Consequences of mitochondrial injury induced by pharmaceutical fatty acid oxidation inhibitors is characterized in human and rat liver slices.

Inhibition of liver mitochondrial beta-oxidation by pharmaceuticals may lead to safety concerns including mitochondrial dysfunction, lipid accumulation, inflammation and necrosis. In this study, the consequences of mitochondrial beta-oxidation inhibition by pharmaceuticals is investigated in human and rat liver slices. The fatty acid oxidation inhibitors Etomoxir and CPI975, inhibit the rate limiting mitochondrial beta-oxidation enzyme carnitine palmitoyltransferase I, while FOX988 and SDZ51-641, sequester mitochondrial coenzyme A to inhibit carnitine palmitoyltransferase II. Mitochondrial dysfunction was evident by a significant decrease of liver slice ATP levels and mitochondrial injury was verified by ultrastructural changes in morphology, manifested as enlarged mitochondria, C- or O-shaped mitochondria, and granular or crystalline inclusions. Gene expression changes were evident prior to changes in mitochondrial morphology. Time- and concentration dependent changes in mitochondrial genes linked with respiration and mitochondrial fatty acid beta-oxidation were associated with an up-regulation of peroxisome fatty acid oxidation genes, likely as a compensatory mechanism for the inhibition of the mitochondrial pathways. Gene expression changes preceding the decline of liver slice ATP and GSH levels included an up-regulation of stress response and oxidative stress gene expression, as well as genes linked with transcription, transporters, proliferation, cell matrix and signaling. In association with the decline of liver slice ATP and GSH was increased apoptosis and inflammation. Caspase activity, a functional indicator of apoptosis, was significantly increased as well as an up-regulation of genes linked with apoptosis. The increased gene and protein expression of the pro-inflammatory cytokine IL-8, produced by endothelial cells, is likely in response to the manifestation of oxidative stress and GSH depletion; further amplifying the oxidative stress response induced by the fatty acid oxidation inhibitors and triggering an inflammatory response. In summary, human and rat liver slices exhibited similar effects to the inhibitors of mitochondrial beta-oxidation, and the mitochondrial injury is associated with apoptosis and inflammation in the liver slices.

In vitro metabolism of tegaserod in human liver and intestine: assessment of drug interactions.

Tegaserod is a selective 5-HT(4) receptor partial agonist with promotile activity in the gastrointestinal tract. This study was designed to describe the metabolic pathways of tegaserod in the human liver and small intestine in vitro, to identify the enzymes involved in tegaserod metabolism, and to investigate the effect of tegaserod on CYP-catalyzed reactions involving other compounds. Tegaserod was metabolized in human liver microsomes to O-desmethyl tegaserod at a low rate. This metabolite was also formed by cDNA expressed CYP2D6, and the reaction in human liver microsomes was inhibited by quinidine. In human liver slices, direct N-glucuronidation of tegaserod at the guanidine nitrogens (M43.2, M43.8, and M45.3) was found, with M43.8 being the major metabolite. Human small intestine slices also metabolized tegaserod to the N-glucuronides, suggesting a contribution of the small intestine to the presystemic metabolism. 5-Methoxyindole-3-carboxylic acid (M29.0), the main metabolite in human plasma, was generated in vitro by a sequence of reactions starting with nonenzymatic acid-catalyzed hydrolysis, followed by enzymatic oxidation and conjugation with glucuronic acid. Tegaserod inhibited CYP2C8, CYP2C9, CYP2C19, CYP2E1, and CYP3A only to a small extent with IC(50) values >30 microM. Tegaserod more effectively inhibited CYP1A2 and CYP2D6 with K(i) values of 0.84 and 0.85 microM, respectively. However, these K(i) values are approximately 140-fold greater than the maximal tegaserod plasma concentrations following the clinically relevant 6-mg oral dose given to healthy volunteers. M29.0, the main circulating metabolite, did not demonstrate any inhibitory potential toward cytochrome P450 enzymes in vitro. Therefore, clinically relevant metabolic drug interactions with tegaserod seem unlikely.

