Drug Metabolism Letters - Volume 3, Issue 1, 2009

Combi-molecules are novel agents designed to be hydrolyzed into two bioactive species: an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor + a DNA alkylating agent. With the purpose of enhancing the tumour concentration of the bioactive species, we synthesized and compared the activities of RB107, a quinazolinotriazene designed to generate the bioactive BJ2000 upon hydrolysis, ZRDM and RB107ZR that require metabolic activation to generate BJ2000. The results showed that RB107 released the highest level of BJ2000 and its degradation product FD105 in vivo and high levels of the DNA alkylating methyl diazonium ion in the brain, kidney, liver and the DU145 tumours as confirmed by 14C-labeling. The results in toto suggest that RB107 was stable enough to deliver the bioactive species to the tumour site and for optimal tumour distribution of the bioactive species, combi-molecules of the triazene class must be designed to be primarily degraded by hydrolytic cleavage and not by metabolic activation.

The potential of various microbes to metabolize the dextromethorphan (DXM) , a CYP 2D6 substrate was studied to investigate similarities between microbial and mammalian metabolism. Eight microbes were screened for their ability to metabolize DXM in a manner comparable to humans with a view to develop alternative systems to study human drug metabolism. The major metabolite of DXM produced by Saccharomyces cerevisiae (NCIM 3090) was characterized by HPLC and Liquid Chromatography Mass Spectrometry (LC/MS). DXM was biotransformed by Saccharomyces cerevisiae into a major metabolite dextrorphan which is a known , active metabolite of DXM in human. The microbial metabolism of DXM was similar to the metabolism in mammals. The Saccharomyces cerevisiae could be used as a suitable model strain in vitro to mimic CYP 2D6 metabolism and to produce dextrorphan an active metabolite of DXM for further pharmacological, toxicological studies.

Cerebral malaria (CM) is the most severe complication of Plasmodium falciparum malaria. The aim of this study was to investigate the influence of CM on the cerebral uptake of mefloquine (MQ), in an experimental model of mice infected with Plasmodium berghei ANKA (PbA). Drug diffusion in brain is closely related to efflux pumps such as P-glycoprotein (P-gp/ABCB1/MDR1) and Breast Cancer Resistant Protein (BCRP/ABCG2), two major components of the blood-brain barrier (BBB) which can be modified by inflammation and/or infection. After a single IP dose, MQ concentrations were measured by liquid chromatography in blood and brains of mice infected with Plasmodium berghei ANKA and compared with that of non-infected mice. Our results show that MQ brain concentrations were decreased in CM mice versus healthy mice (0.77 versus 1.31 for brain/plasma concentrations). Although MQ is transported out of endothelial cells by P-glycoprotein, this result cannot be related to this transporter as we have previously shown that CM does not alter P-gp function (personal data). CM induces a reduction of MQ brain transport and, therefore, an increase of central toxicity due to MQ should not be expected during CM.

The metabolism and biotransformation of the 14C-labeled phenylurea herbicide fluometuron was examined using tobacco cell suspension cultures transformed separately with human cyp1a1, cyp1a2 and cyp3a4, and corresponding non-transformed cultures in order to screen and predict metabolic patterns. Experimental parameters modified were concentration of 14C-fluometuron, incubation period, and additional application of inhibitor carbaryl. Media and cell extracts were analyzed by radio-TLC and radio-HPLC, isolated metabolites by LC-MS, and non-extractable residues by combustion. During 48 hours, the CYP1A1 expressing cultures metabolized 90.0 % of applied fluometuron, while the nontransgenic controls transformed 67.0 %. The CYP1A2 expressing cultures exhibited highest rates (95.1 %), CYP3A4 expressing cultures lowest rates (43.0 %). The primary metabolites identified were mono-demethyl (main metabolite in controls) and di-demethyl fluometuron (mainly in CYP1A2 cultures), besides a non-identified primary product (mainly in CYP1A1 cultures); metabolic profiles differed distinctly among cultures. After addition of carbaryl, rates of fluometuron decreased noticeably in controls and not in CYP3A4 expressing cultures. This may indicate inhibition of endogenous tobacco P450s involved in fluometuron metabolism but not of CYP3A4. Additionally, the P450-transgenic cultures proved to be valuable tools to produce large amounts of metabolites for thorough identification.

