Methylation of captopril in human liver,kidney and intestine

1. The methylation of captopril was studied in the microsomal fraction from 20 human liver, 12 kidney, and 14 intestinal rnucosa specimens.

2. The hepatic methyltransferase activity (mean ± SD) was 477 ± 204 pmol/min per mg. Renal and intestinal methyltransferase activities were 3 and 8 times lower, respectively, than hepatic activity.

3. The kinetics of methyltransferase with captopril as substrate were studied in four specimens of liver, kidney and intestine. The maximum velocities of reaction (mean ± SD; pmol/min per?mg) were 697 ± 219 (liver), 456 ± 120 (renal cortex), 264 ± 77 (renal medulla) and 101 ± 28 (ileum mucosa). K_m values (mean ± SD; mM) were 5.2±2.3 (liver) 4.3±1.7 (renal cortex) 4.1±1.5 (renal medulla) and 5.3 ± 2.0 mM (ileum mucosa). V_max is subjected to a marked tissue dependence whereas K_m is similar in all tissues.

4. Liver is the primary site of captopril methylation whereas the intestine plays only a minor role. Kidney may contribute substantially to the hepatic methylation of captopril.     

Methylation of captopril in human liver, kidney and intestine.

1. The methylation of captopril was studied in the microsomal fraction from 20 human liver, 12 kidney, and 14 intestinal mucosa specimens. 2. The hepatic methyltransferase activity (mean +/- SD) was 477 +/- 204 pmol/min per mg. Renal and intestinal methyltransferase activities were 3 and 8 times lower, respectively, than hepatic activity. 3. The kinetics of methyltransferase with captopril as substrate were studied in four specimens of liver, kidney and intestine. The maximum velocities of reaction (mean +/- SD; pmol/min per mg) were 697 +/- 219 (liver), 456 +/- 120 (renal cortex), 264 +/- 77 (renal medulla) and 101 +/- 28 (ileum mucosa). Km values (mean +/- SD; mM) were 5.2 +/- 2.3 (liver) 4.3 +/- 1.7 (renal cortex) 4.1 +/- 1.5 (renal medulla) and 5.3 +/- 2.0 mM (ileum mucosa). Vmax is subjected to a marked tissue dependence whereas Km is similar in all tissues. 4. Liver is the primary site of captopril methylation whereas the intestine plays only a minor role. Kidney may contribute substantially to the hepatic methylation of captopril.     

Immunoquantitation of FMO1 in human liver, kidney, and intestine.

To determine the level of FMO1 protein present in human liver tissues, a monospecific antibody was prepared and a sensitive Western blotting procedure with enhanced chemiluminescence detection was developed. Human FMO1, purified from insect cells expressing the recombinant protein, was used as a protein standard for absolute quantification. The average concentrations of FMO1 in microsomes prepared from human liver, kidney, intestine, and fetal liver were found to be <1, 47 +/- 9, 2.9 +/- 1.9, and 14.4 +/- 3.5 pmol/mg, respectively. Quantitation in intestinal microsomes was complicated by variable degrees of proteolytic degradation of FMO1, not seen in microsomes prepared from liver or kidney. Recombinant human FMO1 and detergent-solubilized human duodenal microsomes both metabolized p-tolyl methyl sulfide stereoselectively to the (R)-sulfoxide, indicating the expression of functional FMO1 in human intestine. The relatively high levels of immunoquantifiable FMO1 in human kidney and fetal liver complement our previous catalytic studies in these tissues, which also demonstrated preferential (R)-p-tolyl methyl sulfoxide formation. These data demonstrate a profound ontogenic change in expression of hepatic FMO1 in humans, such that in adult life FMO1 is exclusively an extrahepatic drug-metabolizing enzyme. The marked expression levels of FMO1 found in human kidney coupled to the high catalytic activity of this isoform toward a diverse array of sulfides and tertiary amines suggest the possibility that human renal FMO1 is a significant contributor to the metabolic clearance of drugs and other xenobiotics bearing these functionalities.     

