Prediction of Human Pharmacokinetics of E0703, a Novel Radioprotective Agent, Using Physiologically Based Pharmacokinetic Modeling and an Interspecies Extrapolation Approach.

E0703, a new steroidal compound optimized from estradiol, significantly increased cell proliferation and the survival rate of KM mice and beagles after ionizing radiation. In this study, we characterize its preclinical pharmacokinetics (PK) and predict its human PK using a physiologically based pharmacokinetic (PBPK) model. The preclinical PK of E0703 was studied in mice and Rhesus monkeys. Asian human clearance (CL) values for E0703 were predicted from various allometric methods. The human PK profiles of E0703 (30 mg) were predicted by the PBPK model in Gastro Plus software 9.8 (SimulationsPlus, Lancaster, CA, USA). Furthermore, tissue distribution and the human PK profiles of different administration dosages and forms were predicted. The 0.002 L/h of CL and 0.005 L of Vss in mice were calculated and optimized from observed PK data. The plasma exposure of E0703 was availably predicted by the CL using the simple allometry (SA) method. The plasma concentration-time profiles of other dosages (20 and 40 mg) and two oral administrations (30 mg) were well-fitted to the observed values. In addition, the PK profile of target organs for E0703 exhibited a higher peak concentration (Cmax) and AUC than plasma. The developed E0703-PBPK model, which is precisely applicable to multiple species, benefits from further clinical development to predict PK in humans.

Identification of human cytochrome P450 and UGT enzymes involved in the metabolism of ferulic acid, a major bioactive component in traditional Chinese medicines.

Ferulic acid (FA) is an active component of herbal medicines. One of the best documented activities of FA is its antioxidant property. Moreover, FA exerts antiallergic, anti-inflammatory, and hepatoprotective effects. However, the metabolic pathways of FA in humans remain unclear. To identify whether human CYP or UGT enzymes are involved in the metabolism of FA, reaction phenotyping of FA was conducted using major CYP-selective chemical inhibitors together with individual CYP and UGT Supersomes. The CYP- and/or UGT-mediated metabolism kinetics were examined simultaneously or individually. Relative activity factor and total normalized rate approaches were used to assess the relative contributions of each major human CYPs towards the FA metabolism. Incubations of FA with human liver microsomes (HLM) displayed NADPH- and UDPGA-dependent metabolism with multiple CYP and UGT isoforms involved. CYPs and UGTs contributed equally to the metabolism of FA in HLM. Although CYP1A2 and CYP3A4 appeared to be the major contributors in the CYP-mediated clearance, their contributions to the overall clearance are still minor (< 25%). As a constitute of many food and herbs, FA poses low drug-drug interaction risk when co-administrated with other herbs or conventional medicines because multiple phase I and phase II enzymes are involved in its metabolism.

Inhibition of P-Glycoprotein and Multidrug Resistance-Associated Protein 2 Regulates the Hepatobiliary Excretion and Plasma Exposure of Thienorphine and Its Glucuronide Conjugate.

Thienorphine (TNP) is a novel partial opioid agonist that has completed phase II clinical evaluation as a promising drug candidate for the treatment of opioid dependence. Previous studies have shown that TNP and its glucuronide conjugate (TNP-G) undergo significant bile excretion. The purpose of this study was to investigate the roles of efflux transporters in regulating biliary excretion and plasma exposure of TNP and TNP-G. An ATPase assay suggested that TNP and TNP-G were substrates of P-gp and MRP2, respectively. The in vitro data from rat hepatocytes showed that bile excretion of TNP and TNP-G was regulated by the P-gp and MRP2 modulators. The accumulation of TNP and TNP-G in HepG2 cells significantly increased by the treatment of mdr1a or MRP2 siRNA for P-gp or MRP2 modulation. In intact rats, the bile excretion, and pharmacokinetic profiles of TNP and TNP-G were remarkably changed with tariquidar and probenecid pretreatment, respectively. Tariquidar increased the Cmax and AUC0-t and decreased MRT and T1/2 of TNP, whereas probenecid decreased the plasma exposure of TNP-G and increased its T1/2. Knockdown P-gp and MRP2 function using siRNA significantly increased the plasma exposure of TNP and TNP-G and reduced their mean retention time in mice. These results indicated the important roles of P-gp and MRP2 in hepatobiliary excretion and plasma exposure of TNP and TNP-G. Inhibition of the efflux transporters may affect the pharmacokinetics of TNP and result in a drug-drug interaction between TNP and the concomitant transporter inhibitor or inducer in clinic.