In vitro human tissue models in risk assessment: report of a consensus-building workshop.

Advances in the technology of human cell and tissue culture and the increasing availability of human tissue for laboratory studies have led to the increased use of in vitro human tissue models in toxicology and pharmacodynamics studies and in quantitative modeling of metabolism, pharmacokinetic behavior, and transport. In recognition of the potential importance of such models in toxicological risk assessment, the Society of Toxicology sponsored a workshop to evaluate the current status of human cell and tissue models and to develop consensus recommendations on the use of such models to improve the scientific basis of risk assessment. This report summarizes the evaluation by invited experts and workshop attendees of the current status of such models for prediction of human metabolism and identification of drug-drug interactions, prediction of human toxicities, and quantitative modeling of pharmacokinetic and pharmaco-toxicodynamic behavior. Consensus recommendations for the application and improvement of current models are presented.

Multiple cytochrome P-450s involved in the metabolism of terbinafine suggest a limited potential for drug-drug interactions.

Biotransformation pathways and the potential for drug-drug interactions of the orally active antifungal terbinafine were characterized using human liver microsomes and recombinant human cytochrome P-450s (CYPs). The terbinafine metabolites represented four major pathways: 1) N-demethylation, 2) deamination, 3) alkyl side chain oxidation, and 4) dihydrodiol formation. Michaelis-Menten kinetics for the pathways revealed mean K(m) values ranging from 4.4 to 27.8 microM, and V(max) values of 9.8 to 82 nmol/h/mg protein. At least seven CYP enzymes are involved in terbinafine metabolism. Recombinant human CYPs predict that CYP2C9, CYP1A2, and CYP3A4 are the most important for total metabolism. N-demethylation is primarily mediated by CYP2C9, CYP2C8, and CYP1A2; dihydrodiol formation by CYP2C9 and CYP1A2; deamination by CYP3A4; and side chain oxidation equally by CYP1A2, CYP2C8, CYP2C9, and CYP2C19. Additionally, characteristic CYP substrates inhibited pathways of terbinafine metabolite formation, confirming the involvement of multiple enzymes. The deamination pathway was mainly inhibited by CYP3A inhibitors, including troleandomycin and azole antifungals. Dihydrodiol formation was inhibited by the CYP1A2 inhibitor furafylline. Terbinafine had little or no effect on the metabolism of many characteristic CYP substrates. Terbinafine, however, is a competitive inhibitor of the CYP2D6 reaction, dextromethorphan O-demethylation (K(i) = 0.03 microM). In summary, terbinafine is metabolized by at least seven CYPs. The potential for terbinafine interaction with other drugs is predicted to be insignificant with the exception that it may inhibit the metabolism of CYP2D6 substrates. Clinical trials are needed to assess the relevance of these findings.

Metabolism-dependent stimulation of reactive oxygen species and DNA synthesis by cyclosporin A in rat smooth muscle cells.

The clinical use of the immunosuppressive drug cyclosporin A (CsA) is limited by its side effects, namely hypertension and nephrotoxicity. It has been proposed that reactive oxygen species (ROS) could be involved as mediators of the toxic effects of CsA. Here, we have studied the possible interrelationship between CsA metabolism and production of ROS. Using cultures of rat aortic smooth muscle cells (RASMC), CsA (1 microM) produced a rapid (within 10 min) increase in reactive oxygen species, detected by oxidation of the fluorescent probes 2,7-dichlorofluorescin and dihydrorhodamine-123. DNA synthesis was increased in the presence of CsA as assessed by [3H]thymidine incorporation. The superoxide dismutase inhibitor diethyldithiocarbamate (1 mM) and the iron chelator desferal (5 microM), as well as ketoconazole (1 microM) and troleandomycin (10 microM), inhibitors of the cytochrome P-450 3A, were able to block both effects. High-performance liquid chromatography analysis revealed that RASMC were capable to metabolize CsA to its primary metabolites (AM1, AM9 and AM4N), and that their formation was inhibited by ketoconazole and troleandomycin. Furthermore, mRNAs encoding cytochrome P-450 3A1 and 3A2 were detected in RASMC by reverse transcriptase-polymerase chain reaction. Our data suggest that CsA is metabolized by cytochrome P-450 3A in RASMC producing reactive oxygen species, most likely superoxide and the hydroxyl radical, known to damage lipids and DNA.

