Association of drug metabolism gene polymorphisms with toxicities, graft-versus-host disease and survival after HLA-identical sibling hematopoietic stem cell transplantation for patients with leukemia

Individual differences in drug efficacy or toxicity can be influenced by genetic factors. We investigated whether polymorphisms of pharmacogenes that interfere with metabolism of drugs used in conditioning regimen and graft-versus-host disease (GvHD) prophylaxis could be associated with outcomes after HLA-identical hematopoietic stem cell transplantation (HSCT). Pharmacogenes and their polymorphisms were studied in 107 donors and patients with leukemia receiving HSCT. Candidate genes were: P450 cytochrome family (CYP2B6), glutathione-S-transferase family (GST), multidrug-resistance gene, methylenetetrahydrofolate reductase (MTHFR) and vitamin D receptor (VDR). The end points studied were oral mucositis (OM), hemorrhagic cystitis (HC), toxicity and venoocclusive disease of the liver (VOD), GvHD, transplantation-related mortality (TRM) and survival. Multivariate analyses, using death as a competing event, were performed adjusting for clinical factors. Among other clinical and genetic factors, polymorphisms of CYP2B6 genes that interfere with cyclophosphamide metabolism were associated with OM (recipient CYP2B6*4; P=0.0067), HC (recipient CYP2B6*2; P=0.03) and VOD (donor CYP2B6*6; P=0.03). Recipient MTHFR polymorphisms (C677T) were associated with acute GvHD (P=0.03), and recipient VDR TaqI with TRM and overall survival (P=0.006 and P=0.04, respectively). Genetic factors that interfere with drug metabolisms are associated with treatment-related toxicities, GvHD and survival after HLA-identical HSCT in patients with leukemia and should be investigated prospectively.

In vitro metabolism study of a new nitrosyl ruthenium complex [Ru(NH center dot NHq)(terpy)NO](3+) nitric oxide donor using rat microsomes

A new nitrosyl ruthenium complex [Ru(NH center dot NHq)(terpy)NO](3+) nitric oxide donor was recently developed and due to its excellent vasodilator activity, it has been considered as a potential drug candidate. Drug metabolism is one of the main parameters that should be evaluated in the early drug development, so the biotransformation of this complex by rat hepatic microsomes was investigated. In order to perform the biotransformation study, a simple, sensitive and selective HPLC method was developed and carefully validated. The parameters evaluated in the validation procedure were: linearity, recovery, precision, accuracy, selectivity and stability. Except for the stability study, all the parameters evaluated presented values below the recommended by FDA guidelines. The stability study showed a time-dependent degradation profile. After method validation, the biotransformation study was accomplished and the kinetic parameters were determined. The biotransformation study obeyed the Michaelis-Menten kinetics. The V(max) and K(m) were, respectively, 0.1625 +/- 0.010 mu mol/mg protein/min and 79.97 +/- 11.52 mu M. These results indicate that the nitrosyl complex is metabolized by CYP450. (C) 2009 Elsevier Inc. All rights reserved.

Influence of gestational diabetes mellitus on the stereoselective kinetic disposition and metabolism of labetalol in hypertensive patients

Purpose This study investigated the influence of gestational diabetes mellitus on the kinetic disposition and stereoselective metabolism of labetalol administered intravenously or orally. Methods Thirty hypertensive women during the last trimester of pregnancy were divided into four groups: non-diabetic and diabetic women treated with intravenous or oral labetalol. Results The pharmacokinetics of labetalol was not stereoselective in diabetic or non-diabetic pregnant women receiving the drug intravenously. However, oral administration of labetalol resulted in lower values of the area under the plasma concentration versus time curve (AUC) for the beta-blocker (RR) than for the other enantiomers in both diabetic and non-diabetic women. Gestational diabetes mellitus caused changes in the kinetic disposition of the labetalol stereoisomers when administered orally. The AUC values for the less potent adrenoceptor antagonist (SS) and for the alpha-blocking (SR) isomers were higher in diabetic than in non-diabetic pregnant women. Conclusions The approximately 100% higher AUC values obtained for the (SR) isomer in diabetic pregnant women treated with oral labetalol may be of clinical relevance in terms of the alpha-blocking activity of this isomer.

