The influence of the choice of digestion enzyme used to prepare rat hepatocytes on xenobiotic uptake and efflux.

Isolated rat hepatocytes are widely used to assess the metabolism and toxicity of xenobiotics. The choice of digestion enzyme used to prepare the cells has been shown previously to influence their metabolic capability. This study investigates the effect of the digestion enzyme (collagenase II, collagenase A/trypsin inhibitor, or collagenase plus dispase) on the uptake of xenobiotics into, and efflux from, hepatocytes. The choice of digestion enzymes used in this study does not affect uptake of either pravastatin (an organic anion probe substrate for Oatp transporter) or metformin (an organic cation probe substrate for Oct transporter). With regard to efflux transporters, hepatocyte differentiation was better maintained when cells were isolated using collagenase II alone.

The extent of phase I and phase II reactions is affected by the choice of enzyme used to prepare rat hepatocytes.

The preparation of hepatocytes using the two-stage perfusion technique usually involves the use of collagenase (CII) alone or in combination with dispase (C/D) or trypsin inhibitor (CA/TI) as digestion enzymes. The effect of CII, C/D and CA/TI on cell viability, yield, cytochrome P450 mediated oxidation of testosterone, glucuronidation and sulfation of 7-hydroxycoumarin, glutathione content, glutathione-S-transferase activity and glutathione-conjugation capacity of hepatocytes has been assessed. Cytochrome P450 mediated oxidation of testosterone was significantly (p < 0.05) decreased with CII isolated hepatocytes (81.7 +/- 3.3 nmol/10(6) cells, mean +/- S.E.M., n = 3), compared with those isolated using CA/TI (96.6 +/- 1.9 nmol/10(6) cells) or C/D (95.1 +/- 2.1 nmol/10(6) cells). In contrast, glutathione conjugation of the non-specific substrate 1-chloro-2,4-dinitrobenzene was significantly (p < 0.05) increased with CII isolated hepatocytes (56.9 +/- 5.9 nmol/10(6) cells, mean +/- S.E.M., n = 3), compared with those isolated using CA/TI (36.0 +/- 3.7 nmol/10(6) cells) or C/D (31.6 +/- 3.7 nmol/10(6) cells). These findings have significant implications for the interpretation of metabolism data derived from hepatocytes in suspension, particularly in terms of glutathione conjugation of potentially toxic reactive intermediates of xenobiotic metabolism. Indeed, data presented show that the presence of trypsin inhibitor in the preparation of isolated rat hepatocytes significantly affects the formation of glutathione conjugates of reactive intermediate products of troglitazone metabolism.

The determination of iron as its EDTA complex in Helix aspera by hydrophilic interaction liquid chromatography coupled to Fourier transform electrospray ionisation mass spectrometry.

Metal complexes of Fe(III) such as Fe(III) ethylene diamine tetraacetic acid (FeETDA) have been observed to be effective molluscicides. The mechanism of toxicity of FeEDTA complex on molluscs is not clear and it is also not known if Fe(III) in the form FeEDTA is absorbed more effectively by snails than simple iron salts. Snails were fed with molluscicide pellets containing the FeEDTA complex and also with pellets containing FePO(4) after 3-4 days the hearts, kidneys and dart sacs removed and analysed for Fe(III) content. Hydrophilic interaction liquid chromatography (HILIC) coupled with Fourier transform electrospray ionisation mass spectrometry (FT-ESIMS) was used to analyse the sample extracts. The method had a very wide linear range from 2 to 10,000 ngmL(-1), intra- and inter-day precisions of ca. +/-0.5% were observed for the analysis of extracts from snail tissues spiked with Fe(III). The limit of detection was of 0.5 ngmL(-1) for a 20 microL injection. The levels of Fe(III) in tissues from snails fed Fe EDTA pellets were 10-100 times higher than the levels in snails fed FePO(4) pellets. The analysis of Cu, Zn, Ca and Mn could also be carried out using the same analytical procedure.