In vitro biotransformation of SDZ RAD: a new immunosuppressive macrolide in human liver microsomal preparations.

In the present biotransformation study using human in vitro liver samples, metabolites of SDZ RAD were characterized by LC-MS. The major metabolites resulted from single hydroxylation and demethylation pathways and corresponded to the class of first-generation metabolites; 39-O-demethyl-RAD was identified as metabolite. The potential ring-opened degradation product resulting from ester hydrolysis and its dehydrated analogue (seco acid) was detected to comparable amounts in both the incubations and in controls without the presence of NADPH. The direct formation of rapamycin from SDZ RAD could not be detected in this study.

The multidrug resistance modulator valspodar (PSC 833) is metabolized by human cytochrome P450 3A. Implications for drug-drug interactions and pharmacological activity of the main metabolite.

The metabolism of valspodar (PSC 833; PSC), which is developed as a multidrug resistance-reversing agent, was investigated to assess the potential for drug-drug interactions and the pharmacological activity of major metabolites. The primary metabolites of PSC produced by human liver microsomes were monohydroxylated, as revealed by LC/MS. The major site of hydroxylation was at amino acid 9, resulting in M9, as determined by cochromatography with synthetic M9. Dihydroxylated and N-demethylated metabolites were also detected. PSC metabolism in two human livers exhibited KM values of 1.3-2.8 microM. The intrinsic clearance was 9-36 ml/min/kg of body weight. PSC biotransformation was cytochrome P450 (CYP or P450) 3A dependent, based on chemical inhibition and on metabolism by Chinese hamster ovary cells expressing CYP3A. Ketoconazole was a competitive inhibitor (Ki = 0.01-0.04 microM). The inhibition by 27 compounds, including four antineoplastic agents, corresponded to the inhibitory potentials of these compounds toward CYP3A. For vinblastine, paclitaxel, doxorubicin, and etoposide, the IC50 values were 5, 12, 20, and 150 microM, respectively. M9 was also an inhibitor, with a lower apparent affinity for CYP3A (IC50 = 21 microM), compared with that of PSC. M9 was also less active as a multidrug resistance-reversing agent. M9 demonstrated low potency in sensitizing resistant cells to paclitaxel and was a poor inhibitor of rhodamine-123 efflux from paclitaxel-resistant cells. In addition, compared with PSC, a higher concentration of M9 was needed to compete with the photoaffinity labeling of P-glycoprotein. Conversely, PSC inhibited only reactions catalyzed by CYP3A, including cyclosporine A metabolism (IC50 = 6.5 microM) and p-hydroxyphenyl-C3'-paclitaxel formation (Ki = 1.2 microM). Thus, PSC behaves in a manner very similar to that of other cyclosporines, and a comparable drug-drug interaction profile is expected.

Human and rat lung biotransformation of cyclosporin A and its derivatives using slices and bronchial epithelial cells.