Inhibition of P-glycoprotein Gene Expression and Function Enhances Triptolide-induced Hepatotoxicity in Mice.

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.

[The evaluation of efflux transporter model based on RNA interference technology in vitro].

In the present study, the specifically knockdown models of P-gp or MRP2 were constructed by using a series of chemically synthesized small interfering RNA (siRNA) in vitro. The expression of P-gp and MRP2 was measured by real-time PCR and Western blot, and the function was evaluated by applying P-gp and MRP2 substrate, rhodamine and methotrexate. The results showed that MRP2 siRNA-3 or P-gp siRNA-2 significantly decreased the mRNA expression of MRP2 or P-gp, the inhibition ratio was 68% or 84%; MRP2 siRNA-3 or P-gp siRNA-2 at a dose of 80 nmol x L(-1) significantly reduced the protein expression of MRP2 or P-gp at 48 h after treatment, the inhibition ratio was 62% or 70%. Meanwhile, other transporters were not influenced by siRNA. When pretreatment with MRP2 siRNA-3 or P-gp siRNA-2, the efflux of methotrexate or rhodamine decreased significantly and the intra-cellular concentration increased. The results suggested that chemically synthesized siRNA could significantly inhibit the expression and function of MRP2 and P-gp, and the model of RNAi in vitro could be used to evaluate the role of efflux transporters in transportation of drugs.

[Investigation of metabolic kinetics and reaction phenotyping of ligustrazin by using liver microsomes and recombinant human enzymes].

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.

Neutralization of chemokine-like factor 1, a novel C-C chemokine, protects against focal cerebral ischemia by inhibiting neutrophil infiltration via MAPK pathways in rats.

BACKGROUND: Inflammation plays a key role in the pathophysiology of ischemic stroke. Some proinflammatory mediators, such as cytokines and chemokines, are produced in stroke. Chemokine-like factor 1 (CKLF1), as a novel C-C chemokine, displays chemotactic activities in a wide spectrum of leukocytes and plays an important role in brain development. In previous studies, we have found that the expression of CKLF1 increased in rats after focal cerebral ischemia and treatment with the CKLF1 antagonist C19 peptide decreased the infarct size and water content. However, the role of CKLF1 in stroke is still unclear. The objective of the present study was to ascertain the possible roles and mechanism of CKLF1 in ischemic brain injury by applying anti-CKLF1 antibody. METHODS: Male Sprague-Dawley rats were subjected to one-hour middle cerebral artery occlusion. Antibody to CKLF1 was applied to the right cerebral ventricle immediately after reperfusion; infarct volume and neurological score were measured at 24 and 72 hours after cerebral ischemia. RT-PCR, Western blotting and ELISA were utilized to characterize the expression of adhesion molecules, inflammatory factors and MAPK signal pathways. Immunohistochemical staining and myeloperoxidase activity was used to determine the extent of neutrophil infiltration. RESULTS: Treatment with anti-CKLF1 antibody significantly decreased neurological score and infarct volume in a dose-dependent manner at 24 and 72 hours after cerebral ischemia. Administration with anti-CKLF1 antibody lowered the level of inflammatory factors TNF-α, IL-1ß, MIP-2 and IL-8, the expression of adhesion molecules ICAM-1 and VCAM-1 in a dose-dependent manner. The results of immunohistochemical staining and detection of MPO activity indicated that anti-CKLF1 antibody inhibited neutrophil infiltration. Further studies suggested MAPK pathways associated with neutrophil infiltration in cerebral ischemia. CONCLUSIONS: Selective inhibition of CKLF1 activity significantly protects against ischemia/reperfusion injury by decreasing production of inflammatory mediators and expression of adhesion molecules, thereby reducing neutrophils recruitment to the ischemic area, possibly via inhibiting MAPK pathways. Therefore, CKLF1 may be a novel target for the treatment of stroke.

Contribution of carboxylesterase and cytochrome P450 to the bioactivation and detoxification of isocarbophos and its enantiomers in human liver microsomes.

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. Flavin-containing monooxygenase (FMO) did not 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 isocarbophos oxon (ICPO) were found to be inhibitors of acetylcholinesterases at different extents. For example, (±)ICPO is more potent than ICP (IC50 0.031µM vs. 192µM), whereas (+)ICPO is more potent than (-)ICPO (IC50 0.017µM vs. 1.55µM). 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.

