The influence of cisapride and clarithromycin on QT intervals in healthy volunteers.

OBJECTIVE: Recently a few cases of long QT syndrome were reported during treatment with cisapride. In most of these cases, risk factors for cardiac arrhythmias or pharmacologic interactions might have been involved, and the role of cisapride remained unclear. Macrolides such as clarithromycin potentially interact with the metabolic elimination of cisapride and have overlapping indication areas. We therefore studied whether combined treatment with clarithromycin and cisapride leads to pharmacokinetic changes and increased QT intervals. METHODS: The study was an open, randomized, 2-way crossover study with washout periods of 1 week. Twelve healthy volunteers were recruited. Treatments were cisapride (10 mg 4 times a day) for 10 days with concomitant clarithromycin (500 mg twice a day) from days 6 through 10, or clarithromycin (500 mg twice a day) for 10 days combined with cisapride (10 mg 4 times a day) from days 6 through 10. Frequent ECG recordings were performed for 24 hours before drug treatment (baseline). After 5 days of monotherapy and combination therapy, frequent ECG recordings and assessments of plasma drug levels were performed for 24 hours. RESULTS: Clarithromycin alone was associated with a minimal increase in QTc intervals. Monotherapy with 10 mg cisapride 4 times a day led to a concentration-dependent QTc elevation, amounting to 6 ms during steady state. Combination of cisapride and clarithromycin caused an average QTc increase of 25 ms above pretreatment values and 3-fold increases in cisapride concentrations. CONCLUSIONS: QTc elevations after cisapride or clarithromycin alone remained within the normal range of diurnal variation. Coadministration of cisapride and clarithromycin produced a substantial QT prolongation. The data support the recently purported interaction between cisapride and clarithromycin and thus the filed contraindication to combine these drugs.

Hepatic cytochrome P450 induction in goats. Effects of model inducers on the metabolism of alkoxyresorufins, testosterone and ethylmorphine, and on apoprotein and mRNA levels.

Male and female African dwarf goats were treated orally with phenobarbital (PB) or triacetyloleandomycin (TAO), or subcutaneously with beta-naphthoflavone (BNF). Hepatic microsomal cytochrome P450 content was increased by PB and TAO, but not by BNF. PB effects on P450 activities were non-selective: ethoxyresorufin deethylase (EROD) and pentoxyresorufin depentylase (PROD), hydroxylation of testosterone (TST) and demethylation of ethylmorphine (ETM) were all induced by a factor of 2-3. A similar non-selective induction was observed with TAO, except for EROD and PROD (no effects). After PB and TAO treatment, increased levels of a protein cross-reactive with anti-sheep P450 3A and 2B were found. Thus, in dwarf goats, both PB and TAO appeared to be P450 3A inducers. Selective PB effects related to a P450 2B form on PROD are lacking but 16 alpha-hydroxylation of TST was induced markedly. At the mRNA level, PB induced an mRNA that showed good sequence homology with a human P450 3A4 cDNA probe, rather than with a rat 3A1 probe. BNF selectively induced EROD, whereas TST hydroxylation and ETM dealkylation were inhibited. With BNF-treated animals, increased concentrations of a protein cross-reactive with anti-rat P450 1A1/1A2 and of an mRNA that showed homology with a human 1A1 cDNA probe, but not with a mouse 1A1/1A2 probe, were observed.

Cytochrome P450 induction and metabolism of alkoxyresorufins, ethylmorphine and testosterone in cultured hepatocytes from goats, sheep and cattle.