Lung biotransformation of the immunosuppressants, cyclosporin A (CSA), the hydroxyethyl derivative SDZ IMM 125 (IMM), and the methylcarbonate derivative SDZ SCP 764 (SCP), was demonstrated in slices from human and rat. The major biotransformation pathway for CSA and IMM (0.1-10 microM) was hydroxylation at amino acid 1 to form AM1 or IMM1, while for SCP it was an esterase cleavage of the methylcarbonate group to form AM1 in both species. The initial rate (0-1 hr) of human total metabolite formation increased proportionally with substrate concentration. AM1 formation was five times greater from SCP, an esterase pathway, than CSA, an oxidative pathway which was inhibited (50%) by ketoconazole. At 24 hr human lung CSA metabolite formation was greater than IMM (3-fold) or SCP (2-fold), whereas rat lung and liver and human bronchial epithelial cell SCP metabolite formation generally exceeded CSA or IMM metabolism. CSA biotransformation is expected to occur throughout the human lung as demonstrated by the similar metabolite profile and extent of metabolism by slices derived from five different regions. The scaling of slice total metabolism to organ metabolism revealed that initially lung CSA metabolite formation would be equal to liver but with time liver metabolism would exceed lung for human and rat. This study has demonstrated that human and rat lung are metabolically active, exhibiting oxidative and esterase pathways toward cyclosporin derivatives. The lung will play an important role in this metabolism, particularly when administered via inhalation; however, the liver will also be a major organ involved in the total clearance of these compounds.

Estrogen receptor levels and occupancy in hepatic sinusoidal endothelial and Kupffer cells are enhanced by initiation with diethylnitrosamine and promotion with 17alpha-ethinylestradiol in rats.

We report the presence of estrogen receptors (ER) in rat liver sinusoidal endothelial (SEC) and Kupffer cells (KC), which exhibited comparable saturation kinetics and receptor affinity (Kd) for 17alpha-estradiol, as characterized for the rat hepatocyte ER. The ER levels in both cell types were significantly decreased by ovariectomy, indicating a regulatory role of estrogens. Initiation of ovariectomized rats with a single dose (200 mg/kg) of diethylnitrosamine (DEN) or saline (S), followed by chronic exposure to 17alalpha-ethinylestradiol (EE2), 90 microg/kg/day for 30 weeks packed in cholesterol (C) resulted in significant changes of ER levels in both the endothelial and Kupffer cells. The isolation of enriched liver SEC and KC populations by centrifugal elutriation allowed for the evaluation of chronic EE2 exposure and DEN-induced alterations on each cell type. The DEN-EE2 regime significantly enhanced gamma-gluta-myltranspeptidase activity in SEC (5-fold) and KC (6.6-fold) compared to the S/C treated animals. Nuclear ER levels were elevated 5.1-fold in the SEC and 6.5-fold in the KC, and both cell types exhibited significant increases in the proportion of occupied nuclear ER compared to the S/C derived cells, suggesting that exogenous estrogens could influence SEC and KC function through changes in ER levels and occupancy. ER occupancy was approximately 50% of the total ER in SEC and KC from DEN-EE2 rats. Increases in ER and occupancy for SEC and KC were similar to those observed for hepatocytes. Cellular growth was clearly modified in DEN-EE2 animals as indicated by a 4- to 10-fold increase in the proportion of SEC, KC or hepatocytes in S-phase as shown by flow cytometry. However, unlike hepatocytes, the epidermal growth factor receptor (EGFR) was not detected in SEC or KC using a monoclonal EGFR antibody. These findings suggest that the EGFR at 30 weeks is not involved in EE2-mediated stimulation of mitogenesis in SEC and KC which may be different from hepatocytes. In summary, our studies demonstrate that SEC and KC contain significant amounts of high-affinity ER and that ER pathways may modulate some activities of the SEC and KC, but that ER-EGFR interactions may be different in these cells from hepatocytes.

Biotransformation of a somatostatin analogue in precision-cut liver and kidney slices from rat, dog and man.