Simultaneous determination of triptolide and its prodrug MC002 in dog blood by LC-MS/MS and its application in pharmacokinetic studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Tripterygium wilfordii HOOK F (TWHF) is a traditional Chinese medicine used in the treatment of various autoimmune diseases and inflammatory disorders including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and skin diseases. Triptolide (TP) is one of the main active ingredients of this traditional Chinese medicine. MC002 is a novel semi-synthetic derivate of TP which is highly water soluble, acts as a prodrug and is converted to TP in vivo. AIM OF THIS STUDY: A sensitive, rapid method for the simultaneous determination of TP and its chemo-unstable prodrug MC002 in dog blood was developed and validated using electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using this method, a pharmacokinetic study of MC002 and TP following an intravenous drip infusion of 0.2mg/kg MC002 in dogs was performed. MATERIALS AND METHODS: Chemo-degradation of the prodrug in blood samples was inhibited by the addition of a small amount of sodium fluoride solution before using liquid-liquid extraction with ethyl acetate. The concentrations of MC002 and TP in dog blood were determined using the LC-MS/MS method. RESULTS: The quantitative method showed good precision and stability and is suitable for the assay of biological samples. The pharmacokinetic study showed that the elimination of MC002 was faster than that of TP, and the concentrations and AUC0-t values of TP were higher than MC002. MC002 can rapidly convert to TP in vivo. CONCLUSIONS: This validated method was successfully applied in a pharmacokinetic study of MC002 following an intravenous drip infusion in dogs. With the development of this new prodrug of TP as a promising anti-cancer drug, this method is suitable for its further analysis in clinical studies.

Assessment of the roles of P-glycoprotein and cytochrome P450 in triptolide-induced liver toxicity in sandwich-cultured rat hepatocyte model.

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 hepatocyte model was used in this study to identify the involvement of P-glycoprotein (P-gp) in TP disposition and to evaluate TP-induced hepatotoxicity after modulation of P-gp by the known inhibitors, ritonavir and tariquidar, and known inducers, phenobarbital, quercetin, and H(2)O(2). 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 H(2)O(2) (0.5 mM), respectively. The TP-induced hepatotoxicity increased by twofold 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 hepatotoxicity. Thus, the modulation of canalicular P-gp has a potential to cause drug-drug interaction between TP and the coadministered P-gp inhibitors or inducers in the clinic.

Identification and characterization of psoralen and isopsoralen as potent CYP1A2 reversible and time-dependent inhibitors in human and rat preclinical studies.

Naturally occurring furanocoumarin compounds psoralen (PRN) and isopsoralen (IPRN) are bioactive constituents found in herbaceous plants. They are widely used as active ingredients in several Chinese herbal medicines. In this study, the CYP1A2 inhibitory potential of PRN and IPRN was investigated in rats in vitro and in vivo as well as in human liver microsomes. Both compounds exhibited reversible and time-dependent inhibition toward rat microsomal cyp1a2. The IC(50), k(inact), and K(I) values 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, potent reversible CYP1A2 inhibition was observed for both compounds, with IC(50) values of 0.26 ± 0.01 µM and 0.22 ± 0.03 µM for PRN and IPRN, respectively. However, time-dependent inhibition was only observed for IPRN, with kinact and KI values of 0.050 ± 0.002 min(-1) and 0.40 ± 0.06 µM, respectively. Coadministration with PRN or IPRN significantly inhibited cyp1a2 activity in rats, with the area under the curve (AUC) of phenacetin increasing more than 5-fold. Simcyp simulation predicted that PRN would cause 1.71- and 2.12-fold increases in the phenacetin AUC in healthy volunteers and smokers, respectively. IPRN, on the other hand, would result in 3.24- and 5.01-fold increases in phenacetin AUCs in healthy volunteers and smokers, respectively. These findings represent the first detailed report comparing the potential drug-drug interactions of PRN and IPRN, and provide useful information for balancing safe and efficacious doses of PRN and IPRN.

[Evaluation of P-glycoprotein mediated in vitro loperamide biliary excretion with sandwich-cultured rat hepatocytes model].

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.

Metabolism of novel anti-HIV agent 3-cyanomethyl-4-methyl-DCK by human liver microsomes and recombinant CYP enzymes.

AIM: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[In vitro comparison of thienorphine metabolism in liver microsomes of human, Beagle dog and rat].

