP3A4 had the highest enzymatic activity of 466. 1 +/- 32. 1 and 694. 3 +/- 11. 7 pmole/(mg.min), which were 27. 7 and 41. 3 times of CYP2C19 activity, respectively. This method had been applied to the activity determination of the CYP isoforms. It was proved to be simple, rapid, sensitive and suitable for the evaluation of CYP inhibition and induction.
No preview · Article · Apr 2013 · CHINESE JOURNAL OF ANALYTICAL CHEMISTRY
[Show abstract][Hide abstract] ABSTRACT: An in vitro P-glycoprotein mediated drug biliary excretion model (B-Clear model) was developed and validated using sandwich-cultured rat hepatocytes (SCRH) and a model substrate rhodamine 123 (Rh123). SCRH formed functional bile canalicular networks after 5 days of culture. Rh123 (10 micromol x L(-1)) was then incubated with the SCRH in standard Ca+ Hanks buffer or Ca(2+)-free buffer. The cumulative cell uptake and canalicular efflux of Rh123 under Ca2+ and Ca(2+)-free conditions were measured with a LC-MS/MS method. The biliary excretion index (BEI) and instinct biliary clearance (CL(bile, int)) were calculated. To assess the effect of known P-gp inhibitors on the efflux of Rh123, cyclosporine A (CyA), tariquidar (TQD) or quinidine (QND) (10, 50 and 100 micromol x L(-1)) was pre-incubated separately with SCRH for 30 min, then co-incubated with Rh123. The BEI and CL(bile, int) of Rh123 obtained from the SCRH model were (17.8 +/- 1.3) % and (10.7 +/- 0.9) mL x min(-1) x kg(-1), respectively. All the three P-gp inhibitors showed a dose-dependent inhibition on the bile clearance of Rh123, indicating that the B-Clear model with SCRH was functional properly. The biliary excretion of loperamide (LPAD) and the role of P-gp were further investigated with this validated model. The BEI and CL(bile, int) for LPAD (20 micromol x L(-1)) were obtained after it was incubated with SCRH for 30 min, and found to be (12.9 +/- 1.2)% and (6.1 +/- 0.3) mL x min(-1) x kg(-1) respectively. The dose-dependent inhibition on LPAD biliary excretion by CyA, TQD or QND confirmed the major role of P-gp in LPAD canalicular efflux. The results suggested that the B-Clear model with SCRH would be a useful tool for evaluation of P-gp mediated efflux and drug-drug interaction.
No preview · Article · Apr 2012 · Yao xue xue bao = Acta pharmaceutica Sinica
[Show abstract][Hide abstract] ABSTRACT: To investigate the metabolism of 3-cyanomethyl-4-methyl-DCK (CMDCK), a novel anti-HIV agent, by human liver microsomes (HLMs) and recombinant cytochrome P450 enzymes (CYPs).
CMDCK was incubated with HLMs or a panel of recombinant cytochrome P450 enzymes including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5. LC-ion trap mass spectrometry was used to separate and identify CMDCK metabolites. In the experiments with recombinant cytochrome P450 enzymes, specific chemical inhibitors combined with CYP antibodies were used to identify the CYP isoforms involved in CMDCK metabolism.
CMDCK was rapidly and extensively metabolized by HLMs. Its intrinsic hepatic clearance estimated from the in vitro data was 19.4 mL·min(-1)·kg(-1), which was comparable to the mean human hepatic blood flow rate (20.7 mL·min(-1)·kg(-1)). The major metabolic pathway of CMDCK was oxidation, and a total of 14 metabolites were detected. CYP3A4 and 3A5 were found to be the principal CYP enzymes responsible for CMDCK metabolism.
CMDCK was metabolized rapidly and extensively in human hepatic microsomes to form a number of oxidative metabolites. CYP3A4 and 3A5 were the predominant enzymes responsible for the oxidation of CMDCK.