1. Cleavage of the glucopyranosyl moiety of the somatostatin analogue SDZ CO 611 results in the formation of the major metabolite, SDZ CO 610, in liver and kidney slices of rat, dog and man, as well as in liver S9 and cytosol of rat and man. 2. The rates of SDZ CO 610 formation (nmol/h/mg slice protein) for all three species were determined in liver slices for 24 h and the relative order was: rat (0.12) > dog (0.096) = man (0.095). The rates of SDZ CO 610 formation (nmol/h/mg slice protein) for all three species in kidney were determined, and the relative order was: rat (0.29) > dog (0.16) > man (0.10). 3. SDZ CO 610 was rapidly formed by rat gut contents in the absence of NADPH, possibly by disaccharide-splitting enzymes. 4. Biotransformation of SDZ CO 611 to SDZ CO 610 in human and rat liver S9 and cytosol was similar to that found in liver slices cultures indicating that cleavage of the glucopyranosyl moiety of SDZ CO 611 could occur in the presence and in the absence of cytochrome P450, possibly by glucosidases in liver cytosol. 5. Rat intestinal homogenate also formed SDZ CO 610 but metabolism was dependent upon NADPH, suggestive of a cytochrome P450-dependent reaction.

Biotransformation of the antiemetic 5-HT3 antagonist tropisetron in liver and kidney slices of human, rat and dog with a comparison to in vivo.

Species differences in the biotransformation of the antiemetic tropisetron, a potent 5-hydroxytryptamine type 3 (5-HT3) receptor antagonist, were evident in liver slice incubates of human, rat and dog, and reflected the species differences observed in vivo with respect to the relative importance of individual pathways. The dominant biotransformation pathway of tropisetron (10 microM) in human liver slices was formation of 6-hydroxy-tropisetron, whereas in rat liver slices it was 5-hydroxy-tropisetron, and in dog liver slices N-oxide formation. Initial rates of tropisetron metabolite formation in the liver slices (8 mm in diameter, 200 +/- 25 microns thickness) of human (83 +/- 61 pmol/h/mg slice protein), rat (413 +/- 98 pmol/h/mg slice protein) and dog (426 +/- 38 pmol/h/mg slice protein) would predict less of a first-pass effect in humans compared to the rat or the dog. For human and rat, the prediction matched well with the species ranking of tropisetron bioavailability; however, for dog the in vitro data overestimated the apparent first-pass effect. The jejunum is not expected to contribute to the first-pass effect in humans, since human jejunum microsomes did not metabolize tropisetron. The major organ of excretion for tropisetron and its metabolites is the kidney, but the contribution of the kidney to the overall metabolism of tropisetron would be small. Species independent N-oxide formation (2-12 pmol/h/mg slice protein) was the major pathway in human, rat and dog kidney slices, and was comparable to N-oxide formation in the rat and human liver slices but was 1/10 the rate in dog liver slices. This study has demonstrated that the liver is the primary site of tropisetron biotransformation, and the usefulness of organ slices to characterize cross species differences in the dominant biotransformation pathways.

Hydroxyethyl cyclosporin A induces and decreases P4503A and P-glycoprotein levels in rat liver.

1. A new immunosuppressant SDZ IMM 125 (IMM), the hydroxyethyl derivative of D-serine8-cyclosporin (cyclosporin A, CSA), induced or decreased the liver P450s of rat, in particular the 3A proteins, depending on the dose and duration of exposure. Doses of 20 mg/kg/day for 2 weeks and 10 mg/kg/day for 26 weeks induced the rat liver 3A levels 2- and 1.8-fold respectively, whereas 52 weeks of 24 mg/kg/day decreased the 3A levels by 22%. High doses of IMM, 100 mg/kg/day for 26 weeks, significantly decreased the 3A levels by 56%. 2. Changes in the rate of IMM biotransformation paralleled the changes in the levels of liver 3A indicating that liver 3A levels could influence the clearance of IMM. 3. Both IMM and CSA affected liver and kidney P-glycoprotein (Pgp) levels. The increases measured after short-term treatment (20 mg/kg/day for 2 weeks) in the liver (1.8-fold) and kidney (1.3-fold) were less pronounced in the long-term studies in which liver Pgp levels were increased 1.4-fold (48 mg/kg/day for 52 weeks). At higher doses (100 mg/kg/day for 26 weeks) Pgp levels were significantly decreased. The modulation of Pgp levels by IMM did not parallel the changes in 3A levels, indicating that Pgp regulation is most likely due to a direct effect of the cyclosporin rather than a co-regulation mechanism linked to 3A or P4501A modulation. 4. Increased arachidonic metabolism to the 19- and 20-HETE metabolites, a possible mechanism of the cyclosporin-induced renal hypertension, occurred in the liver microsomes and not the kidney S9 fraction of the 2-week study, and only at very high doses (100 mg/kg/day) in the longer studies (26 weeks).