Drug transport in intestine,liver and kidney

Drug transport in intestine, liver and kidney is similar, because in each case transport occurs across a barrier of epithelial cells. However, the physiological conditions differ in each organ: intestinal drug absorption is largely influenced by physicochemical conditions in the intestinal lumen; actual transport across the epithelial barrier occurs mainly by diffusion; carrier-mediated transport plays a subordinate role. In contrast, hepatic uptake is mediated by specific carriers, which transport a wide variety of drugs into the liver cell and then release them either into bile, or back into the portal blood. It is unclear how many carrier systems are involved, how they are organized in the liver cell membrane, and to what extent their substrate specificities overlap. Renal secretion and reabsorption of drugs is mediated by highly active carrier systems for cations and anions. Their cooperative action results in either active reabsorption or active secretion of drugs.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

Drug-metabolizing activity of human and rat liver,lung, kidney and intestine slices

1. Organ-specific biotransformation was studied in human and rat liver, lung, kidney and small intestine slices and compared on a protein basis, using four model substances. 2. Deethylation of lidocaine was highest in liver slices from both man and rat, followed by the small intestine. 3. Metabolism of testosterone was highest in liver slices, but a different overall metabolic pattern was found between the different organs. 4. Lung, kidney and intestine slices prepared from human and rat organs showed mainly an unknown metabolite of 7-ethoxycoumarin identified as 4-ethoxy-2-hydroxyphenyl propionic acid (EPPA). 5. The maximal metabolism of 7-ethoxycoumarin in slices was equal with in vivo V_max in the rat. 6. Phase II metabolism of 7-hydroxycoumarin in kidney and intestinal slices was about 60% of the activity in liver slices. 7. In conclusion, organs other than the liver show a surprisingly high drug-metabolizing activity. Thus, the use of precision-cut slices of a combination of drug metabolizing organs in an in vitro test system from both animal and human origin is required for a proper systematic prediction of drug metabolism in man.     

Drug-metabolizing activity of human and rat liver,lung, kidney and intestine slices

1. Organ-specific biotransformation was studied in human and rat liver, lung, kidney and small intestine slices and compared on a protein basis, using four model substances. 2. Deethylation of lidocaine was highest in liver slices from both man and rat, followed by the small intestine. 3. Metabolism of testosterone was highest in liver slices, but a different overall metabolic pattern was found between the different organs. 4. Lung, kidney and intestine slices prepared from human and rat organs showed mainly an unknown metabolite of 7-ethoxycoumarin identified as 4-ethoxy-2-hydroxyphenyl propionic acid (EPPA). 5. The maximal metabolism of 7-ethoxycoumarin in slices was equal with in vivo V(max) in the rat. 6. Phase II metabolism of 7-hydroxycoumarin in kidney and intestinal slices was about 60% of the activity in liver slices. 7. In conclusion, organs other than the liver show a surprisingly high drug-metabolizing activity. Thus, the use of precision-cut slices of a combination of drug metabolizing organs in an in vitro test system from both animal and human origin is required for a proper systematic prediction of drug metabolism in man.     

Biotransformation of methyl parathion by human foetal liver glutathione S-transferases: an in vitro study

The role of human foetal liver glutathione S-transferases in the detoxification of methyl parathion was investigated. Glutathione S-transferases were partially purified by affinity chromatography utilizing reduced glutathione as the ligand coupled to epoxy-activated Sepharose 4B. This resulted in the isolation of material with an average activity (mean +/- S.E.) of 58.90 +/- 4.83 mumol 1-chloro-2,4-dinitrobenzene conjugate formed/min per mg, representing a purification of 70-fold. These partially purified foetal liver transferases catalysed the metabolism of methyl parathion exclusively to desmethyl parathion via O-dealkylation. High-performance liquid chromatography, radiometric analysis of the enzymic reaction, and co-chromatography with reference standard on thin-layer chromatography confirmed the sole metabolite as desmethyl parathion. The range of foetal liver activity towards methyl parathion was from 30 to 122 nmol desmethyl parathion formed/min per mg. Analysis of the kinetic parameters of three partially purified foetal liver preparations with gestational ages of 14, 16 and 21 weeks resulted in Km values for methyl parathion of 0.24, 0.38 and 0.86 mM, respectively; whereas, the Km values assessed for glutathione were 0.20, 0.10 and 0.18 mM. The ability of human foetal liver glutathione S-transferases to catalyse the metabolism of methyl parathion exclusively to desmethyl parathion via O-dealkylation represents a major qualitative biochemical difference from the rat-liver isozymes.     

