In vivo pharmacokinetic interaction by ethanolic extract of Gymnema sylvestre with CYP2C9 (Tolbutamide), CYP3A4 (Amlodipine) and CYP1A2 (Phenacetin) in rats.

Gymnema sylvestre (GS) is a medicinal herb used for diabetes mellitus (DM). Herbs are gaining popularity as medicines in DM for its safety purpose. The aim of the present study was to evaluate in vivo pharmacokinetic (PK) interaction between allopathic drugs tolbutamide (TOLBU), amlodipine (AMLO), and phenacetin (PHENA) at low (L) and high (H) doses with ethanolic extract (EL) from GS. EL was extracted and subjected to TLC, total triterpenoid content (19.76 ± 0.02 W/W) and sterol content (0.1837 ± 0.0046 W/W) estimation followed by identification of phytoconstituents using HRLC-MS and GC-MS. PK interaction study with CYP2C9, CYP3A4 and CYP1A2 enzymes were assessed using TOLBU, AMLO and PHENA respectively to index cytochrome (CYP) mediated interaction in rats after concomitant administration of EL extract (400 mg/kg) from GS for 7 days. The rats were divided into four groups for each PK study where, group I and II were positive control for low and high dose of test drugs (CYP substrates) while group II and IV were orally administered EL. The PK study result of PHENA indicated that area under the plasma concentration-time curve (AUC0-24) was significantly (P < 0.0001) increased by 1.4 (L) and 1.3-fold (H), plasma concentration (Cmax) was significantly (P < 0.001) increased by 1.6 (L) and 1.4-fold (H). Whereas for TOLBU; clearance rate (CL) was significantly (P < 0.0001) decreased by 2.4 (L) and 2.3-fold (H), Cmax, was significantly (P < 0.001) decreased by 26.5% (L) and 50.4% (H) and AUC0-24 was significantly (P < 0.0001) decreased by 59.8% (L) and 57.5% (H). Thus, EL is seen to be interacting with CYP1A2 by inhibiting its metabolic activity. HRLC-MS and GC-MS helped identify the presence of gymnemic acid (GA), triterpenoids and steroids in EL which could be the reason for PK interaction of CYP1A2 and CYP2C9. Also, in silico structure based site of metabolism study showed Fe accessibility and intrinsic activity for GA-IV, GA-VI, GA-VII and GA-X with CYP2C9. PK parameters of AMLO were not significantly affected by pre-treatment of EL. Thereby our findings indicate that co-administration of GS with drugs that are metabolized by CYP2C9 and CYP1A2 could lead to potential HDI.

Effect of Evodiamine on CYP Enzymes in Rats by a Cocktail Method.

The aim of this study was to assess the influence of evodiamine on the activities of the drug-metabolizing enzymes cytochrome P450 (CYP) 1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 in rats. The activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 were measured using specific probe drugs. After pretreatment for 1 week with evodiamine or physiological saline (control group) by oral administration, probe drugs phenacetin (5.0 mg/kg; CYP1A2 activity), tolbutamide (1.0 mg/kg; CYP2C9 activity), omeprazole (10 mg/kg; CYP2C19 activity), metoprolol (20 mg/kg; CYP2D6 activity) and midazolam (10 mg/kg; CYP3A4 activity) were administered to rats by oral administration. The blood was then collected at different times for ultra-performance liquid chromatography-tandem mass spectrometry analysis. The data showed that evodiamine exhibits an inhibitory effect on CYP1A2, CYP2C9 and CYP2D6 by increasing t(1/2), Cmax and AUC(0-∞), and decreasing CL/F compared with those of the control group. However, no significant changes in CYP2C19 and CYP3A4 activities were observed. In conclusion, the results indicated that evodiamine could inhibit CYP1A2, CYP2C9 and CYP2D6, which may affect the disposition of medicines primarily dependent on these pathways. Our work may be the basis of related herb-drug interactions in the clinic.

Effects of suberoylanilide hydroxamic acid on rat cytochrome P450 enzyme activities.

Vorinostat (suberoylanilide hydroxamic acid, SAHA) is the first approved histone deacetylase (HDAC) inhibitor for the treatment of cutaneous T-cell lymphoma after progressive disease following two systemic therapies. The rats were randomly divided into SAHA groups (low, medium and high dosage) and control group. The SAHA group rats were given 12.3, 24.5, and 49 mg/kg SAHA, respectively, by continuous intragastric administration for 7 days. The influence of SAHA on the activities of CYP450 isoforms CYP2B6, CYP1A2, CYP2C19, CYP2D6 and CYP2C9 were evaluated by cocktail method, they were responsed by the changes of pharmacokinetic parameters of bupropion, phenacetin, tolbutamide, metroprolol and omeprazole. The five probe drugs were given to rats through intragastric administration, and the plasma concentration were determined by UPLC-MS/MS. The result of SAHA group compared to control group, there were statistical pharmacokinetics difference for bupropion, phenacetin, tolbutamide and metroprolol. Continuous intragastric administration for 7 days may induce the activities of CYP2C19 of rats, inhibit CYP1A2 and slightly inhibit CYP2B6 and CYP2D6 of rats. This may give advising for reasonable drug use after co-used with SAHA. The results indicated that drug co-administrated with SAHA may need dose adjustment. Furthermore, continuous intragastric administration of SAHA for 7 days, liver cell damaged, causing liver cell edema, in liver metabolism process.