Human liver cytochrome P4503A biotransformation of the cyclosporin derivative SDZ IMM 125.

In humans, cytochrome P4503A (CYP3A) is the major cytochrome P450 gene family that metabolizes SDZ IMM 125 (IMM) to its primary metabolites. Human liver microsomes could be used for this study, because the metabolite profile matched that found in human blood. The apparent affinity (KM) of IMM for the cytochrome P450 proteins (5.1 +/- 1.8 microM) is similar to that of cyclosporin A (CSA). CSA competitively inhibited the metabolism of IMM, increasing the KM 2- and 4.6-fold in the presence of 4 and 10 microM CSA, respectively (Ki 3.8 +/- 1.1 microM). Ketoconazole exhibited competitive inhibition kinetics toward IMM biotransformation, increasing the KM of IMM 1.8-fold at 0.5 microM ketoconazole and 3.5-fold at 1 microM ketoconazole, with no effect on Vmax (Ki of 0.5 +/- 0.4 microM). These results indicate that both CSA and ketoconazole would cause drug interactions, interfering with the biotransformation of IMM. The metabolism of IMM was also greatly inhibited (approximately 80%) by the CYP3A suicide substrate triacetyloleandomycin and a CYP3A inhibitory antibody, indicating the involvement of CYP3A proteins in the biotransformation of IMM. Confirmation of CYP3A4 involvement in the formation of the three primary IMM metabolites was demonstrated with recombinant cells expressing human CYP3A4. Therefore, compounds interacting with CYP3A proteins are expected to cause drug-drug interactions (i.e. the antimycotics ketoconazole and clotrimazole, the steroids ethinylestradiol and testosterone, the ergots, the calcium channel blocker nifedipine, and the immunosuppressants FK-506 and rapamycin).(ABSTRACT TRUNCATED AT 250 WORDS)

Sites of biotransformation for the cyclosporin derivative SDZ IMM 125 using human liver and kidney slices and intestine. Comparison with rat liver slices and cyclosporin A metabolism.

SDZ IMM 125 (IMM), the hydroxyethyl derivative of cyclosporin A (CSA), is metabolized by human liver slices to analogous primary metabolites, hydroxylated IMM1 and IMM9 and N-demethylated IMM4N, as for CSA (M17/AM1, M1/AM9, and M21/AM4N), but the rate and extent of IMM biotransformation is less than for CSA. Initial rates of IMM metabolite formation in the human liver slice cultures are 6.6 +/- 2.8 nmol/hr/g liver at 1 microM IMM and 24.3 +/- 22.9 nmol/hr/g liver at 10 microM IMM, whereas the rate of CSA metabolite formation is 1.8-fold faster at both concentrations. The percentage of unchanged IMM is 73% at 1 microM and 80% at 10 microM after 24 hr, reflecting the lower extent of IMM metabolism, about one-third (1 microM) and one-half (10 microM) that of CSA. In rat liver slices, IMM is metabolized to the same primary metabolites as in human liver slices, but more slowly and remains 90% unchanged at 24 hr. Human jejunum formed the same primary metabolites of IMM and CSA as in liver. Upscaling the slice rate of biotransformation revealed that human jejunum would contribute considerably to the first-pass of IMM and CSA, being approximately 2 to 3-fold slower than the rate in liver. The inhibition of both IMM and CSA biotransformation by triacetyloleandomycin implicates the involvement of cytochrome P4503A proteins. Human kidney cortex slices metabolized IMM to IMM1 and IMM9, accounting for approximately 75% of the total metabolites. Total metabolite formation represented approximately 64% of liver metabolite formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Uptake and metabolism of cyclosporin A and SDZ IMM 125 in the human in vitro skin2 dermal and barrier function models.