Biotransformation of methyl parathion by human foetal liver glutathione S-transferases: an in vitro study

1. The role of human foetal liver glutathione S-transferases in the detoxification of methyl parathion was investigated.

2. Glutathione S-transferases were partially purified by affinity chromatography utilizing reduced glutathione as the ligand coupled to epoxy-activated Sepharose 4B. This resulted in the isolation of material with an average activity (mean ± S.E.) of 58.90 ± 4.83 µmol 1 -chloro-2,4-dinitrobenzenceo njugate formed/min per mg. representing a purification of 70-fold.

3. These partially purified foetal liver transferases catalysed the metabolism of methyl parathion exclusively to desmethyl parathion via O-dealkylation.

4. High-performance liquid Chromatography, radiometric analysis of the enzymic reaction, and co-chromatography with reference standard on thin-layer chromatography confirmed the sole metabolite as desmethyl parathion.

5. The range of foetal liver activity towards methyl parathion was from 30 to 122 nmol desmethyl parathion formed/min per mg.

6. Analysis of the kinetic parameters of three partially purified foetal liver preparations with gestational ages of 14, 16 and 21 weeks resulted in K_M values for methyl parathion of 0.24, 0.38 and 0.86mM, respectively; whereas, the K_M values assessed for glutathione were 0.20, 0.10 and 0.18mM.

7. The ability of human foetal liver glutathione S-transferases to catalyse the metabolism of methyl parathion exclusively to desmethyl parathion via O-dealkylation represents a major qualitative biochemical difference from the rat-liver isozymes.     

Expression of thirty-six drug transporter genes in human intestine, liver, kidney, and organotypic cell lines.

This study was designed to quantitatively assess the mRNA expression of 36 important drug transporters in human jejunum, colon, liver, and kidney. Expression of these transporters in human organs was compared with expression in commonly used cell lines (Caco-2, HepG2, and Caki-1) originating from these organs to assess their value as in vitro transporter system models, and was also compared with data obtained from the literature on expression in rat tissues to assess species differences. Transporters that were highly expressed in the intestine included HPT1, PEPT1, BCRP, MRP2, and MDR1, whereas, in the liver, OCT1, MRP2, OATP-C, NTCP and BSEP were the main transporters. In the kidney, OAT1 was expressed at the highest levels, followed by OAT3, OAT4, MCT5, MDR1, MRP2, OCT2, and OCTN2. The best agreement between human tissue and the representative cell line was observed for human jejunum and Caco-2 cells. Expression in liver and kidney ortholog cell lines was not correlated with that in the associated tissue. Comparisons with rat transporter gene expression revealed significant species differences. Our results allowed a comprehensive quantitative comparison of drug transporter expression in human intestine, liver, and kidney. We suggest that it would be beneficial for predictive pharmacokinetic research to focus on the most highly expressed transporters. We hope that our comparison of rat and human tissue will help to explain the observed species differences in in vivo models, increase understanding of the impact of active transport processes on pharmacokinetics and distribution, and improve the quality of predictions from animal studies to humans.     

Synthesis and pharmacology of a series of new organic nitrate esters

New organic nitrate esters, derived from structurally different (cyclo)aliphatic templates, were synthesized and pharmacologically investigated. Theirin vitro vascular smooth muscle relaxing activities and, occasionally,in vivo haemodynamic profiles were studied and compared to those of the clinically important nitrates, glyceryl trinitrate, isosorbide dinitrate and isosorbide-5-mononitrate. A number of compounds appeared to be even more potent than glyceryl trinitrate. Qualitative structure-activity relationships within the series of new compounds are discussed. In flexiblen-alkylene dinitrates, lipophilicity as well as chain length appears to affectin vitro activity. In semi-rigid cyclohexylene dinitrates, the number of atoms between and the configuration of the nitrate groups may play an important role. Finally, in cycloalkylene mononitrates neither the number of ring carbon atoms nor the lipophilicity clearly affects thein vitro activity. It is suggested that, apart from a limited involvement of compound lipophilicity, other factors such as differences in enzymatic conversion to a common putative bioactive species, nitric oxide, are responsible for the observed differences in activity.     

Syntheses of organic preparations and intermediate products based on nitrate esters

A new method of making 5-nitrofurfural and furacillin from furfuryl alcohol has been worked out.Translated from Khimiko-Farmatsevticheskii Zhurnal, No. 1, pp. 9–11, January, 1967.     