Development and validation of an improved HPLC method for the control of potentially counterfeit isometamidium products.

Isometamidium, a mixture of related substances of which 8-(3-m-amidinophenyl-2-triazeno)-3-amino-5-ethyl-6-phenylphenanthridinium chloride hydrochloride (M&B4180A) is the principal active component, is the only chemical agent available for prophylaxis of veterinary trypanosomiasis. A method for the simultaneous quantitation of the major constituents M&B4180A, 3-(3-m-amidinophenyl-2-triazeno)-8-amino-5-ethyl-6-phenylphenanthridinium chloride hydrochloride (M&B38897), 7-(m-amidinophenyldiazo)-3,8-diamino-5-ethyl-6-phenylphenanthridinium chloride hydrochloride (M&B4250) and 3,8-di(3-m-amidinophenyltriazeno)-5-ethyl-6-phenylphenanthridinium chloride dihydrochloride (M&B4596) is described. The related substances are resolved on a Gemini C18 column (150 mm x 4.6 mm, 5 microm) using a mobile phase composed of a mixture of acetonitrile and 50 mM ammonium formate buffer pH 2.8 (25:75 v/v) at a flow rate of 1 ml/min with UV detection at 320 nm. The method is compatible with electrospray ionisation mass spectrometry and provides a tool for the control of substandard and counterfeit commercial preparations of isometamidium.

Determination of vancomycin in serum by liquid chromatography-high resolution full scan mass spectrometry.

A liquid chromatography-mass spectrometry (LC-MS) method was developed for the analysis of vancomycin (VCM) in human serum. The method was based on full scan data with extracted ions for the accurate masses of VCM and the atenolol internal standard obtained by Fourier transform MS. VCM was extracted from serum using strong cation exchange (SCX) solid phase extraction (SPE). The method was found to be linear in the range 0.05-10 microg/ml, which was adequate for quantification of VCM in serum samples, with a limit of quantification (LOQ) of 0.005 microg/ml and a limit of detection (LOD) of 0.001 microg/ml. Intra-day precision (n=5) was +/-3.5%, +/-2.5%, +/-0.7% at 0.05, 0.5 and 5 microg/ml, respectively. Inter-day precision (n=5) was +/-7.6%, +/-6.4%, +/-3.9% at 0.05, 0.5 and 5 microg/ml, respectively. The process efficiency for VCM was in the range 89.2-98.1% with the recovery for the atenolol internal standard (IS) being 97.3%. The method was used to determine VCM levels in patients during peri-operative infusion of the drug, which was found to result in drug levels within the required therapeutic window.

Determination of rifampicin in human plasma and blood spots by high performance liquid chromatography with UV detection: a potential method for therapeutic drug monitoring.

A high performance liquid chromatography method has been developed that allows quantification of concentrations of rifampicin in human plasma and blood spots. Rifampicin and papaverine hydrochloride (internal standard) were extracted from plasma using a Strata-X-CW extraction cartridge. These analytes were also extracted into acetonitrile from blood spots dried onto a specimen collection card. The recovery of rifampicin from plasma and blood spots was 84.5% and 65.0%, respectively. Separation was achieved by HPLC on a Kromasil C(18) column with a mobile phase composed of ammonium acetate (20 mM, pH 4.0) and acetonitrile, delivered on a gradient programme. Optimum detection was at 334 nm. The assay was linear over the concentration range of 0.5-20 microg/ml. The limit of quantification was 0.5 microg/ml in plasma; 1.5 microg/ml in blood spots. Both intraday and interday precision data showed reproducibility (R.S.D.< or =8.0, n=9). Stability studies showed rifampicin was stable in plasma for up to 9h after thawing; the samples were also stable for up to 9h after preparation. Five patient samples were analysed using the methods described. A correlation was found between the concentrations of RIF in plasma and blood spots (r(2)=0.92). This method is proposed as a means of therapeutic drug monitoring of rifampicin in patients with tuberculosis.

