Drug Metabolism Letters - Volume 1, Issue 4, 2007

We investigated the effects of Cymbopogon schoenanthus herb on experimental induced kidney stones in male Wistar albino rats. Oxalate nephrotoxicity was experimentally induced by 200 mg single dose of glycolic acid given orally (gavage). Rats were divided into three groups, glycolic acid, glycolic acid plus Cymbopogon schoenanthus, and control (D. water). Urine analysis of blood urea nitrogen (BUN), creatinine, and calcium revealed significant differences comparing to the control. In addition, significant pathological changes were found in the kidney revealed by histopathological studies. Daily oral treatment with Cymbopogon schoenanthus (1 ml of the extract) significantly corrected the incidence of nephrotoxicity, BUN, creatinine, and calcium level differences. Moreover, optimization studies showed highly potent diuretic activity of Cymbopogon schoenanthus. After three days of experiments, four rats treated with the glycolic acid only died. The rest of animal survived and looked healthy.

The roles of alkylation and redox cycling in quinone toxicity were investigated. In general the more cytotoxic quinones produced the highest responses in an assay monitoring redox activity. No evidence of alkylation of high molecular weight protein thiols was detected. We conclude quinone toxicity is dominated by redox cycling.

Acute lung injury is accompanied by an increased endothelial chemokine production and adhesion molecule expression, which may result in an extensive neutrophil infiltration. Moreover, a destruction of the alveolar epithelium and capillary endothelium may result in permeability edema. As such, the search for novel anti-inflammatory substances, able to downregulate these parameters as well as the tissue damage holds therapeutic promise. We therefore describe here the use of human endothelial cell-based in vitro assays for the detection of anti-inflammatory and wound-healing metabolites from cyanobacteria.

Sera from AIH (autoimmune hepatitis) type 2 patients contain an autoantibody against the UGT1A subtype, called anti-LKM3. Previously, we reported that sera in AIH type 1 patients contained autoantibodies against drugmetabolizing enzymes (Shinoda et al. (2004) Autoimmunity, 37, 473). In this report, we showed that AIH-1 sera did not react with some peptides in the C-terminal half of the UGT1A subtype but reacted with a peptide P1(33-42) among several common peptides in the N-terminal half of the UGT1A subtype. This result suggests that the P1 site (33-42) presents on the outside of the UGT1A molecule to be recognized by lymphocytes of the immune system to produce an autoantibody. To detect a key recognition site on peptide P1(33-42), we studied the reactivity of two peptides, M1(28-37) and M2(38-47), containing the N-terminal and C-terminal half of peptide P1. Peptide M2 did not react with AIH-1 serum but peptide M1 did. Thus, the common peptide sequence 33-37 in the positive peptide M1(28-37) and P1(33-42) is a key recognition sequence. Next, we studied the reactivity of some other synthetic peptides, in which some amino acids in the sequence 33-37 in peptide M1 changed to Ala. The peptides changing to Ala (PQ33-34AA) or (DGS35-37AAA) did not react with AIH-1 sera. Meanwhile, these AIH sera did not inhibit the glucuronidation of p-nitrophenol by UGT1A6, suggesting that the key sequence 33-37 might not be contained in active sites of glucuronidation by UGT1A6. In conclusion, sera from AIH-1 patients reacted with the amino acids in the sequence 33-37 (PQDGS) of the N-terminal of UGT1A6.

Background: Sirolimus is the latest immunosuppressive agent used to prevent rejection, and may have less nephrotoxicity than calcineurin inhibitor (CNI)-based regimens. To date there has been little documentation of clinically significant proteinuria linked with the use of sirolimus. We have encountered several patients who developed substantial proteinuria associated with sirolimus use. In each patient, the close temporal association between the commencement of sirolimus therapy and proteinuria implicated sirolimus as the most likely etiology of the proteinuria. Methods: We analyzed the clinical and laboratory information available for all 119 patients transplanted at the Washington Hospital Center between 1999-2003 for whom sirolimus was a component of their immunosuppressant regimen. In these patients, the magnitude of proteinuria was assessed on morning urine samples by turbidometric measurement or random urine protein:creatinine ratios, an estimate of grams of proteinuria/day. Laboratory results were compared between prior, during and following sirolimus use. Results: Twenty-eight patients (24%) developed increased proteinuria from baseline during their post-transplantation course. In 21 patients an alternative cause of proteinuria was either obvious or insufficient data was available to be conclusive. In 7 of the 28 patients there was a striking temporal association between the initiation of sirolimus and the development of nephrotic-range proteinuria. Proteinuria correlated most strongly with sirolimus therapy when compared to other demographic and clinical variables. In most patients, discontinuation of sirolimus resulted in a decrease, but not resolution, of proteinuria. Conclusions: Sirolimus induces or aggravates pre-existing proteinuria in an unpredictable subset of renal allograft recipients. Proteinuria may improve, but does not resolve, when sirolimus is withdrawn.

Our policy of conducting biotransformation studies with extended chromatography prior to pharmacokinetic bioanalyses allowed us to quickly detect an unusual, cis/trans metabolite in rat plasma that was inseparable using a short chromatographic method. We caution investigators that short methods invite unknown isobaric metabolites to cause inaccuracies in plasma concentration measurements.