Very little is known of cytochrome P450 (P450) patterns and enzyme characteristics in food-producing animal species. Oxidative metabolism of alkoxyresorufins, ethylmorphine (EtM) and testosterone (TST) was used to monitor the effects of the P450 inducers phenobarbital (PB), beta-naphthoflavone (BNF), dexamethasone (DEX) and triacetyloleandomycin (TAO) in primary cultured hepatocytes from goats, sheep and cattle. BNF effectively and specifically induced ethoxyresorufin deethylase (> 20-fold), indicating the presence of an inducible P450 1A form, and down-regulated EtM demethylation and most selected TST hydroxylations. In non-induced hepatocyte cultures, TST was metabolized to 6 beta-, 2 beta-, 12 beta-, and 11 alpha-hydroxy-TST (OHT). PB and, to a lesser extent, DEX non-specifically induced all OHT formations, and EtM demethylation. TAO almost completely inhibited OHT formation and EtM demethylation. These results indicate the involvement of principally one P450 form, or a restricted number of related P450 forms, presumably belonging to the P450 3A subfamily. In western blot analysis, cross reactivity was found with rat anti-P450 3A1 and anti-sheep P450 3A. A more specific PB effect was observed for 16 alpha-OHT, which may be formed though a ruminant P450 2B form. None of the inducers influenced pentoxyresorufin depentylase (PROD) or EtM O-deethylation. Metabolite patterns and inducibility of selected activities in ruminant hepatocytes are in accordance with previous findings in goats in vivo. Cytochrome P450 characteristics in ruminants appear to differ from those in rats whereas similarities to the situation in humans appear to exist.

Primary bovine hepatocytes in the study of cytokine induced acute-phase protein secretion in vitro.

To investigate the utility of primary cultures of bovine hepatocytes for compartmentalized acute phase protein studies the secretion of serum amyloid-A (SAA) and haptoglobin (Hp) was measured after stimulation by pro-inflammatory cytokines (recombinant human IL-6 (rhIL-6) and recombinant human tumour necrosis factor-alpha (rhTNF-alpha)). During the incubation period of the experiment, the SAA and Hp secretion into the culture medium increased (P < 0.05). SAA concentrations showed an additional increase following treatment with each of the cytokines (P < 0.01). Hp concentrations remained unchanged, whereas incubation with a combination of both resulted in a significant increase of the medium concentration of both SAA (P < 0.01) and Hp (P < 0.05). From these findings it is concluded that primary bovine hepatocytes can be used for in vitro studies on acute-phase protein secretion.

The influence of nitrite, nitrate and nitric oxide on ammonia use and urea production in primary cultures of sheep hepatocytes.

Inorganic nitrite introduced into the living organism is rapidly converted into nitrate and nitric oxide (NO). It is known that nitrite decreases ammonia use and urea formation in vitro and it seems that nitrite also influences these processes in vivo. However, the mechanism underlying this effect is not known. Therefore, we decided to compare the influence of sodium nitrite (NaNO(2)), sodium nitrate (NaNO(3)) and NO on ammonia use and ureagenesis in monolayer cultures of sheep hepatocytes during 18 hr of cultivation. It was found that 0.5 and 2.5 mmNaNO(2) significantly reduced ammonia use in a dose-dependent manner for the first 6 hr of incubation; at higher concentrations (2.5 mm), it also decreased urea formation. Also, the presence of nitrite did not affect the lactate dehydrogenase (LDH) activity in the medium which indicates that the nitrite effect did not result from its cytotoxic action. NaNO(3) (0.5 and 2.5 mm) did not induce any changes in ammonia use and urea synthesis in hepatocytes. With sodium nitroprusside (SNP) (0.001, 0.01, 0.1, 0.5 and 1.0 mm) a decrease of ammonia use and urea production was observed corresponding to the nitrite effect, but contrary to nitrite exposure these changes in metabolism were persistent during the whole cultivation period. On the other hand, potassium cyanide (KCN) (0.1 and 0.5 mm) did not influence either urea formation or ammonia use. Thus, it can be concluded that in isolated hepatocytes nitrate and/or NO are not the mediators of nitrite effects on nitrogen metabolism. Furthermore, there is no evidence that nitrite effects are mediated by impaired mitochondrial respiration and energy production.