Metabolism of isometamidium in hepatocytes isolated from control and inducer-treated rats.

Little is known about the metabolism and mechanism of action of the trypanocide, isometamidium (ISM), the major drug used for prophylaxis of trypanosomiasis. We have investigated its metabolism and distribution in isolated rat hepatocytes using liquid chromatography-mass spectrometry and confocal laser scanning microscopy (CLSM). Two putative metabolites were formed, which were proposed to be a mono-acetyl derivative and an oxidized metabolite (SII). This is the first demonstration of the hepatic metabolism of ISM, as previous in vivo studies were hampered by dose-limiting toxicity and insensitive analytical methods. The intrinsic fluorescence of the drug enabled its intracellular uptake to be followed by CLSM. It is taken up rapidly into the nucleolus, nuclear membrane and endoplasmic reticulum within 5 min, and retained in the nucleus for at least 24 h. Persistent binding of ISM to cellular macromolecules may contribute to its prophylactic effect in vivo. Pretreatment of rats with 3-methylcholanthrene, phenobarbitone (PB) or the widely used pyrethroid pesticide, deltamethrin, resulted in an increase in metabolism of ISM to the proposed SII after 1 h incubation with hepatocytes. 3-methylcholanthrene was the most potent inducer, causing a maximal 19.5-fold induction of SII formation after exposure of hepatocytes to ISM for 1 h compared with formation by control hepatocytes. In comparison, at the 1 h timepoint deltamethrin pre-treatment caused a 10.2-fold induction, and PB only 8.2 fold.

Metabolism of troglitazone in hepatocytes isolated from experimentally induced diabetic rats.

Troglitazone (TGZ), the prototype 2,4-thiazolidinedione antidiabetic agent, is associated with hepatotoxicity in patients with Type 2 diabetes. Although the mechanism of toxicity has not been established, alterations in the clearance of TGZ from in-vitro hepatocyte cultures through metabolic conjugation reactions are believed to modulate the toxicity of the compound. In this study, the metabolism of TGZ in freshly isolated hepatocytes from the fat-fed streptozotocin-treated rat model of Type 2 diabetes is described. Biochemical parameters such as cellular reduced glutathione content, content of cytochromes P450 and b5, and the expression of glutathione-S-transferase alpha (subunits Ya and Yc2) were not affected by the induced diabetes. TGZ was metabolized primarily to a sulfonate, a quinone and a glucuronide in both control and experimentally diabetic animals. However, metabolism after induction of diabetes was characterized by a moderate increase in sulfation, a decrease in the elimination half-life of TGZ and the absence of the minor metabolites of TGZ, notably the glutathione adduct of the putative reactive intermediate (m/z = 747 (M + H)+; m/z = 745 (M - H)-).

A diminazene-resistant strain of Trypanosoma brucei brucei isolated from a dog is cross-resistant to pentamidine in experimentally infected albino rats.

Trypanosomosis is a major cause of mortality for dogs in Nigeria and treatment with diminazene aceturate has steadily become less effective, either as a result of low quality of the locally available diminazene preparations or of drug resistance. To investigate these alternatives, samples of locally obtained drugs were analysed for diminazene aceturate content and a strain of Trypanosoma brucei brucei was isolated from a diminazene-refractory dog in Nsukka, south-eastern Nigeria, and used to infect albino rats. The quality of diminazene aceturate-based preparations was variable, with two preparations containing less than 95% of the stated active compound. Rats infected with T. brucei isolated from the dog were treated 7 and 10 days after infection either with 7 mg/kg diminazene aceturate (intraperitoneally, once) or with 4 mg/kg pentamidine isethionate (intramuscularly, 7 consecutive days). Relapse rates were 100% for both trypanocides in the groups of rat treated 10 days post-infection, and 83% and 50% of rats treated 7 days after infection relapsed to diminazene aceturate and pentamidine isethionate, respectively. Careful consideration of physiological parameters showed that pentamidine was only marginally superior to diminazene aceturate as applied in this study. It was concluded that dogs in Nigeria are infected with genuinely diminazene aceturate-resistant trypanosomes that appear to be cross-resistant to pentamidine isethionate.

