Therapeutic prospect of tetrandrine against SARS-CoV-2 based on its pulmonary pharmacology and exposure character / 药学学报

As the main active compound of Stephania tetrandra S. Moore, tetrandrine (TET) has been used to treat silicosis for nearly 50 years. TET has clear therapeutic effect on pulmonary fibrosis and lung cancer. A recent study suggests that TET may inhibit the replication of SARS-CoV-2 by blocking the two-pore channel 2 (TPC2), revealing its potential as a natural medicine to treat COVID-19. To explore the material basis of TET targeting lung efficacy and its potential toxicity, available literatures related to the pharmacological activity on pulmonary, dosage, toxicity and pharmacokinetics of TET are systemically reviewed. The prospect and current problems of TET to be a therapeutic agent for COVID-19 are further investigated on this basis.

Identification of human cytochrome P450 and UGT enzymes involved in the metabolism of ferulic acid, a major bioactive component in traditional Chinese medicines / 中国天然药物

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.

Phenoconversion of drug-metabolizing enzymes and its mechanism during inflammatory conditions / 国际药学研究杂志

Inflammatory conditions are associated with most diseases. Phenoconversion of drug-metabolizing enzymes (DME) leads to altered drug metabolism and disposition. It has profound impact on the pharmacotherapy of widely used clinically relevant medications in terms of safety and efficacy. More and more evidence has proved that elevated levels of proinflammatory cytokines may downregulate the expression and the activity of many Phase I and Phase “DME, which are involved in complex regulation mechanisms of drug disposition. The aim of this review is to present the recent findings in this area. Clinical practice based on personalized medicine according to DME phenotype with improved safety and efficiency can yield robust efficacy outcomes of drug treatment and has promising future prospects.

The evaluation of efflux transporter model based on RNA interference technology in vitro / 药学学报

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 / 药学学报

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.

Evaluation of P-glycoprotein mediated in vitro loperamide biliary excretion with sandwich-cultured rat hepatocytes model / 药学学报

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 3-cyanomethyl-4-methyl-DCK, a new anti-HIV candidate, in human intestinal microsomes / 药学学报

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.

In vitro comparison of thienorphine metabolism in liver microsomes of human, Beagle dog and rat / 药学学报

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 / 药学学报

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.