Drug Metabolism Letters - Volume 2, Issue 1, 2008

The aim of this article is to focus on the implementation and the application of matrix-assisted laser desorption/ ionization-imaging mass spectrometric system (MALDI-IMS) to determine the disposition or biotransformation pathway of terfenadine and its active metabolite, fexofenadine in mouse and rat whole-body tissue sections. Whole-body MALDI-IMS data showed that the poor oral bioavailability of terfenadine was largely due to high first-pass metabolism in the intestines and the liver before the compound reached systemic circulation.

CYP3A isoforms account for about 30% of total hepatic P450s. Most statins have been accepted probes for CYP3A4. The metabolic ratio of atorvastatin/ortho-hydroxyatorvastatin showed a bimodal distribution with respect to metabolism of atorvastatin. These observations showed that the frequency of occurrence of the poor metabolizer phenotype is 2.4 % in the Gujarat subjects.

Membrane transporters are critical for the uptake as well as elimination of chemicals and by-products of metabolism from the liver and kidneys. Since these proteins are important determinants of chemical disposition, changes in their expression in different disease states can modulate drug pharmacokinetics. The present study investigated alterations in the renal and hepatic expression of organic anion and cation transporters (Oats/Octs), multidrug resistance-associated proteins (Mrps), breast cancer resistance protein (Bcrp), P-glycoprotein (Pgp), and hepatic Na+-taurocholate cotransporting polypeptide (Ntcp) in type 2 diabetic rats. For this purpose, type 2 diabetes was induced by feeding male Sprague- Dawley rats a high fat diet followed by a single dose of streptozotocin (45 mg/kg, i.p., in 0.01 M citrate buffer pH 4.3) on day 14. Controls received normal diet and vehicle. Kidney and liver samples were collected on day 24 for generation of crude plasma membrane fractions and Western blot analysis of Oat, Oct, Mrp, Bcrp, Pgp, and Ntcp proteins. With regards to renal uptake transporters, type 2 diabetes increased levels of Oat2 (2.3-fold) and decreased levels of Oct2 to 50% of control kidneys. Conversely, efflux transporters Mrp2, Mrp4, and Bcrp were increased 5.4-fold, 2-fold, and 1.6-fold, respectively in type 2 diabetic kidneys with no change in levels of Mrp1, Mrp5, or Pgp. Studies of hepatic transporters in type 2 diabetic rats reveal that the protein level of Mrp5 was reduced to 4% of control livers with no change in levels of Bcrp, Mrp1, Mrp2, Mrp4, Ntcp, or Pgp. The changes reported in this study may have implications in type 2 diabetic patients.

Methylenetetrahydrofolate reductase (MTHFR) gene located on chromosome 1p36.3 catalyses the conversion of 5,10-methylenetetrahydrofolate to 5,methyltetrahydrofolate, the major methyl donor for the conversion of homocysteine to methionine. Two common polymorphisms in the MTHFR gene have been identified, 677C>T in exon 4, leading to substitution of alanine by valine and 1298A>C in exon 7 which leads to the replacement of glutamic acid by alanine resulting into reduced enzyme activity. The potential influence of MTHFR activity on DNA methylation and on the availiblity of uridylates and thymidylates for DNA synthesis and repair makes MTHFR an attractive candidate for cancer predisposing gene. In order to elucidate the role of MTHFR polymorphism in cervical cancer, both the exons for 677C>T and 1298A>C mutations were analyzed among 219 females, including 77 females with normal cervical cytology, 80 with cervical dysplasia and 62 with squamous cell carcinoma of uterine cervix. Females with mutant allele at 677 position (CT/TT genotypes) were found to be almost three times the risk of cervical dysplasia than females with CC genotype [OR, 2.9; (CI, 1.5-5.7)], but were less likely to develop squamous cell carcinoma [OR, 1.5 (CI, 0.7-3.2)]. Similar findings were observed for mutation at 1298 position, females with AC/CC genotypes were almost four times the risk of cervical dysplasia [OR, 4.3 (CI, 2.1-9.0)], as compared to AA genotype. Our study lends further support to the hypothesis that the MTHFR polymorphism (677C>T or 1298A>C) is involved in susceptibility to cervical dysplasia.

The pharmacokinetic profile of ceftriaxone was studied in female healthy goats, induced hepatopathic and nephropathic goats after a single intravenous dose at 50 mg kg-1. Ceftriaxone persisted for 2 h in plasma of hepatopathic goats compared to 1 h of healthy goats, but the kinetic behaviour followed ‘one-compartment open model’ in both healthy and hepatopathic goats. Mean value of t½β (0.32 ± 0.008 h) was significantly higher in hepatopathic goats compared to healthy goats (0.19 ± 0.002 h). Ceftriaxone was recovered at 24 h in urine of hepatopathic goats but it could not be detected in urine of healthy goats. However, its metabolite ceftizoxime was present in urine of healthy goats but not in urine of hepatopathic goats. On the other hand ceftriaxone persisted for 2 h in plasma of kidney damaged goats with significant higher concentration compared to healthy goats but kinetic behaviour followed ‘one Compartment open model’. Ceftizoxime was identified with an adequate plasma concentration from 8 h to 12 h post dosing in nephropathic goats. Elimination halflife (t½β) of Elimination ceftriaxone (0.38 ± 0.01 h) in nephropathic goats increased significantly compared to healthy goats (0.19 ± 0.002 h). Ceftriaxone, not the metabolite ceftizoxime was recovered at 24 h and 48 h post dosing in urine of nephropathic goats, while only ceftizoxime not ceftriaxone was detected in urine of healthy goats.