Characterization of cytochrome P450 isoenzymes in primary cultures of pig hepatocytes.

Despite the fact that pigs are increasingly used in pharmacological and toxicological studies, knowledge on the enzymes which metabolize xenobiotics, in particular cytochrome P450 (CYP) enzymes, in pigs is still very limited. Primary cultures of pig hepatocytes were used to characterize CYP enzymes. The characterization was performed at the level of enzymatic activities, apoprotein and mRNA analyses. Enzyme inducers investigated were beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) and rifampicin (RIF). After 48hr of BNF treatment, CYP1A protein and mRNA levels were increased, and ethoxyresorufin O-deethylation and caffeine 3-demethylation were strongly induced. PB and RIF increased the levels of CYP3A apoprotein and mRNA, whereas BNF down-regulated CYP3A and related activities. PB and RIF treatment resulted in increased ethylmorphine N-demethylation and testosterone hydroxylation, which appears to be the result of CYP3A induction. Hybridization of pig RNA with a human CYP2C9 cDNA probe showed a PB and RIF inducible CYP, which was down-regulated by BNF. Similar inducing effects were observed for tolbutamide, a marker substrate for CYP2C. DEX was not a potent inducer, although some induction of CYP3A mRNA was observed. The present results indicate the absence of CYP2B and probably CYP2D enzymes and activities in pig liver. Despite some dissimilarities, the results indicate that pigs, apart from their very human-like physiology, might represent a more appropriate model species for oxidative drug metabolism in humans than rats.

Comparative aspects and sex differentiation of plasma sulfamethazine elimination and metabolite formation in rats, rabbits, dwarf goats, and cattle.

Plasma disposition and urinary recovery of sulfamethazine (SMZ), its N4-acetylated metabolite (N4AcSMZ), and 2 of its hydroxylated metabolites--5-hydroxysulfamethazine (5OHSMZ) and 6-hydroxymethylsulfamethazine (6CH2OHSMZ)--were determined in either sex of 4 animal species: rats, dwarf goats, rabbits, and cattle. Rats, rabbits, and dwarf goats had significant (P < 0.01) sex difference in SMZ plasma clearance. Male rats had higher plasma clearance than did female rats, and excreted higher amounts of the hydroxy metabolites and lower amounts of N4AcSMZ. The N4AcSMZ metabolite was predominant in plasma and urine of rabbits. Male rabbits had higher plasma clearance than did female rabbits, but differences in metabolite profile were not apparent. With regard to plasma SMZ elimination, the situation in goats was opposite to that in rats. Male goats had considerably lower clearance than did female goats. This was associated with a lower hydroxylation rat in males. Plasma half-life of SMZ in cows was lower than that in bulls, probably because of a smaller distribution volume in cows. Compared with elimination via urine, elimination via milk was negligible in cows. Significant differences in metabolite profiles were not found between bulls and cows. Similar to those in rats and mice, hormone-dependent xenobiotic metabolic pathways may exist in other species. Depending on species and xenobiotic compound residue concentrations of xenobiotics, their metabolites, or both may differ with sex of the animal, or may be altered after treatment with anabolic hormones.

Improved high-performance liquid chromatographic method for the determination of ethylmorphine and its metabolites in microsomal incubations and cell culture media.

Ethylmorphine N-demethylation is used as a marker pathway in studies of rat cytochrome P450 3A and 2C11 biotransformations. At present, microsomal activities are generally measured by a colorimetric determination of the formed formaldehyde. In the present study, a high-performance liquid chromatographic method of separating and quantifying both the N-demethylated (norethylmorphine) and the O-de-ethylated (morphine) metabolites is described. Either samples are extracted with ethyl acetate or proteins are precipitated with zinc sulphate-barium hydroxide. Separation is achieved on a CN reversed-phase column, using a mobile phase of phosphate buffer (pH 4.5)-acetonitrile (90:10, v/v). At a flow-rate of 1.5 ml/min, the analysis time is 30 min. The limit of detection (ultraviolet, 210 nm) for ethylmorphine and its metabolites is 0.5 micrograms/ml.