Full-text · Article · Oct 2011 · Acta Pharmacologica Sinica
[Show abstract][Hide abstract] ABSTRACT: The biotransformation, CYP reaction phenotyping, the impact of CYP inhibitors and enzyme kinetics of 3-cyanomethyl-4-methyl-DCK (CMDCK), a new anti-HIV preclinical candidate belonging to DCK analogs, were investigated in human intestinal microsomes and recombinant cytochrome P450 (CYP) enzymes. CMDCK (4 micromol L(-1)) was incubated with a panel of rCYP enzymes (CYP1A2, 2C9, 2C19, 2D6 and 3A4) in vitro. The remaining parent drug in incubates was quantitatively analyzed by a LC-MS method. CYP3A4 was identified as the principal CYP isoenzyme responsible for its metabolism in intestinal microsomes. The major metabolic pathway of CMDCK was oxidation and a number of oxidative metabolites were screened with LC-MS. The Km, Vmax, CLint and T1/2 of CMDCK obtained from human intestinal microsome were 45.6 micromol L(-1), 0.33 micromol L(-1) min(-1), 12.1 mL min(-1) kg(-1) and 25.7 min, respectively. Intestinal clearance of CMDCK was estimated from in vitro data to be 3.3 mL min(-1) kg(-1), and was almost equal to the intestinal blood flow rate (4.6 mL min(-1) kg(-1)). The selective CYP3A4 inhibitors, ketoconazole, troleandomycin and ritonavir demonstrated significant inhibitory effects on CMDCK intestinal metabolism, which suggested that co-administration of CMDCK with potent CYP3A inhibitors, such as ritonavir, might decrease its intestinal metabolic clearance and subsequently improve its bioavailability in body.
No preview · Article · Sep 2010 · Yao xue xue bao = Acta pharmaceutica Sinica
[Show abstract][Hide abstract] ABSTRACT: Rotundine (1 micromol L(-1)) was incubated with a panel of rCYP enzymes (1A2, 2C9, 2C19, 2D6 and 3A4) in vitro. The remained parent drug in incubates was quantitatively analyzed by an Agilent LC-MS. CYP2C19, 3A4 and 2D6 were identified to be the isoenzymes involved in the metabolism of rotundine. The individual contributions of CYP2C19, 3A4 and 2D6 to the rotundine metabolism were assessed using the method of total normalized rate to be 31.46%, 60.37% and 8.17%, respectively. The metabolites of rotundine in incubates were screened with ESI-MS at selected ion mode, and were further identified using MS2 spectra and precise molecular mass obtained from an Agilent LC/Q-TOF-MSMS, as well as MS(n) spectra of LC-iTrap-MS(n). The predominant metabolic pathway of rotundine in rCYP incubates was O-demethylation. A total 5 metabolites were identified including 4 isomerides of mono demethylated rotundine and one di-demethylated metabolite. The results also showed that CYP2C19, 2D6 and 3A4 mediated O-demethylation of methoxyl groups at different positions of rotundine. Furthermore, the ESI-MS cleavage patterns of rotundine and its metabolites were explored by using LC/Q-TOF-MSMS and LC/iTrap-MS(n) techniques.
No preview · Article · Mar 2010 · Yao xue xue bao = Acta pharmaceutica Sinica
[Show abstract][Hide abstract] ABSTRACT: The inter-species differences of thienorphine metabolism were investigated in human, Beagle dog and rat liver microsomes, by comparing enzyme kinetics of the parent drug and the formation of its major metabolites. The incubation systems of thienorphine with liver microsomes of the three species were optimized in terms of thienorphine concentration, microsomal protein content and incubation time. The concentrations of thienorphine and its metabolites in incubates were measured by a LC-MS/MS method. The biotransformation of thienorphine by human liver microsomes was the lowest among the three species. The K(m), V(max), CL(int) and T1/2 of thienorphine obtained from human liver microsomes were (4.00 +/- 0.59) micromol x L(-1), (0.21 +/- 0.06) micromol x L(-1) x min(-1), (117 +/- 3.19) mL x min(-1) x kg(-1) and (223 +/- 6.10) min, respectively. The corresponding kinetic parameters for dog and rat liver microsomes were (3.57 +/- 0.69) and (3.28 +/- 0.50) micromol x L(-1), (0.18 +/- 0.04) and (0.14 +/- 0.04) micromol x L(-1) x min(-1), (213 +/- 1.06) and (527 +/- 7.79) mL x min(-1) x kg(-1), (244 +/- 1.21) and (70.7 +/- 1.05) min, respectively. A total of six phase I metabolites were observed in liver microsomes, including one N-dealkylated metabolite, three oxidative metabolites and two N-dealkylated oxidation metabolites. All these six metabolites were detected in the liver microsomes of the three species. However, the relative amounts of the metabolites generated were different in three species. The results indicated that the major phase I metabolic pathway of thienorphine was similar in the liver microsomes from all three species. However, the inter-species differences observed were relative amounts of the metabolites as well as the metabolic characteristics of thienorphine in liver microsomal incubates.
