Chunhua Xia

Herb-Drug Interactions: In Vivo and In Vitro Effect of Shenmai Injection, a Herbal Preparation, on the Metabolic Activities of Hepatic Cytochrome P450 3A1/2, 2C6, 1A2, and 2E1 in Rats

Shenmai injection (SMI), a mixture of Radix Ginseng and Radix Ophiopogonis, is one of the most popular herbal medicinal products and is widely used for the treatment of coronary atherosclerotic cardiopathy and viral myocarditis. The purpose of this study was to investigate the effect of SMI, in vivo and in vitro, on the metabolic activities of hepatic cytochrome CYP450 3A1/2, 2C6, 2E1, and 1A2 in rats. After a single or multiple pretreatment with SMI, the rats were administrated intravenously a cocktail containing midazolam (1 mg/kg), diclofenac (0.5 mg/kg), theophylline (1 mg/kg), and chlorzoxazone (0.5 mg/kg) as probe substrates of rat CYP450 3A1/2, 2C6, 1A2, and 2E1, respectively. Single and multiple SMI pretreatment to rats resulted in a rise of 33.8 % (p < 0.01) and 25.6 % (p < 0.01) in AUC for midazolam, and an increase in AUC for diclofenac by 14.7 % (p < 0.05) and 31.2 % (p < 0.01), respectively. However, the pharmacokinetics of chlorzoxazone and theophylline in rats was not altered markedly. In rat liver microsomes, linear mixed-type inhibition of SMI against the enzyme activities of CYP3A1/2, CYP2C6, and CYP1A2 was shown with IC(50) values of 3.3 %, 2.0 %, and 3.1 % and K(i) values of 3.8 %, 1.5 %. and 1.9 %, respectively. These in vivo and in vitro results demonstrated that SMI had the potential to inhibit the activities of hepatic CYP3A1/2 and CYP2C6, but might not significantly affect CYP1A2 and CYP2E1-mediated metabolism in rats.

A sensitive liquid chromatography–electrospray ionization–mass spectrometry method for the simultaneous determination of pentoxyverine citrate and guaifenesin in human plasma—application to pharmacokinetic and bioequivalence studies

A sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry method for the identification and quantification of pentoxyverine citrate and guaifenesin in human plasma has been developed. After extraction from plasma samples by ethyl acetate, the internal standard and analytes were separated by high-performance liquid chromatographic on a Shim-pack VP-ODS C(18) column (150 x 2.0 mm) using a mobile phase consisting of A (methanol) and B (0.4% glacial acetic acid and 4 mmol/L ammonium acetate) (A:B, 43 : 57). Analysis was performed on a Shimadzu LC/MS-2010A in selected ion monitoring mode with a positive electrospray ionization interface. The method was linear in the concentration range of 1.0-640.0 ng/mL for pentoxyverine citrate and 0.025-6.4 microg/mL for guaifenesin. The inter- and intra- precision were all within 12% and accuracy ranged from 85 to 115%.The lower limits of quantification were 1.0 ng/mL for pentoxyverine citrate and 25.0 ng/mL for guaifenesin. The extraction recovery was on average 81.95% for pentoxyverine citrate and 89.03% for guaifenesin. This is the first assay method reported for the simultaneous determination of pentoxyverine citrate and guaifenesin in plasma using one chromatographic run.

Identification of the active components in Shenmai injection that differentially affect Cyp3a4-mediated 1'-hydroxylation and 4-hydroxylation of midazolam.