Raloxifene glucuronidation in human intestine, kidney, and liver microsomes and in human liver microsomes genotyped for the UGT1A1*28 polymorphism

Raloxifene, a selective estrogen receptor modulator, exhibits quite large interindividual variability in pharmacokinetics and pharmacodynamics. In women, raloxifene is metabolized extensively by different isoforms of UDP-glucuronosyltransferase (UGT) to its glucuronides. To gain an insight into intestine, kidney, liver, and lung glucuronidation of raloxifene, human microsomes of all tested organs were used. Raloxifene-6-β-glucuronide (M1) formation followed the Michaelis-Menten kinetics in intestinal, kidney, and liver microsomes; meanwhile, raloxifene-4'-β-glucuronide (M2) formation followed the substrate inhibition kinetics. Human lung microsomes did not show any glucuronidation activity. The tissue intrinsic clearances for kidney, intestine, and liver were 3.4, 28.1, and 39.6 ml · min(-1) · kg(-1), respectively. The aim of our in vitro study was to explain the mechanism behind the observed influence of UGT1A1*28 polymorphism on raloxifene pharmacokinetics in a small-sized in vivo study (Br J Clin Pharmacol 67:437-444, 2009). Incubation of raloxifene with human liver microsomes genotyped for UGT1A1*28 showed a significantly reduced metabolic clearance toward M1 in microsomes from donors with *28 allele. On the contrary, no significant genotype influence was observed on the formation of M2 because of the high variability in estimated apparent kinetic parameters, although a clear trend toward lower glucuronidation activities was observed when UGT1A1*28 polymorphism was present. The liver intrinsic clearances of both homozygotes differed significantly, whereas the clearance of heterozygotes did not differ from the wild-type and the mutated homozygotes. In conclusion, our results show the high importance of the liver and intestine in raloxifene glucuronidation. Moreover, the significant influence of UGT1A1*28 polymorphism on metabolism of raloxifene was confirmed.     

Dose-dependent protein adduct formation in kidney, liver, and blood of rats and in human blood after perchloroethene inhalation

Perchloroethene (PER) was a widely used solvent and is an environmental contaminant. In bioassays for carcinogenicity, PER was found to increase the incidence of liver tumors in mice and of renal tumors in male rats. Toxic effects of PER after repeated administration are likely caused by bioactivation. PER bioactivation occurs by two pathways. Oxidation by cytochrome P450 results in trichloroacetyl chloride, which binds to lipids and proteins. Glutathione S-conjugate formation from PER and further processing of the formed S- (trichlorovinyl)glutathione to S-(trichlorovinyl)-L-cysteine, followed by cysteine conjugate beta-lyase catalyzed cleavage, resulted in the reactive dichlorothioketene, which binds to proteins under formation of N epsilon-(dichloroacetyl)-L-lysine in proteins. The objective of this study was to comparatively quantify the dose-dependent formation of protein adducts from PER in rats and humans using antibodies with high specificity for either N epsilon-(trichloroacetyl)-L-lysine or N epsilon-(dichloroacetyl)-L-lysine in proteins. Male and female rats (n = 2, per concentration and time point) were exposed to 400, 40, and 10 ppm PER for 6 h and killed at various time points. Formation of N epsilon-(dichloroacetyl)-L-lysine and N epsilon-(trichloroacetyl)-L- lysine in proteins was comparatively quantified in subcellular fractions from liver and kidney and in blood. In addition, three male and three female human volunteers were exposed to 10 and 40 ppm PER, and formation of protein adducts in blood was analyzed using the antibodies and GC/MS after immunoaffinity enrichment of modified proteins. In liver and kidney subcellular fractions and blood of PER- exposed rats, dose-dependent formation of N epsilon-(dichloroacetyl)-L- lysine and N epsilon-(trichloroacetyl)-L-lysine in proteins was observed. Highest concentrations of N epsilon-(dichloroacetyl)-L-lysine in proteins were formed in kidney mitochondria, followed by kidney cytosol. Only low concentrations of N epsilon-(dichloroacetyl)-L-lysine in proteins were present in liver proteins; blood concentrations of N epsilon-(dichloroacetyl)-L-lysine in proteins were 5 to 10 fold lower than in kidney mitochondria. Highest concentrations of N epsilon- (trichloroacetyl)-L-lysine were found in microsomal and cytosolic proteins from the liver of rats exposed to PER. A higher protein adduct formation was seen in PER-exposed-male than -female rats for N epsilon- (dichloroacetyl)-L-lysine in renal mitochondrial proteins, after exposure to 400 ppm PER. In human blood samples taken 0 and 24 h after the 6 h exposures to PER, N epsilon-(trichloroacetyl)-L-lysine- containing proteins were present in low concentrations. N epsilon- (Dichloroacetyl)-L-lysine-containing proteins were not detected either by Western blotting or GC/MS after immunoaffinity chromatography. The obtained results indicate a dose-dependent covalent binding of PER metabolites to proteins in rat liver, kidney, and blood and suggest that the concentration of covalent protein adducts is much lower in blood of humans as compared to the blood of rats exposed under identical conditions.      