Metabolism of antipyrine and sulphadimidine in dwarf goats: effects of the enzyme-inducing agents phenobarbital, troleandomycin and rifampicin.

1. Antipyrine (AP) and sulphadimidine (SDD) plasma elimination and metabolite formation were studied in dwarf goats before and after treatment with phenobarbital (PB), triacetyloleandomycin (TAO), and rifampicin (RIF). 2. PB treatment significantly increased AP plasma clearance in both male and female goats. With SDD, only male goats were studied, which showed a significant increase of SDD plasma clearance following PB treatment. 3. After PB treatment, partial clearance values of four AP metabolites, 3-hydroxymethylantipyrine (HMA), norantipyrine (NORA), 4-hydroxyantipyrine (OHA) and 4,4'-dihydroxyantipyrine (DOHA), were significantly increased. This induction effect was different for the individual metabolites and also showed sex-dependency. 4. In PB-induced male goats the formation of the hydroxylated SDD metabolites, 6-hydroxymethyl-SDD and 5-hydroxy-SDD, was significantly increased. 5. After TAO treatment, female goats showed a slightly reduced AP plasma clearance and a decreased partial clearance of two AP metabolites, HMA and DOHA. There was no effect on SDD plasma elimination or metabolite excretion. 6. In male goats, RIF had no effect on plasma elimination of AP and SDD. With SDD, it decreased the urinary excretion of the unchanged drug and its N4-acetylated metabolite. 7. Induction/inhibition studies of drug metabolism in food-producing animal species are desirable to gain more insight into the regulation of enzymes involved in the metabolism of xenobiotics.

Rapid method for the determination and quantification of bromosulphophthalein and metabolites in cultured hepatocytes, culture media and bile by high-performance liquid chromatography.

An ion-pair high-performance liquid chromatographic method for the rapid, selective and sensitive analysis of samples containing bromosulphophthalein (BSP) and its conjugates is presented. The method is useful for analysis in bile, culture media and cultured hepatocytes. Two sample preparation methods are described. Even though BSP recovery from albumin binding is complete, only a small percentage of free BSP can be detected in cells, possibly owing to a conjugation-related pool of BSP in cells. As BSP-glutathione recovery is complete, the method offers a useful tool to investigate impairment of glutathione conjugation.

Sulfadimidine metabolism in vitro: II. Comparative studies in cultured rat, goat, sheep and cattle hepatocytes.

Hydroxylation and acetylation of sulphadimidine (SDD) and the deacetylation of N4-acetyl SDD was investigated in cultured hepatocytes from male and female rats, from male and female goats and from female sheep and cattle. Significant sex differences were observed for hydroxylation of SDD in hepatocytes from rat and goat. In goat, sheep and cow hepatocytes, the hydroxylation pathway is relatively important, whereas in rat hepatocytes, acetylation is predominant. Hepatocytes of all four species deacetylated N4-acetyl SDD. In ruminant hepatocytes, deacetylating activity was of considerable importance, whereas in rat hepatocytes, it appeared a minor pathway of metabolism. Similar to the in vivo situation, formation of N4-acetyl SDD in cultured hepatocytes results from an equilibrium of acetylation and deacetylation. A good correlation was found between results in isolated hepatocytes and previous findings in vivo, both in levels of species-related activities and in acetylation-hydroxylation ratios. In conclusion, cultured hepatocytes appear a useful in vitro model to study comparative sulfonamide metabolism.

High-performance liquid chromatographic method for the routine determination of sulphadimidine, its hydroxy metabolites and N4-acetylsulphadimidine in body fluids and cell culture media.