Effects of hydroxysafflor yellow A on the activity and mRNA expression of four CYP isozymes in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional therapy with Chinese medicine, hydroxysafflor yellow A (HSYA), a main active component isolated from the dried flower of Carthamus tinctorius L., is the principal efficiency ingredient of Safflor Yellow Injection. Now HSYA has been demonstrated to have good pharmacological activities of antioxidation, myocardial and cerebral protective and neuroprotective effects. The purpose of this study was to find out whether HSYA influences the effect on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C11, CYP2D4 and CYP3A1) by using cocktail probe drugs in vivo; the influence on the levels of CYP mRNA was also studied. MATERIALS AND METHODS: A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg), dextromethorphan (20 mg/kg) and midazolam (10 mg/kg), was given as oral administration to rats treated with short or long period of intravenous HSYA via the caudal vein. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. In addition, real-time RT-PCR was performed to determine the effect of HSYA on the mRNA expression of CYP1A2, CYP2C11, CYP2D4 and CYP3A1 in rat liver. RESULTS: HSYA had significant inhibition effects on CYP1A2 and CYP2C11 in rats as oriented from the pharmacokinetic profiles of the probe drugs. Furthermore, HSYA had no effects on rat CYP2D4. However, CYP3A1 enzyme activity was induced by HSYA. The mRNA expression results were in accordance with the pharmacokinetic results. CONCLUSIONS: HSYA can either inhibit or induce activities of CYP1A2, CYP2C11 and CYP3A1. Therefore, co-administration of some CYP substrates with HSYA may need dose adjustment to avoid an undesirable herb-drug interaction.

Reduction of hydroxamic acids to the corresponding amides catalyzed by rabbit blood.

1. The hydroxamic acids N-hydroxyphenacetin and N-hydroxy-2-acetylaminofluorene were reduced to the corresponding amides, phenacetin and 2-acetylaminofluorene respectively by rabbit blood supplemented with both NAD(P)H and FAD. These reducing activities were found in erythrocytes but not in plasma, and were sensitive to inhibition by carbon monoxide and oxygen. When blood or erythrocytes were boiled, these activities were not abolished. 2. Haemoproteins such as haemoglobin and catalase exhibited the reductase activity in the presence of both NAD(P)H and FAD under anaerobic conditions. The activity was not abolished when the haemoproteins were boiled. 3. Haematin showed a significant reducing activity in the presence of these cofactors. The activity of haematin was also observed with the photochemically reduced form of FAD. 4. The reduction system in blood was composed of NAD(P)H, FAD and haemoglobin. Reduction appears to proceed in two steps, i.e. the reduction of FAD by NADH or NADPH, followed by the non-enzymatic reduction of the hydroxamic acids to the amides by reduced FAD, catalyzed by the haem group of haemoglobin in rabbit erythrocytes.

Possible enhancement of the first-pass metabolism of phenacetin by ingestion of grape juice in Chinese subjects.

AIMS: This serendipitous study revealed an unexpected effect of Jufeng grape juice on the CYP1A2-mediated metabolism of phenacetin. Investigation of the inhibition of CYP1A2 by grapefruit juice was involved but a translation error led to the grape juice substitution. METHODS: Twelve healthy subjects took a single oral dose of phenacetin (900 mg) on two randomized occasions together with 200 ml water or grape juice. Plasma phenacetin and paracetamol concentrations were assessed by h.p.l.c. RESULTS: Ingestion of grape juice was associated with reduced plasma phenacetin concentrations, while paracetamol levels were unaffected. Paracetamol to phenacetin AUC ratios increased from 13.9+/-3.1 to 24.3+/-3.8 after ingestion of grape juice. CONCLUSIONS: These findings suggest enhanced first-pass metabolism of phenacetin, due to CYP1A2 activation by grape juice or to desaturation of CYP1A2 isoenzymes secondary to a slower rate of phenacetin absorption.

Dose selection for carcinogenicity studies of pharmaceuticals: systemic exposure to phenacetin at carcinogenic dosage in the rat.

A systemic exposure-based alternative to the MTD (maximally tolerated dose) for high-dose selection in carcinogenicity studies of pharmaceuticals has been accepted by the ICH (International Conference on Harmonisation of Technical Requirements for the Registration of Pharmaceuticals for Human Use). As a result of a retrospective analysis performed by the U.S. FDA (United States Food and Drug Administration), a rat/human relative systemic exposure ratio of 25 is proposed by the ICH as an acceptable pharmacokinetic endpoint for high-dose selection. For use as a dose selection criterion, it is particularly important that the magnitude of the relative systemic exposure ratio should be sufficient to detect human pharmaceuticals classified by IARC (International Agency for Research on Cancer, World Health Organization) as known (category 1) or probable (category 2A) human carcinogens. For one of these, phenacetin (an IARC 2A compound and a rat carcinogen), a systemic exposure ratio of 15 was calculated by the FDA. This calculation was based on a number of extrapolations. The present study reports the actual systemic exposure to phenacetin in the rat under conditions mimicking the conditions in the carcinogenicity study used by the FDA to calculate the relative systemic exposure ratio of 15. The ratio was found to be 7, indicating that the carcinogenic potential of this particular probable human carcinogen could be detected at a considerably lower systemic exposure ratio than that proposed by the ICH.