The chemical and pharmaceutical equivalence of sulphadoxine/pyrimethamine tablets sold on the Tanzanian market.

This study investigated chemical and pharmaceutical equivalence of 11 brands of pyrimethamine-sulphadoxine combination tablets sold on the Tanzanian market. Physical and chemical tests were performed for all the 11 brands. These tests included hardness test, friability, disintegration, dissolution, weight uniformity and assay for the active components. All the brands passed all the quality specifications of the United States Pharmacopoeia (USP) and British Pharmacopoeia (BP) in terms of hardness, friability, disintegration, assay and dissolution test, except for three brands that failed the hardness, disintegration or friability tests. One brand failed both the hardness and disintegration test; one failed the hardness test, whereas another one failed the friability test. The percentage content of pyrimethamine in the brands was in the range of 91.04-100.20% whereas that of sulphadoxine ranged from 91.53% to 99.88%. There were no major differences between the different brands of tablets containing pyrimethamine and sulphadoxine and the innovator product (Fansidar), and all brands were physically and chemically equivalent. The results indicate that the post-market surveillance and registration process in Tanzania is having an impact on product quality as there was no brand which could be considered of very poor quality. Impurity profiling of all the locally produced brands indicated that they all contained the same sulphadoxine impurity, which was absent in the innovator product, suggesting a common source of generic raw material.

Metabolism of the dihydropyridine calcium channel blockers mebudipine and dibudipine by isolated rat hepatocytes.

The prototype 1,4-dihydropyridine (1,4-DHP) nifedipine, indicated for the management of hypertension and angina pectoris, has drawbacks of rapid onset of vasodilating action and a short half-life. Several newer analogues have been designed to offset these problems and these include mebudipine and dibudipine. These analogues contain t-butyl substituents that have been selected to alter the fast metabolism without altering pharmacological activity. In this study, the metabolism of mebudipine and dibudipine by isolated rat hepatocytes has been investigated. These compounds were extensively metabolized in 2 h by oxidative pathways, analogous to those known for nifedipine, and by O-glucuronidation after hydroxylation of the t-butyl substituents. The in-vitro half-lives of mebudipine (22 +/- 7.1 min) and dibudipine (40 +/- 9.8 min) were significantly longer than that of nifedipine (5.5 +/- 1.1 min), which was investigated in parallel in this study. These newer 1,4-DHPs address the problem of the short half-life of nifedipine and have potential for further development in view of their comparable potency to nifedipine.

Determination of diminazene aceturate in pharmaceutical formulations by HPLC and identification of related substances by LC/MS.

A validated, reversed-phase, isocratic high-performance liquid chromatographic method for the simultaneous assay of diminazene aceturate, antipyrine (excipient) and diminazene impurities in pharmaceutical formulations is described. The chromatographic system consisted of a Lichrospher-60 RP-select B column with a mobile phase composition of acetonitrile-methanol-ammonium formate (pH 4.0, 20 mM) (10:10: 80 v/v/v) and UV detection at 254 nm. The method is specific, precise and accurate for the determination of diminazene in the presence of its manufacturing and degradation impurities with a limit of detection and quantification of 50 ng/ml and 10 microgram/ml (RSD<3.0%), respectively. The major manufacturing impurity [1-(4 amidino phenyl)3-(4 carbamoyl phenyl)-triazene] and a degradant (p-aminobenzamidine) of diminazene aceturate have been resolved and identified by liquid chromatography/electrospray ionization-mass spectrometry operated in a positive ion mode.