A simple high-performance liquid chromatographic method is presented for the determination of trace amounts of sulphadimidine (SDD), its hydroxylated metabolites and N4-acetyl-SDD in blood plasma, urine, hepatocyte culture media and microsomal incubations. The synthesis of 5-hydroxy-SDD and an improved method for the isolation of 4-methylhydroxy-SDD from urine are described and their respective specific absorption coefficients at 265 nm are calculated by on-line radiochemical and ultraviolet detection. The limit of detection of the analytical method is 0.05 micrograms/ml for SDD and its hydroxy metabolites and 0.2 micrograms/ml for N4-acetyl-SDD. Linear calibration graphs for SDD and its metabolites were constructed from 0.2 to 50 micrograms/ml. The method has been applied to biotransformation studies in vivo and in vitro.

Determination of aditoprim and its oxidative metabolites in plasma and microsomal incubation mixtures by high-performance liquid chromatography.

A high-performance liquid chromatographic method is presented for the determination of aditoprim (ADP) and its two oxidative metabolites in biological fluids including sheep plasma and the incubation medium of liver microsomes. The compounds were separated using a highly deactivated C18 column; the mobile phase consisted of an 0.05 M phosphate buffer (pH 6) and acetonitrile in a ratio of 90:10 (v/v). The eluate was quantified by ultraviolet detection at 230 nm. Calibration curves were linear from 0.25 to 5 micrograms/ml. The limit of detection was 0.05 microgram/ml. In an in vivo kinetic study in sheep, N-monodesmethyl-ADP and N-didesmethyl-ADP appeared to be equally important metabolites. In contrast, in in vitro metabolism studies using liver microsomes from different animal species including sheep, N-didesmethyl-ADP was formed predominantly.

Determination of trimethoprim and its oxidative metabolites in cell culture media and microsomal incubation mixtures by high-performance liquid chromatography.

A high-performance liquid chromatographic method is presented for the determination of trimethoprim (TMP), 3'-hydroxy-TMP, 4'-hydroxy-TMP, alpha-hydroxy-TMP and two TMP N-oxides. The last two metabolites appear to decompose on liquid extraction. TMP and its oxidative metabolites are separated using a C18 radial-compression column and quantified by UV detection at 230 nm. Calibration curves are linear from 0.5 to at least 50 microM. The limit of detection is 0.05-0.15 micrograms/ml. In in vitro rat liver metabolism studies. 3'- and 4'-hydroxylation of TMP appear to be important metabolic pathways whereas TMP N-oxides are minor metabolites.

Effects of various medium formulations and attachment substrata on the performance of cultured ruminant hepatocytes in biotransformation studies.

1. A procedure for the isolation and primary culture of hepatocytes from goat and cattle is described. Hepatocyte culture performance was monitored for 51 h by measuring viability, cytochrome P-450 maintenance, dealkylation of scoparone and ethylmorphine, and glucuronidation of phenol red. 2. Culture medium composition is discussed in relation to differences between splanchnic blood composition of ruminant and monogastric animal species. Main differences are in glucose and volatile fatty acid concentrations. Modified Williams' E culture medium did not yield higher culture performance than non-modified Williams' E. 3. Coating of culture dishes with either collagen or fibronectin did not improve culture performance. 4. Williams' E, although developed for rodent cells, proves to be a suitable basal medium for ruminant hepatocytes. In this medium, culture quality is high for at least several days. 5. In cultured goat hepatocytes, biotransformation rate for scoparone amounted to 20 nmol/mg protein per h, for ethylmorphine 96 nmol/mg protein per h and for phenol red 2 nmol/mg protein per h. Biotransformation activity in cow hepatocytes is approximately half that in goat hepatocytes.

Applicability of cultured hepatocytes derived from goat, sheep and cattle in comparative drug metabolism studies.