Omeprazole preferentially inhibits the metabolism of (+)-(S)-citalopram in healthy volunteers

center dot Citalopram (CITA) pharmacokinetics are enantioselective in healthy volunteers and the metabolism of (+)-(S)-CITA to (+)-(S)-DCITA is dependent on CYP2C19. Omeprazole is a potent CYP2C19 inhibitor. WHAT THIS STUDY ADDS center dot This study indicates that omeprazole induces a loss of enantioselectivity in the CITA pharmacokinetics because of the selective inhibition of (+)-(S)-CITA metabolism. AIM The study assessed the influence of omeprazole on the kinetic disposition of the (+)-(S)-citalopram (CITA) and (-)-(R)-CITA enantiomers in healthy volunteers. METHODS In a cross-over study, healthy volunteers (n = 9) phenotyped as extensive metabolizers of CYP2C19 and CYP2D6 and with an oral midazolam clearance ranging from 10.9 to 149.3 ml min-1 kg-1 received a single dose of racemic CITA (20 mg orally) in combination or not with omeprazole (20 mg day-1 for 18 days). Serial blood samples were collected up to 240 h after CITA administration. CITA and demethylcitalopram (DCITA) enantiomers were analyzed by LC-MS/MS using a Chiralcel (R) OD-R column. RESULTS The kinetic disposition of CITA was enantioselective in the absence of treatment with omeprazole, with the observation of a greater proportion of plasma (-)-(R)-CITA [AUC S : R ratio of 0.53 (95% CI 0.41, 0.66) for CITA and 1.08 (95% CI 0.80, 1.76) for DCITA] than (+)-(S)-CITA. Racemic CITA administration to healthy volunteers in combination with omeprazole showed a loss of enantioselectivity in CITA pharmacokinetics with an increase of approximately 120% in plasma (+)-(S)-CITA concentrations [AUC S : R ratio of 0.95 (95% CI 0.72, 1.10) for CITA and 0.95 (95% CI 0.44, 1.72) for DCITA]. CONCLUSIONS The administration of multiple doses of omeprazole preferentially inhibited (+)-(S)-CITA metabolism in healthy volunteers. Although omeprazole increased plasma concentrations of (+)-(S)-CITA by approximately 120%, it is difficult to evaluate the clinical outcome because the range of plasma CITA concentrations related to maximum efficacy and minimum risk of adverse effects has not been established.

In vitro characterization of rosiglitazone metabolites and determination of the kinetic parameters employing rat liver microsomal fraction

Rosiglitazone (RSG), a thiazolidinedione antidiabetic drug, is metabolized by CYP450 enzymes into two main metabolites: N-desmethyl rosiglitazone (N-Dm-R) and rho-hydroxy rosiglitazone (rho-OH-R). In humans, CYP2C8 appears to have a major role in RSG metabolism. On the other hand, the in vitro metabolism of RSG in animals has not been described in literature yet. Based on these concerns, the kinetic metabolism study of RSG using rat liver microsomal fraction is described for the first time. Maximum velocity (V (max)) values of 87.29 and 51.09 nmol/min/mg protein were observed for N-Dm-R and rho-OH-R, respectively. Michaelis-Menten constant (K (m)) values were of 58.12 and 78.52 mu M for N-Dm-R and rho-OH-R, respectively. Therefore, these results demonstrated that this in vitro metabolism model presents the capacity of forming higher levels of N-Dm-R than of rho-OH-R, which also happens in humans. Three other metabolites were identified employing mass spectrometry detection under positive electrospray ionization: ortho-hydroxy-rosiglitazone (omicron-OH-R) and two isomers of N-desmethyl hydroxy-rosiglitazone. These metabolites have also been observed in humans. The results observed in this study indicate that rats could be a satisfactory model for RSG metabolism.

Primidone oxidation catalyzed by metalloporphyrins and Jacobsen catalyst

Primidone (PRM) oxidation by various oxidants such as iodosylbenzene (PhIO), tert-butyl hydroperoxide 70wt.% (t-BOOH), 3-chloroperoxybenzoic acid (m-CPBA) and hydrogen peroxide 30wt.%, mediated by either a salen complex or metalloporphyrins, was investigated. The catalytic systems led to phenylethyl-malondiamide (PEMA) and phenobarbital (FEND), the same metabolites obtained in vivo with P450 enzymes, although three other products were also detected. Product formation was highly dependent on the oxidant, co-catalyst (imidazole), pH and dioxygen. These biomimetic chemical models have potential application in the synthesis of drug metabolites. which should provide samples for pharmacological tests. They can also be employed in studies that pursue the elucidation of in vivo drug metabolism. (C) 2008 Elsevier B.V. All rights reserved.