GyrATPase is a cellular enzyme that has been used as an antibacterial target for treatment of nosocomial and community acquired bacterial infections. The leading chemical series targeted at inhibiting this enzyme, indazoles, were rapidly cleared in rats (CL > 70 mL/min/kg). The predominant metabolite identified in both urine and bile samples from a bile duct-cannulated study corresponded to direct glucuronidation of the parent compound and was excreted rapidly. Subsequently, a carefully designed analog was used to pinpoint the site of glucuronidation (N-glucuronidation) by incubation with rat hepatocytes and followed by mass spectrometry analysis. Reaction mapping with an array of recombinant UGT isozymes revealed that N-glucuronidation was predominantly catalyzed by the UGT1A family of enzymes. Based on the results, the following approaches were considered to reduce or eliminate glucuronidation: 1) adding sterically hindered substitutions on the phenyl ring of the indazole core; 2) changing the electron distribution by substituting with electrondonating or – withdrawing groups; 3) replacing the site of glucuronidation. The resulted compounds were evaluated in vitro in rat hepatocytes to assess their metabolic stabilities followed by in vivo efficacy studies in the murine peritonitis sepsis model (at 50 mg/kg) for selected compounds.

Anti-proliferative effects are described for newly synthesised copper (II) complexes of two triazolo-pyrimidine derivatives (1,2,4-triazolo-[1,5-a]pyrimidine, tp, and 5,7-dimethyl 1,2,4-triazolo-[1,5-a]pyrimidine, dmtp) against to Trypanosoma cruzi and Leishmania (Viannia) peruviana. Of the compounds assayed, those that presented the ligand tp and auxiliary ligand 1,10-phenanthroline (C24b, C49) were most highly active against to T. cruzi with IC50 within the range of the reference drug benznidazole. These compounds, together with C35 were the most effective against L. (V.) peruviana with an IC50 greater than that presented by reference drugs (Pentostam and Glucantim). These compounds were not toxic to the host cell. IC25 diminished the infection capacity and severely reduced the multiplication of intracellular forms of T. cruzi, and L. (V.) peruviana. In the case of T. Cruzi, the transformation to trypomastigote was seriously depressed. Copper (II) complexes C24b, C49 and C35, acted on the energy metabolism of the parasites at the level of the NAD/NADH balance and at the level of the organelle membranes, causing degradation and cell death.

Enzyme kinetic parameters for midazolam were time-dependent in human liver microsomes, under initial velocity conditions. Vmax and Km decreased up to 3.7 and 3.1-fold, respectively, for 10 min compared to 1 min incubations. Mathematical models describing the relationship between inactivation and the time-dependency of enzyme kinetic parameter estimates were derived and discussed.

Reactivation with bis-quaternary aldoxime HI-6, chemical formula 1-(2-hydroxyaminomethylpyridinium)-3-(4- carbamoylpyridinium)-2-oxapropane dichloride of immobilized enzyme acetylcholinesterase inhibited by nerve agent type “G” was studied. This aldoxime is effective in reactivation of sarin-inhibited acetylcholinesterase. Substantially lower reactivation potency was observed with cyclosarin-inhibited enzyme and almost no effect was found for that acetylcholinesterase is the enzyme complex. HI-6 is completely ineffective towards the soman-inhibited enzyme: After a 2-minute inhibition of the enzyme with soman no ability to define reactivator the inhibited enzymes and complexes.

Codeine is a common drug widely used in some countries as a pain reliever. The effect of codeine on yeast sucrase activity was studied in this report. Non-competitive inhibition was observed using double reciprocal plot. The Km of enzyme did not change in the presence of different concentrations of codeine (0.5- 1.5 mM) and was determined about 11.5 mM. The Vmax of enzyme was determined about 8.8 mM/min, and the Vmax decreased in the presence of codeine. The Ki of codeine was measured by using the reaction rate and the initial concentration of the inhibitor according to the Dixon plot. The Ki was found to be 0.42 mM and the IC50 of codeine was determined about 0.875 mM.

The induction of dog CYP3A12 and CYP3A26 mRNA levels was evaluated in liver slices after treatment with 22 xenobiotics. Eleven of the 22 xenobiotics increased 3A12 mRNA by more than four-fold, while nine did the same for 3A26 mRNA. A four-fold increase in the mRNA level was used as the cut-off for indication of induction based on the noise level of the real time-PCR. A good correlation was found between the mRNA levels for 3A12 and 3A26 after treatment with compounds, suggesting that these two CYPs may be co-induced. Induction of CYP3A4 in human hepatocytes was evaluated after treatment with the same 22 compounds. Thirteen out of the 22 compounds increased the 3A4 mRNA levels by more than four-fold. When the mRNA levels of 3A4 and 3A12 were compared after treatment with compounds, no correlation was found. The regulation of CYP3A expression has been demonstrated to be controlled by pregnane X receptor (PXR). Upon examination of the sequence homology and the three-dimensional structures of human PXR and a dog PXR model, only two different amino acids (met323/val and arg410/lys) were found in the ligand-binding domain. This finding suggests that these two amino acids may play a role in the binding specificity of ligands.