Flubendazole (FLU) is a widely administered benzimidazole anthelmintic indicated for the control of parasitic diseases in farm animals including pigs and pheasants. This study was designed to test the biotransformation of FLU in control animals and animals treated with FLU in recommended therapeutic doses. The activities of several pheasant and porcine hepatic and intestinal carbonyl reducing enzymes and their modulation by FLU were also studied. Twelve adult pheasant hens, approximately 1 year old, were divided into two groups and treated for 7 days with placebo or 6 mg of FLU/kg of body weight. Eight male hog weaners, approximately 3 month old, were divided into two groups and treated for 5 days with placebo or 1.57 mg of FLU/kg of body weight. Subcellular fractions, prepared from livers and small intestines of control and FLU treated animals, were incubated with FLU. In vitro formation of two main FLU metabolites, reduced FLU, and hydrolyzed FLU were analyzed using HPLC. While FLU was reduced significantly more intensively in FLU-treated pheasants than in control animals, no differences were observed in pigs. These results were confirmed by measuring the enzyme activities: carbonyl reducing enzyme activities were increased in pheasants treated by FLU, whereas FLU did not affect these enzymes in pigs.

High resolution accurate MS with an LTQ-Orbitrap identified two novel metabolites of diclofenac in rat bile and rat and human hepatocyte incubations: a benzyl-S-glutathione conjugate and 2-(2,6-dichlorophenylamino) benzoic acid. A mechanism for the bioactivation of diclofenac involving decarboxylation is proposed.

This study evaluated the reproducibility, sensitivity, accuracy, and precision of stop-flow liquid radiochromatographic detection (RFD). Stop-flow RFD was about 10-fold more sensitive compared with traditional RFD and had a good reproducibility for quantification and HPLC retention time. Stop-flow RFD was applied to determine enzyme kinetics of radiolabeled drugs. The enzyme kinetic parameters of muraglitazar glucuronidation determined by stop-flow RFD were comparable with those determined by microplate scintillation counting (MSC).

Doxorubicin is one of the most effective anti-tumour agent. To clarify whether a single dose produces its effects soon after dosing or only after hours or days and whether the effects are brief or prolonged, time-dose relationships were explored by calculating lethal time (LT50) values which is a statistical estimate of the time from dosage to death of 50% of the organisms/ or cells in a very large population subjected to a toxicant under specific conditions. This was achieved by the log-time log-dose curve which may be used to predict the proper doses to be used in long-term studies. Drug chemosensitivity using DU-145 cell lines have been investigated for this purpose. The results shows that the effects of doxorubicin started only after 24hrs; however, resistance was developed, 40 hrs was the time required to kill 50% of cells , and post-incubation with fresh media (F.M.) exhibited more cell damages. It is concluded that doxorubicin is effective only after 24 hrs with resistance developed and post-incubation with F.M after treating cells with doxorubicin causes more damage than continuous incubation with the drug.

The relationship between time-dependent inactivation (TDI) and IC50 is examined using a consolidated method for evaluating CYP450 inhibition during drug discovery. An IC50 fold-shift of >1.5 indicated significant TDI potency. Further, the “shifted IC50” could be used to estimate, the KI and TDI potency ratio kinact/KI to within 2-fold in most cases.

Ginkgo biloba is one of the most popular herbal medicines in the world, due to its purported pharmacological effects, including memory-enhancing, cognition-improving, and antiplatelet effects. The study aimed to investigate the activity and expression of cytochrome P450 (CYP) 3A in human and rat primary hepatocytes treated with standardized G. biloba extract (100, 500, and 2500 ng/ml) for 72 hr, and to measure the protein expression of CYP3A in human and rat primary hepatocytes treated with bilobalide (2, 10, and 50 ng/ml) and ginkgolides B (2, 10, and 50 ng/ml). The activity of CYP3A was measured by the quantification of dehydronifedipine formation using a validated tandem liquid chromatography mass spectrometry (LC/MS/MS) method. The levels of mRNA and protein of CYP3A were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western-blotting analysis, respectively. The G. biloba extract at 100-2,500 ng/ml significantly induced the activity, protein and mRNA expression of CYP3A in a dose-dependent manner in human and rat primary hepatocytes. Bilobalide at 2-50 ng/ml significantly increased CYP3A protein expression in a dose-dependent manner in human and rat primary hepatocytes. However, ginkgolide B did not affect CYP3A protein expression in vitro. The results indicate that G. biloba extract pretreatment significantly induced the expression of CYP3A protein and mRNA and increased CYP3A activity, and there was no significant species difference between human and rat. G. biloba may cause potential interactions with substrate drugs of CYP3A. Bilobalide might play a key role in the enzyme- inducing effects of G. biloba extract. Further study is needed to identify the substances in GBE that induce CYPs in vivo, and elucidate the molecular mechanism of CYP3A induction by GBE and bilobalides.

The new analgesic tapentadol was evaluated for induction and inhibition of several cytochrome P450 enzymes in vitro, and protein binding was assessed. It was concluded that no clinically relevant drug-drug interactions are likely to occur through either mechanism.