Biomimetic Oxidation of Piperine and Piplartine Catalyzed by Iron(III) and Manganese(III) Porphyrins

Synthetic metalloporphyrins, in the presence of monooxygen donors, are known to mimetize various reactions of cytochrome P450 enzymes systems in the oxidation of drugs and natural products. The oxidation of piperine and piplartine by iodosylbenzene using iron(III) and manganese(III) porphyrins yielded mono- and dihydroxylated products, respectively. Piplartine showed to be a more reactive substrate towards the catalysts tested. The structures of the oxidation products were proposed based on electrospray ionization tandem mass spectrometry.

Chronic Acetonemia Alters Liver Oxidative Balance and Lipid Content in Rats. A Model of Nash?

Acetone is considered to be a substance that can disturb cellular oxidative status, being also associated with the production of glucose during its metabolization. The objective of the present study was to determine the effects of chronic treatment with acetone in oxidative stress and metabolic parameters in rats. Twenty male Wistar rats were divided into two groups: control (CG) and chronic acetone group (CAG). After 28 days of acetone ingestion in a 5% aqueous solution (CAG) or water (CG) the animals were euthanized and urine, plasma and liver were collected for the determination of acetone, glucose, lipemia, hepatic fat, malondialdehyde (MDA), reduced glutathione (GSH), and vitamin E. As expected, urinary and plasma acetone levels were higher in CAG. There was no difference in hepatic MDA values between groups, whereas hepatic GSH was lower in CAG than in CG and hepatic vitamin E was higher in CAG than in CG. There was also an increase in glycemia, cholesterolemia and hepatic fat in CAG compared to CG. Chronic treatment with a 5% acetone solution produced an increase in acetonemia that was able to promote changes in hepatic oxidative metabolism and in lipid content in rats similar to those observed in nonalcoholic steatohepatitis.

Influence of Glomerular Filtration Rate on the Pharmacokinetics of Cyclophosphamide Enantiomers in Patients With Lupus Nephritis

The pharmacokinetics of cyclophosphamide (CYC) enantiomers were evaluated in patients with lupus nephritis distributed in 2 groups according to creatinine clearance: group 1 (90.6-144.6 mL/min/1.73 m(2)) and group 2 (42.8-76.4 mL/min/ 1.73 m(2)). All patients were treated with 0.75 to 1.3 g of racemic CYC as a 2-hour infusion and with 1 mg intravenous midazolam as a drug-metabolizing marker. CYC enantiomers and midazolam concentrations in plasma were measured by liquid chromatography/tandem mass spectrometry (LC/MS/MS). The following differences (Wilcoxon test, P <= .05) were observed between the (S)-(-) and (R)-(+) enantiomers: AUC(0-infinity) 152.41 vs 129.25 mu g.h/mL, CL 3.28 vs 3.89 L/h, Vd 31.38 vs 29.74 L, and t(1/2) 6.79 vs 5.56 h for group 1 and AUC(0-infinity) 167.20 vs 139.08 mu g.h/mL, CL 2.99 vs 3.59 L/h, and t(1/2) 6.15 vs 4.99 h for group 2. No differences (Mann test, P <= .05) were observed between groups 1 and 2 in the pharmacokinetic parameters of both enantiomers. No significant relationship was observed between midazolam clearance (2.92-16.40 mL/min.kg) and clearance of each CYC enantiomer. In conclusion, CYC kinetic disposition is enantioselective, resulting in higher exposures of the (S)-(-) enantiomer in lupus nephritis patients, and the pharmacokinetic parameters of both enantiomers are not altered by the worsening of renal condition.