Shenmai injection (SMI) is a popular herbal preparation that is widely used for the treatment of atherosclerotic coronary heart disease and viral myocarditis. In our previous study, SMI was shown to differentially affect CYP3A4-mediated 1′-hydroxylation and 4-hydroxylation of midazolam (MDZ). The present study was conducted to identify the active components in SMI responsible for the differential effects on MDZ metabolism, using in vitro incubation systems (rat and human liver microsomes and a recombinant CYP3A4 system) to measure 1′-hydroxylation and 4-hydroxylation of MDZ. First, different fractions of SMI were obtained by gradient elution on an solid phase extraction system and individually tested for their effects on MDZ metabolism. The results demonstrated that lipid-soluble constituents were likely to be the predominant active components of SMI. Second, the possible active components were gradually separated on an high-performance liquid chromatography system under different conditions and individually tested in vitro for their effects on MDZ metabolism. Third, the active component obtained in the above experiment was collected and subjected to structural analysis, and identified as panaxytriol (PXT). Finally, it was validated that PXT had significant differential effects on 1′-hydroxylation and 4-hydroxylation of MDZ in various in vitro systems that were similar to those of SMI. We conclude that PXT is the constituent of SMI responsible for the differential effects on CYP3A4-mediated 1′-hydroxylation and 4-hydroxylation of MDZ.

In vitro inhibition of UGT1A3, UGT1A4 by ursolic acid and oleanolic acid and drug–drug interaction risk prediction

Abstract 1. Ursolic acid (UA) and oleanolic acid (OA) may have important activity relevant to health and disease prevention. Thus, we studied the activity of UA and OA on UDP-glucuronosyltransferases (UGTs) and used trifluoperazine as a probe substrate to test UGT1A4 activity. Recombinant UGT-catalyzed 4-methylumbelliferone (4-MU) glucuronidation was used as a probe reaction for other UGT isoforms. 2. UA and OA inhibited UGT1A3 and UGT1A4 activity but did not inhibit other tested UGT isoforms. 3. UA-mediated inhibition of UGT1A3 catalyzed 4-MU-β-d-glucuronidation was via competitive inhibition (IC50 0.391 ± 0.013 μM; Ki 0.185 ± 0.015 μM). UA also competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC50 2.651 ± 0.201 μM; Ki 1.334 ± 0.146 μM). 4. OA offered mixed inhibition of UGT1A3-mediated 4-MU-β-d-glucuronidation (IC50 0.336 ± 0.013 μM; Ki 0.176 ± 0.007 μM) and competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC50 5.468 ± 0.697 μM; Ki 6.298 ± 0.891 μM). 5. Co-administering OA or UA with drugs or products that are substrates of UGT1A3 or UGT1A4 may produce drug-mediated side effects.

Contribution of cytochrome P450 isoforms to gliquidone metabolism in rats and human

Abstract 1. Gliquidone, a second generation sulfonylurea, is a widely used oral antidiabetic drug. Due to the differences in its rate of metabolism, gliquidone shows inter-subject variability in pharmacokinetic and pharmacodynamic profiles. 2. Cytochrome P450 (CYP450) isoforms are involved in the metabolism of a majority of drugs in clinical use and plays a significant role in reducing possible drug interactions. This research aimed to systematically study the contribution of various human CYP450 isoforms to gliquidone metabolism in vitro in rats and human. 3. In rat liver microsomes, gliquidone was metabolized mainly by the most abundant CYP2C. The other isoforms involved in the metabolism included CYP3A, CYP2D, CYP1A and CYP2E. 4. Further investigation of rat recombinant enzymes showed that CYP3A1 and CYP2C11 played a major role in gliquidone metabolism in vitro, while CYP2D1, CYP1A2 and CYP2E1 were also involved. 5. But the metabolism of gliquidone in the human liver microsomes was mainly mediated by CYP3A4. The other isoforms involved in this process were CYP2C9, CYP2C19 and CYP2D6. 6. The further study of human recombinant enzymes demonstrated that CYP3A4 was the principal isoform enzyme for the metabolism of gliquidone. The intrinsic clearance (Vmax/Km) of CYP3A4 during gliquidone metabolism was 3–12 times greater than that of other CYP450 isoforms including CYP2C9, CYP2D6 and CYP2C19. 7. These findings may assist in valuable prediction of potential interactions of gliquidone with other drugs that are CYP3A4 inhibitors or inducers and help to design more efficacious and safer pharmacotherapy for patients of diabetes mellitus.