In vitro studies in microsomes from rat and human liver, kidney, and intestine suggest that perfluorooctanoic acid is not a substrate for microsomal UDP-glucuronosyltransferases

Perfluorooctanoic acid (PFOA) is a fluorinated fatty acid analogue used as a surfactant in the manufacture of fluoropolymers. Previous studies have indicated that PFOA was metabolically inert in mammals, but recent metabolism studies with related fluorochemicals suggested that PFOA might form a glucuronide conjugate. [(14)C(1)]-PFOA was incubated with male and female human and rat liver, kidney, and small intestine microsomes. Incubations were carried out in the presence of alamethicin and beta-saccharolactone to increase access of PFOA to the enzyme active site and to inhibit potential hydrolysis of PFOA-glucuronide by microsomal beta-glucuronidase, respectively. Although positive control experiments using p-nitrophenol demonstrated significant UDP-glucuronosyltransferase (UDPGT) activity in all of the tested microsomal preparations, no evidence for formation of a PFOA-glucuronide was obtained, either by high-sensitivity radiochromatography or by LC/MS. These data suggest that PFOA is not a substrate for human or rodent microsomal UDPGTs.     

Biotransformation of cyclosporin in primary rat,porcine and human liver cell co-cultures

The purpose of this study was to investigate the species-specific cyclosporin biotransformation in primary rat, human, and porcine liver cell cultures, and to investigate the suitability of a modified sandwich culture technique with non-purified liver cell co-cultures for drug metabolism studies. A sandwich culture was found to enhance hepatocellular metabolic activity and improve cellular morphology and ultrastructure. The cyclosporin metabolites AM9 and AM1 were formed in porcine and human liver cell sandwich co-cultures at levels corresponding to the respective in vivo situations. In contrast, metabolite profiles in rat hepatocytes were at variance with the in vivo situation. However, for all cell types, the overall metabolic activity was positively influenced by sandwich co-culture. The initial levels of albumin synthesis were higher in sandwich cultures than in those without matrix overlay. It is hypothesized that the sandwich culture system provides an improved microenvironment and is, therefore, an advantageous tool for in vitro studies of drug metabolism.     

Biotransformation of lovastatin--III. Effect of cimetidine and famotidine on in vitro metabolism of lovastatin by rat and human liver microsomes

The effects of the H2-receptor antagonists, cimetidine and famotidine, on the microsomal metabolism of [14C]lovastatin were investigated. Liver microsomes were prepared from control, phenobarbital- and 3-methylcholanthrene-pretreated rats and humans (male and female). Concentration-dependent inhibition of the metabolism of lovastatin (0.1 mM) was observed with cimetidine (0.1 to 1.0 mM). In contrast, famotidine at a similar concentration was a very weak inhibitor. The formation of 6'beta-hydroxy-lovastatin, the major microsomal metabolite of lovastatin, was similarly inhibited. The results suggest that in vivo metabolic interaction with concomitantly administered lovastatin is less likely with famotidine than with cimetidine. Phenobarbital pretreatment produced 58% stimulation in overall metabolism, whereas 3-methylcholanthrene pretreatment had no effect relative to control rats (5.4 nmol/mg protein/min). Liver microsomes from phenobarbital-pretreated rats produced 67% more of the 6'beta-hydroxy-lovastatin but 63-66% less of the 3'-hydroxy and 6'-exomethylene metabolites. Liver microsomes from 3-methylcholanthrene-treated rats also produced less 3"-hydroxy-lovastatin (49%) but similar quantities of the other two metabolites. 6'beta-Hydroxy-lovastatin was a major metabolite with human liver microsomes. Interestingly with these microsomes, hydroxylation at the 3'-position of the molecule was a negligible pathway and hydrolysis to the hydroxy acid form was not observed. The formation of 6'-exomethylene-lovastatin was also catalyzed by human liver microsomes (0.5 to 0.8 nmol/mg protein/min).