Role of the CYP2D6, EPHX1, MPO, and NQO1 Genes in the Susceptibility to Acute Lymphoblastic Leukemia in Brazilian Children

Polymorphic variations of several genes associated with dietary effects and exposure to environmental carcinogens may influence susceptibility to leukemia development. The objective of the present study was to evaluate the effect of the polymorphisms of debrisoquine hydroxylase (CYP2D6), epoxide hydrolase (EPHX1), myeloperoxidase (MPO), and quinone-oxoreductase (NQO1), which have been implicated in xenobiotic metabolism, on the risk of childhood acute lymphoblastic leukemia (ALL). We evaluated the frequency of polymorphisms in the CYP2D6 (*3 and *4), EPHX1 (*2 and *3), MPO (*2), and NQO1 (*2) genes in 206 patients with childhood ALL and in 364 healthy individuals matched for age and gender from a Brazilian population separated by ethnicity (European ancestry and African ancestry), using the PCR-RFLP method. The CYP2D6 polymorphism variants were associated with an increased risk of ALL. The EPHX1, NQO1, and MPO variant genotypes were significantly associated with a reduced risk of childhood ALL. A significantly stronger protective effect is observed when the EPHX1, NQO1, and MPO variant genotypes are combined suggesting that, CYP2D6 polymorphisms may play a role in the susceptibility to pediatric ALL, whereas the EPHX1, NQO1, and MPO polymorphisms might have a protective function against leukemogenesis. Environ. Mal. Mulagen. 51:48-56, 2010. (C) 2009 Wiley-Liss, Inc.

Chemoprotective effect of the tetrahydrofuran lignan grandisin in the in-vivo rodent micronucleus assay

Objectives The chemoprotective effect of the tetrahydrofuran lignan grandisin against DNA damage induced by cyclophosphamide (200 mg/kg) has been evaluated using the in vitro rodent micronucleus assay. Methods The effects of a daily oral administration of grandisin (2, 4, or 8 mg/kg) for five days before exposure to cyclophosphamide on the frequency of micronucleus in the bone marrow of normal mice exposed and unexposed to cyclophosphamide were investigated (n = 5 per group). Electrochemical measurements were applied to investigate whether the antimutagenic effects of grandisin could be, at least in part, a consequence of its or its metabolite`s antioxidant properties. Key findings Grandisin did not show mutagenic effects on the bone marrow cells of exposed mice. On the other hand, the oral administration of grandisin (2, 4, or 8 mg/kg) per day reduced dose-dependently the frequency of micronucleus, induced by cyclophosphamide, in all groups studied. Cyclic voltammograms showed two peaks for a grandisin metabolite, which were absent for grandisin. Conclusions Under the conditions tested herein, this study has shown that mice treated with grandisin presented, in a dose-dependent manner, a protective effect against cyclophosphamide-induced mutagenicity. This effect could be, at least in part, associated to grandisin bioactivation. These data open new perspectives for further investigation into the toxicology and applied pharmacology of grandisin.

Novel Application of 2D and 3D-Similarity Searches To Identify Substrates among Cytochrome P450 2C9, 2D6, and 3A4

Cytochrome P450 (CYP450) is a class of enzymes where the substrate identification is particularly important to know. It would help medicinal chemists to design drugs with lower side effects due to drug-drug interactions and to extensive genetic polymorphism. Herein, we discuss the application of the 2D and 3D-similarity searches in identifying reference Structures with higher capacity to retrieve Substrates of three important CYP enzymes (CYP2C9, CYP2D6, and CYP3A4). On the basis of the complementarities of multiple reference structures selected by different similarity search methods, we proposed the fusion of their individual Tanimoto scores into a consensus Tanimoto score (T(consensus)). Using this new score, true positive rates of 63% (CYP2C9) and 81% (CYP2D6) were achieved with false positive rates of 4% for the CYP2C9-CYP2D6 data Set. Extended similarity searches were carried out oil a validation data set, and the results showed that by using the T(consensus) score, not only the area of a ROC graph increased, but also more substrates were recovered at the beginning of a ranked list.