Modulation of transporter activity of OATP1B1 and OATP1B3 by the major active components of Radix Ophiopogonis

Abstract Radix Ophiopogonis is often an integral part of many traditional Chinese formulas, such as Shenmai injection used to treat cardio-cerebrovascular diseases. This study aimed to investigate the influence of the four active components of Radix Ophiopogonis on the transport activity of OATP1B1 and OATP1B3. The uptake of rosuvastatin in OATP1B1-HEK293T cells were stimulated by methylophiopogonanone A (MA) and ophiopogonin D′ (OPD′) with EC50 calculated to be 11.33 ± 2.78 and 4.62 ± 0.64 μM, respectively. However, there were no remarkable influences on rosuvastatin uptake in the presence of methylophiopogonanone B (MB) or ophiopogonin D (OPD). The uptake of atorvastatin in OATP1B1-HEK293T cells can be increased by MA, MB, OPD and OPD′ with EC50 calculated to be 6.00 ± 1.60, 13.64 ± 4.07, 10.41 ± 1.28 and 3.68 ± 0.85 μM, respectively. The uptake of rosuvastatin in OATP1B3-HEK293T cells was scarcely influenced by MA, MB and OPD, but was considerably increased by OPD′ with an EC50 of 14.95 ± 1.62 μM. However, the uptake of telmisartan in OATP1B3-HEK293T cells was notably reduced by OPD′ with an IC50 of 4.44 ± 1.10 μM, and barely affected by MA, MB and OPD. The four active components of Radix Ophiopogonis affect the transporting activitives of OATP1B1 and OATP1B3 in a substrate-dependent manner.

Interaction of deoxyschizandrin and schizandrin B with liver uptake transporters OATP1B1 and OATP1B3

Abstract 1. Deoxyschizandrin and schizandrin B have diverse pharmacological effects, including hepatoprotective activity. We aim to study their hepatic uptake and their effects on the hepatic uptake of other clinical drugs mediated by OATP1B1 and OATP1B3. 2. Deoxyschizandrin exhibited a high affinity for OATP1B1 with Km of 17.61 ± 0.43 μM but a low affinity for OATP1B3. Similarly, schizandrin B also showed a strong affinity for OATP1B1 with Km of 18.45 ± 1.23 μM but a weak affinity for OATP1B3. 3. Atorvastatin and rifampicin could inhibit the uptake of deoxyschizandrin and schizandrin B mediated by OATP1B1. 4. Intriguingly, both deoxyschizandrin and schizandrin B significantly promoted the uptake of atorvastatin (with EC50 of 50.58 ± 8.08 and 24.70 ± 5.82 µM, respectively) and rosuvastatin (with EC50 of 13.46 ± 2.70 and 8.99 ± 4.73 µM, respectively) mediated by OATP1B1. Deoxyschizandrin could markedly promote the uptake of fluvastatin but inhibit the uptake of sodium taurocholate (TCNa) mediated by OATP1B1. 5. The promotion on hepatic uptake of statins mediated by OATP1B1 might lead to enhanced efficacy of cholesterol lowering and reduced risk of myopathy for hyperlipidemia patients when given statins together with deoxyschizandrin or schizandrin B.

CYP2C9 and OATP1B1 genetic polymorphisms affect the metabolism and transport of glimepiride and gliclazide

The therapeutic use of glimepiride and gliclazide shows substantial inter-individual variation in pharmacokinetics and pharmacodynamics in human populations, which might be caused by genetic differences among individuals. The aim of this study was to assess the effect of CYP2C9 and OATP1B1 genetic polymorphisms on the metabolism and transport of glimepiride and gliclazide. The uptake of glimepiride and gliclazide was measured in OATP1B1*1a, *5 and *15-HEK293T cells, and their metabolism was measured using CYP2C9*1, *2 and *3 recombinase by LC-MS. Glimepiride in OATP1B1*1a, *5 and *15-HEK293T cells had Vmax values of 155 ± 18.7, 80 ± 9.6, and 84.5 ± 8.2 pmol/min/mg, while gliclazide had Vmax values of 15.7 ± 4.6, 7.2 ± 2.5, and 8.7 ± 2.4 pmol/min/mg, respectively. The clearance of glimepiride and gliclazide in OATP1B1*5 and *15 was significantly reduced compared to the wild-type. Glimepiride in the presence of CYP2C9*1, *2 and *3 recombinase had Vmax values of 21.58 ± 7.78, 15.69 ± 5.59, and 9.17 ± 3.03 nmol/min/mg protein, while gliclazide had Vmax values of 15.73 ± 3.11, 10.53 ± 4.06, and 6.21 ± 2.94 nmol/min/mg protein, respectively. The clearance of glimepiride and gliclazide in CYP2C9*2 and *3 was significantly reduced compared to the wild-type. These findings collectively indicate that OATP1B1*5 and *15 and CYP2C9*2 and *3 have a significant effect on the transport and metabolism of glimepiride and gliclazide.