Epidemiological studies have identified a number of risk factors that contribute to the development of cervical cancer precursors and cervical cancer. These include infection with certain oncogenic types of human papillomaviruses (HPVs) and other socio-economic factors. Tobacco smoking is an independent risk-factor for cervical neoplasia. It has been found that polymorphism at loci that encode carcinogen-metabolizing enzyme such as cytochrome P450 2D6 (CYP2D6) catalyzing the detoxification of carcinogens may determine susceptibility to cervical cancer. Therefore, it is likely that an understanding of these allelic differences is important for determining an individual's risk of cancer and susceptibility to potentially toxic agents. The aim of the present study was to elucidate the role of CYP2D6 polymorphism and susceptibility to squamous cell carcinoma of the uterine cervix in Indian population. Therefore, the genotype frequencies at this locus in females suffering with low-grade CIN, high-grade CIN and squamous cell carcinoma were compared. The control group consisted of 77 females with normal cervical cytology and the cases comprised of 61 mild/moderate dysplasia, 48 severe dysplasia and 45 cases of squamous cell carcinoma of uterine cervix. The individuals were divided into poor metabolizers (PM) and extensive metabolizers (EM) on the basis of their ability to metabolize certain drugs and carcinogens. Comparison of the frequency distribution for the combination of CYP2D6 EM genotype and smoking between mild/moderate and severe dysplasia was statistically significant (p=0.047) suggesting that women with cervical intraepithelial neoplasia I/II (CIN I/ CIN II) and CYP2D6 EM genotype who smoke appears to have more chances for the lesions to progress to CIN III. Whereas, frequency distribution for the same combination between severe dysplasia and squamous cell carcinoma failed to attain any statistical significance suggesting that CIN III with CYP2D6 EM genotype has less chance to progress to cervical cancer. Increased frequency of CYP2D6 EM and tobacco smoking show strong association with CIN III, indicating that not all lesions with the histopathological high grade CIN are premalignant. Conversely some squamous cell carcinomas may not be preceded by CIN.

Cytochrome P450 isoforms from male rat liver microsomes were comprehensively identified using nano liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). The enrichment of P450, an endomembrane-anchored heme protein, was achieved by solubility-based protein fractionation, and greatly improved the total number of identified P450 isoforms. LC-MS/MS analysis of fractions resulted in the identification of total 36 P450 isoforms. The combination of proteomic analysis and the solubility-based fractionation would provide powerful tool for the expression analysis of the superfamily proteins having great similarities between the amino acids sequences.

This study describes the application of the mass defect filter method for the detection of two unpredicted oxazole- ring opened metabolites of muraglitazar in the feces of humans following oral administration. Unlike other muraglitazar metabolites, these metabolites formed little to no protonated ions, and the NH4 + or Na+ adduct ions that were formed were weak and not discernible from fecal interferences even after background subtraction. With mass defect filtering on high resolution LC/MS data, the resulting total ion chromatogram and the simplified mass spectra allowed for the identification and characterization of these metabolite ions, and their structures were confirmed by synthesis.

An LC-MS/MS-based approach that employs authentic radioactive metabolites as reference standards was developed to estimate metabolite exposures in early drug development studies. This method is useful to estimate metabolite levels in studies done with non-radiolabeled compounds where metabolite standards are not available to allow standard LC-MS/MS assay development. A metabolite mixture obtained from an in vivo source treated with a radiolabeled compound was partially purified, quantified, and spiked into human plasma to provide metabolite standard curves. Metabolites were analyzed by LC-MS/MS using the specific mass transitions and an internal standard. The metabolite concentrations determined by this approach were found to be comparable to those determined by valid LC-MS/MS assays. This approach does not requires synthesis of authentic metabolites or the knowledge of exact structures of metabolites, and therefore should provide a useful method to obtain early estimates of circulating metabolites in early clinical or toxicological studies.

Leflunomide was found to be metabolized predominantly to A77-1726 and two novel hydroxylated metabolites, M1 and M2, in microsomes while A77-1726 was only biotransformed to M1. M1 and M2 were proposed to be the hydroxylated α-cyanoenol form of A77-1726 and the hydroxylated 5 methyl-isoxazole form of leflunomide, respectively.

We have developed an easy and sensitive method to measure P-glycoprotein (P-gp) activity using [γ-32P]ATP and charcoal. This method utilizes the nature of adsorption of organic phosphate to charcoal. The standard assay method is as follows: the reaction mixture (20 μl) of 1 mM [γ-32P]ATP (1 Ci/mol), 2.5 μg P-gp membranes, and the drug was incubated for 30 min, and 50 μl of 10% charcoal suspension in 0.1 M phosphate buffer at pH7.3 was then added to the mixture. The solution was centrifuged and the supernatant (20 μl) in duplicate containing inorganic 32P was spotted onto filter paper, and radioactivity was measured by radio-image analyzer. This method can reduce the amount of P-gp membrane compared to the conventional method utilizing coloring of the inorganic phosphate-molybdate reaction. This method is also applicable to other ATP-binding cassette (ABC) transporters in phosphate buffer.

We used 2,3,8,9-tetrachlorodibenzo-p-dioxin (TCDD) and dexamethasone (DEX) to examine their effects on aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR) in HeLa cells. TCDD (5 nM), DEX (100 nM) and their combination down-regulated GR after 24 h. DEX reversed AhR mRNA increase and AhR protein decrease caused by TCDD. Since AhR-GR cross-talk occurs in cell-type and species-specific manner, the presented data may serve as the basis in the understanding of mechanisms underlying mutual interactions between AhR and GR.