Splanchnic non-hepatic hemodynamics and metabolism during liver transplantation

Background/Aims: The aim of this study is to compare the splanchnic non-hepatic hemodynamics and the metabolic changes during orthotopic liver transplantation between the conventional with bypass and the piggyback methods. Methodology: A prospective, consecutive series of 59 primary transplants were analyzed. Oxygen consumption, glucose, potassium, and lactate metabolism were quantitatively estimated from blood samples from the radial artery and portal vein, collected up to 120 minutes after graft reperfusion. Mean arterial pressure, portal venous pressure, portal venous blood flow, and splanchnic vascular resistance were also measured or calculated at postreperfusion collection times. Results: There was a greater increase in portal venous blood flow (p=0.05) and lower splanchnic vascular resistance (p=0.04) in the piggyback group. Mean arterial pressure and portal venous pressure were similar for both groups. Oxygen, glucose and potassium consumption were higher in the piggyback group, but none of the metabolic parameters differed significantly between groups. Conclusions: In conclusion, the study detected a higher portal venous blood flow and a lower and splanchnic vascular resistance associated with the piggyback technique. After graft reperfusion, no difference in the splanchnic non-hepatic metabolic parameters was observed between the conventional with bypass and the piggyback methods of orthotopic liver transplantation.

Hepatotrophic factors reduce hepatic fibrosis in rats

CONTEXT: Hepatic fibrosis occurs in response to several aggressive agents and is a predisposing factor in cirrhosis. Hepatotrophic factors were shown to stimulate liver growth and to restore the histological architecture of the liver. They also cause an improvement in liver function and accelerate the reversion of fibrosis before it progresses to cirrhosis. OBJECTIVE: To test the effects of hepatic fibrosis solution composed by amino acids, vitamins, glucose, insulin, glucagon and triiodothyronine on hepatic fibrosis in rats. METHODS: Fibrosis was induced in rats by gastric administration of dimethylnitrosamine (10 mg/kg) for 5 weeks. After liver biopsy, the rats received either hepatotrophic factors solution (40 mg/kg/day) or saline solution for 10 days by intraperitoneal injection. Blood samples and liver fragments were collected for hepatic function analysis, standard histopathology evaluation, and morphometric collagen quantification. RESULTS: Rats in the hepatotrophic factors group showed a decrease of the histopathological components of fibrosis and an increase of their hepatic mass (12.2%). There was no development of neoplasic lesions in both groups. Compared with the saline group, the hepatotrophic factors group also had a decrease of blood levels of hepatic-lesion markers (AST, ALT) and a decrease of collagen content in the portal spaces (31.6%) and perisinusoidal spaces (42.3%), as well as around the hepatic terminal vein (57.7%). Thus, hepatotrophic factors administration in the portal blood promoted a regenerative hepatic response, with an overall reduction of the volumetric density of collagen, improved hepatic function, and a general improvement in the histopathological aspects of fibrosis. CONCLUSION: Taken together, these results suggest the potential therapeutic use of this hepatotrophic factors solution to treat chronic liver diseases.

Reduction of enantioselectivity in the kinetic disposition and metabolism of verapamil in rats exposed to toluene

Toluene and verapamil are subject to extensive oxidative metabolism mediated by CYP enzymes, and their interaction can be stereoselective. In the present study we investigated the influence of toluene inhalation on the enantioselective kinetic disposition of verapamil and its metabolite, norverapamil, in rats. Male Wistar rats (n = 6 per group) received a single dose of racemic verapamil (10 mg/kg) orally at the fifth day of nose-only toluene or air (control group) inhalation for 6 h/day (25, 50, and 100 ppm). Serial blood samples were collected from the tail up to 6 h after verapamil administration. The plasma concentrations of verapamil and norverapamil enantiomers were analyzed by LC-MS/MS by using a Chiralpak AD column. Toluene inhalation did not influence the kinetic disposition of verapamil or norverapamil enantiomers (p > 0.05, Kruskal-Wallis test) in rats. The pharmacokinetics of verapamil was enantioselective in the control group, with a higher plasma proportion of the S-verapamil (AUC 250.8 versus 120.4 ng.h.mL(-1); p <= 0.05, Wilcoxon test) and S-norverapamil (AUC 72.3 versus 52.3 ng.h.mL(-1); p <= 0.05, Wilcoxon test). Nose-only exposure to toluene at 25, 50, or 100 ppm resulted in a lack of enantioselectivity for both verapamil and norverapamil. The study demonstrates the importance of the application of enantioselective methods in studies on the interaction between solvents and chiral drugs.