Ginsenoside Rb1 and Rd Remarkably Inhibited the Hepatic Uptake of Ophiopogonin D in Shenmai Injection Mediated by OATPs/oatps

Shenmai injection (SMI) is derived from traditional Chinese herbal prescription Shendong yin and widely used for treating cardiovascular diseases. Ophiopogonin D (OPD) is one of the main active components of SMI. The hepatic uptake of OPD is mediated by organic anion-transporting polypeptides (OATPs/oatps) and inhibited by some other components in SMI. This study aimed to identify the active components of SMI responsible for the inhibitory effects on hepatic uptake of OPD in rats and explore the compatibility mechanisms of complex components in SMI based on OATPs/oatps. The known effective fractions, the known components in Shenmai Formula, and the fractions obtained from SMI by HPLC gradual-separation technology were individually/combinedly tested for their effects on OPD uptake in rat primary hepatocytes and recombinant OATP1B1/OATP1B3-expressing HEK293T cells. The results indicated that the OPD uptake was inhibited by panaxadiol-type ginsenosides (ginsenoside Rb1 and Rd), but slightly influenced by panaxatriol-type ginsenosides in rat primary hepatocytes and recombinant cells. The fractions of SMI-3-1 (0–11 min) and SMI-3-3 (15–20 min) obtained by HPLC gradual-separation technology were proved to be the major effective fractions that influenced the OPD uptake, and subsequently identified as ginsenoside Rb1 and Rd, respectively. The plasma concentrations of OPD in rats given OPD+ginsenoside Rb1+ginsenoside Rd were higher compared to rats given OPD alone at the same dose. In conclusion, ginsenoside Rb1 and Rd are the major effective components in SMI that remarkably inhibited the hepatic OPD uptake mediated by OATPs/oatps. The interaction of complex components by OATPs/oatps may be one of the important compatibility mechanisms in SMI.

Interaction of Sulfonylureas with Liver Uptake Transporters OATP1B1 and OATP1B3.

Sulfonylureas (SUs) such as glibenclamide, gliclazide, glimepiride, glipizide and gliquidone are one of the first oral medicines available for the treatment of type 2 diabetes and are widely used for the treatment of hyperglycaemia. The hepatic transporters, organic anion transporting polypeptide 1B1 (OATP1B1) and organic anion transporting polypeptide 1B3 (OATP1B3), play an important role in the disposition of a variety of drugs by mediating their uptake from blood into hepatocytes. Drug–drug interactions mediated by OATP1B1/1B3 may result in the hepatic transporting change for drug substrates. The inhibitory effects of glibenclamide and glimepiride on sulfobromophthalein (BSP) uptake have been previously studied, and glibenclamide has been reported as the substrate of OATP1B3, but it remains unclear whether other SUs such as gliclazide, glipizide and gliquidone are substrates of OATP1B1 and OATP1B3. Here, we investigated the relationship between the five most commonly applied SUs (glibenclamide, gliclazide, glimepiride, glipizide, gliquidone) and OATP1B1 and OATP1B3. We performed uptake and inhibition assays in HEK293T cells stably expressing OATP1B1 or OATP1B3, respectively, and established a liquid chromatography–mass spectrometry (LC‐MS) method for the simultaneous measurement of five SUs. We demonstrated that gliclazide and glimepiride are substrates of OATP1B1 and glibenclamide and glipizide are substrates of OATP1B3. We also confirmed the interaction between these SUs and rosuvastatin. No transporting was observed for gliquidone, suggesting that it is not a substrate of either transporter.