1. Using trimethoprim (TMP), scoparone (SCOP), ethylmorphine (EtM), 1-naphthol (1-N) and phenol red (PhR) as test substrates, biotransformation activities were investigated in cultured hepatocytes from male and female rat, male and female goat, and female sheep and cattle. 2. As compared with rat hepatocytes, the total culture cytochrome P450 content was relatively well maintained in ruminant hepatocytes. In 72 h, it decreased to approximately half the initial content, whereas in rat hepatocytes only 30% was maintained. In ruminant hepatocytes, sulphation of 1-N remained fairly stable, glucuronidation of PhR decreased gradually, and glucuronidation of 1-N increased during the 72-h culture period. 3. Oxidative metabolism of TMP was rapid in goat and sheep hepatocytes, as compared with rat hepatocytes, reflecting species differences in TMP pharmacokinetics in vivo. In contrast with rat hepatocytes, 6-O-demethylation was by far the major pathway of scoparone metabolism in ruminant hepatocytes. The glucuronidation and sulphation activities were similar among the species. 4. In goat liver cells, sex differences in some oxidative biotransformations were observed, females being more active than males. In rat hepatocytes, a reverse sex difference was observed. 5. In conclusion, cultured hepatocytes from agricultural target species appear a useful in vitro model to study comparative metabolism of veterinary drugs and other xenobiotics. Comparing rat and ruminant, sex and species differences and similarities in drug metabolism can be observed that reflect the in vivo situation.

cDNA and deduced amino acid sequence of a dwarf goat liver cytochrome P450 fragment belonging to the CYP2C gene subfamily.

From a liver cDNA library derived from a phenobarbital treated dwarf goat a 1176 bp cDNA-fragment, coding for 285 amino acids, has been isolated. Northern blot analysis reveals detection of a 2 kb mRNA which is inducible by phenobarbital, triacetyloleandomycin and to an lesser extent by beta-naphthoflavone. Analysis of the deduced amino acid sequence shows a 72.5% and a 71.1% homology with rabbit CYP2C3 and human CYP2C17, respectively. The nucleotide sequence reveals homologies of 79.8% and 78.7%, respectively. Comparison of the amino acid sequences between CYP2C forms of various species reveals a high homology around the Cys436, the so-called C-terminal cysteine containing peptide, which is assigned to be the heme-binding region.

Hormonal regulation of oxidative drug metabolism in the dwarf goat. The effect of sex and hormonal treatment on plasma disposition and metabolite formation of sulphadimidine.

Sulphadimidine (20 mg/kg i.v.) plasma elimination and metabolite formation were studied in intact male, castrated male, and female dwarf goats. Plasma pharmacokinetics and urinary metabolite patterns were first studied in untreated animals. Afterward, females and castrated were treated with a combination of testosterone-propionate (1 mg/kg) and 17 beta-oestradiol-benzoate (0.02 mg/kg) once every 3 days, for a period of 4 weeks. In untreated animals, males showed a considerably lower plasma clearance than females or castrates. This was accompanied by lower partial clearances for the production of two hydroxylated sulphadimidine metabolites. After hormonal treatment of females and castrates, sulphadimidine plasma clearance was significantly reduced, to values corresponding with those observed in control males. Furthermore, hydroxylation was significantly inhibited after treatment. The results indicate that sulphadimidine hydroxylation in the goat is performed by enzymes of the cytochrome P450 complex which are strongly influenced by gonadal hormones. Androgens seem to play a central role in this respect.

Sulphadimidine metabolism in vitro: I. Sex differences in acetylation and hydroxylation in cultured rat hepatocytes.

The hydroxylation and acetylation of 0.5 mM sulphadimidine (SDD) was studied in primary cultures of hepatocytes from male and female rats, and from castrated male and sham operated male rats. In addition, SDD metabolism was investigated in hepatocytes from castrated male rats treated with testosterone, prior to liver cell isolation. In male rat hepatocytes a significantly higher hydroxylation activity was observed than in hepatocytes from female and castrated male rats. Acetylation activity was higher in females. Testosterone induced hydroxylation but did not affect acetylation. These results correlate well with data from previous in vivo studies, showing the relevance of this in vitro model.