Full-text · Article · Jan 2010 · Yao xue xue bao = Acta pharmaceutica Sinica
[Show abstract][Hide abstract] ABSTRACT: In prior investigation, we discovered that (3'R,4'R)-3-cyanomethyl-4-methyl-3',4'-di-O-(S)-camphanoyl-(+)-cis-khellactone (4, 3-cyanomethyl-4-methyl-DCK) showed promising anti-HIV activity. In these current studies, we developed and optimized successfully a practical 10-step synthesis for scale-up preparation to increase the overall yield of 4 from 7.8% to 32%. Furthermore, compound 4 exhibited broad-spectrum anti-HIV activity against wild-type and drug-resistant viral infection of CD4+ T cell lines as well as peripheral blood mononuclear cells by both laboratory-adapted and primary HIV-1 isolates with distinct subtypes and tropisms. Compound 4 was further subjected to in vitro and in vivo pharmacokinetic studies. These studies indicated that 4 has moderate cell permeability, moderate oral bioavailability, and low systemic clearance. These results suggest that 4 should be developed as a promising anti-HIV agent for development as a clinical trial candidate.
Full-text · Article · Dec 2008 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: TNP, [N-cyclopropylmethyl-7(-[(R)-1-hydroxy-1-methyl-3-(thien-2-yl)-propyl]-6,14- endo-ethanotetrahydronororipavine], is a potent and long-acting partial opioid agonist and currently under development as a promising drug candidate for treatment of opium addiction. The animal PK data showed that, TNP was metabolized into several metabolites via multiple biotransformation pathways in rat and undergone a significant first-pass metabolism after oral administration. The concentrations of TNP glucuronide conjugate were much higher than that of the parent in plasma in orally dosed rats and Beagles. Despite the extensive metabolism of TNP in liver, it was assumed that intestine may also play an important role in quick metabolic clearance of TNP, as a number of metabolites were detected in rat feces. In this study the contribution of intestine to the first-pass metabolism of TNP was evaluated using in situ perfused rat intestinal preparation and intestine-liver preparation.
The in situ perfused rat intestinal preparation and intestine-liver preparation were made as per Pang’s method. TNP (3 mg/kg) was dosed via duodenum of the preparations. The parent compound and its metabolites were quantitatively analyzed by a validated LC-MS/MS method.
On the in situ perfused rat intestinal preparation the absorption rate of TNP was 0.95 1·h-1 and the Cmax (1250 ng·mL-1) was observed at 150 min of perfusion. The conjugate and oxidative metabolites were detected. The Cmax of the conjugate in perfusate was 174 ng·mL-1. When perfused through the in situ intestine-liver preparation, TNP was detected in the perfusate in a constantly low level (200 ng·mL-1), while the concentration of its glucuronide conjugate was higher (250 ng·mL-1). The absorption and metabolism of the parent reached a steady state at 10 min of perfusion, indicating that the absorbed TNP could immediately metabolized by the liver. The AUC of the conjugate in the intestine preparation was counted as 70% of that in the intestine-liver preparation, suggesting that the majority of the conjugate was formed in the intestine. The intestine was the primal organ for phase II metabolism of TNP. The first-pass effect of the drug was intensified by its slow and incomplete absorption in intestine, resulting in the low oral bioavailability.
1. KS Pang et al: Disposition of enalapril in the perfused rat intestine-liver preparation:absorption,metabolism and first-pass effect. J Pharmacol Exp Ther. 1985, March,Vol.233.No.3:788-795.