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.

[In vitro O-demethylation of rotundine by recombinant human CYP isoenzymes].

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.

[Metabolism of 3-cyanomethyl-4-methyl-DCK, a new anti-HIV candidate, in human intestinal microsomes].

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.

Mechanistic studies of C-C bond cleavage of nitriles by dinuclear metal cryptates.

We previously reported that dinuclear copper(II) cryptate [Cu(2)L](4+) cleaves the C-C bond of acetonitrile at room temperature to produce a cyano-bridged dinuclear cryptate and methanol, whereby the reaction mechanism has not yet become clear. We have now systemically investigated this reaction, and four cryptates, [Cu(2)L](ClO(4))(4) (1), [Zn(2)L](ClO(4))(4) (2), [Cu(2)L(H(2)O)(2)](CF(3)SO(3))(4) (5), and [Cu(2)L(OH)(OH(2))](ClO(4))(3) (6) are reported here. Cryptates 1 and 2 can cleave the C--C bonds of acetonitrile, propionitrile, and benzonitrile at room temperature under open atmospheric conditions to give cyano-bridged cryptates [Cu(2)L(CN)](ClO(4))(3) (3) and [Zn(2)L(CN)](ClO(4))(3) (4), respectively, and the corresponding alcohol. In contrast, 5 and 6 do not show any C-C bond activation of nitriles, as the interior axial positions of Cu(II) in 5 and 6 are occupied by water/OH(-). The C-C bond cleavage of (S)-(+)-2-methylbutyronitrile by 2 produced (R)-(-)-2-butanol only; that is, the cleavage reaction proceeds through an S(N)2 pathway (Walden inversion).

Development and preclinical studies of broad-spectrum anti-HIV agent (3'R,4'R)-3-cyanomethyl-4-methyl-3',4'-di-O-(S)-camphanoyl-(+)-cis-khellactone (3-cyanomethyl-4-methyl-DCK).

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.

Determination of 4-dimethylaminophenol concentrations in dog blood using LC-ESI/MS/MS combined with precolumn derivatization.

A sensitive and reproducible LC-ESI/MS/MS method, which was combined with the precolumn dansyl chloride derivatization to enhance the signal intensity of analytes, was developed to determine blood 4-dimethylaminophenol (DMAP) concentrations. The linearity of the method was observed within the concentration range of 2-2000 ng/mL. The precision, accuracy, stability, recovery and matrix effect of the method were also investigated and found to meet the requirements for pharmacokinetic studies of the drug. By using this method, pharmacokinetic studies were conducted in dogs after i.m. and i.v. administrations. The results showed that DMAP could not only be absorbed into blood quickly after i.m., but also can be eliminated rapidly. Both i.m. and i.v. routes are appropriate for DMAP to be used in field first-aid. It has been proved that this LC-MS/MS combined with precolumn derivatization method can be used as a routine analytical method to provide enhanced measurements for blood DMAP concentrations. It is also useful for DMAP pharmacokinetic evaluation.

Anion recognition of chloride and bromide by a rigid dicobalt(II) cryptate.

The crystal structures of [Co 2L(Cl)](ClO 4) 3 ( 1), [Co 2L(Br)](ClO 4) 3 ( 2), [Co 2L(OH)(OH 2)]I 3 ( 3), and [Co 2L (1)(Cl)](ClO 4) 3 ( 4), the density functional theory calculations, as well as the binding constants of [Co 2L] (4+) toward Cl (-) and Br (-) and of [Co 2L (1)] (4+) toward Cl (-), are reported in this paper (L = N[(CH 2) 2NHCH 2(C 6H 4- p)CH 2NH(CH 2) 2] 3N, L (1) = N[(CH 2) 2NHCH 2(C 6H 4- m)CH 2NH(CH 2) 2] 3N). The rigid dicobalt(II) cryptate [Co 2L] (4+) shows the recognition of Cl (-) and Br (-) but not of F (-) and I (-), because of the size matching to its rigid cavity. We also found that the relative rigid tripodal skeleton of L than that of L (1) results in the higher affinity of [Co 2L] (4+) toward Cl (-). Magnetic susceptibility measurements of 1 and 2 indicate that the two Co(II) atoms in the cryptates are antiferromagnetically coupled through the Cl (-)/Br (-) bridge, with g = 2.19, J = -13.7 cm (-1) for 1, and g = 2.22, J = -17.1 cm (-1) for 2.