Treatment of psoriasis with the immunosuppressant cyclosporin A (CSA) is beneficial orally but topical treatment is less efficacious. A comparison of CSA to its hydroxyethyl derivative SDZ IMM 125 (IMM) as to bioavailability to epidermal and dermal cells and the potential for inactivation by biotransformation was investigated using a human dermal skin model (skin2 ZK1100) and a barrier function model (skin2 ZK 1300). The dermal ZK1100 model demonstrated that both cyclosporins could be metabolized by human dermis to the known primary hydroxylated metabolite, M17/AM1 for CSA and the hydroxylated analogue of IMM, IMM1. Application of the cyclosporins to the stratum corneum of the barrier function model ZK 1300 demonstrated that both CSA and IMM would be bioavailable to the epidermal and dermal skin cells. Systemic concentrations would be expected to be low due to the slow permeation of the compounds and because mostly metabolites would reach the circulation. The difference between the two cyclosporins was the rate and extent of biotransformation with IMM metabolite formation being about 1/4 that of CSA.

Use of human organ slices to evaluate the biotransformation and drug-induced side-effects of pharmaceuticals.

Human liver and kidney organ slices were used to investigate the biotransformation competence of the slices in combination with several markers of cell viability and function. The immunosuppressant cyclosporin A (CSA) is extensively metabolized in liver slices to the three known primary metabolites and many secondary metabolites. In kidney cortex slices the biotransformation of CSA is far more pronounced in humans than in rats. In human liver slices, levels of CYP3A, the proteins metabolizing CSA, are depressed about 25% by 1 and 10 mumol/L CSA within 24 h, indicating that high blood or tissue concentrations will inhibit CSA clearance. A clinical marker for liver damage is the release of cellular alpha-glutathione-S-transferases (alpha GST). In this study the alpha GST levels were used to assess donor organ quality, organ slice incubation conditions, and compound exposure. A marker for cell death in human cells is the solubilization and release of nuclear matrix proteins (Numa). Increases were apparent only after 48 h of culture. A side-effect of CSA is that it induces hypertension and perturbs the lipid profile of transplant recipients. A potential marker for lipid disturbances is levels of serum lipoprotein (a) (Lp(a)), which is synthesized in the liver and found only in humans, apes, and nonhuman primates. CSA increases Lp(a) levels in the human liver slice cultures about 2-fold. This study has demonstrated that the biotransformation capability of the organ slices contributes to the optimization of the in vitro system and to the evaluation of markers for drug induced side-effects or toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

The polymorphic cytochrome P-4502D6 is involved in the metabolism of both 5-hydroxytryptamine antagonists, tropisetron and ondansetron.

Tropisetron and ondansetron, which are potent and selective 5-hydroxytryptamine (5-HT3) receptor antagonists, were both metabolized by human liver microsomes to several metabolites. These metabolites include the major metabolites found in humans, which are the 5-, 6-, and 7-hydroxy tropisetron and the 7- and 8-hydroxy ondansetron. The cytochrome P-450 (CYP) 2D6 inhibitor quinidine (1 microM) reduced the hydroxylation of tropisetron (67%) and ondansetron (18%). Confirmation of CYP2D6 involvement in the hydroxylation of tropisetron and ondansetron was obtained by the formation of these metabolites in recombinant V79 cells expressing human CYP2D6. The CYP3A substrate/inhibitor, cyclosporine A (CsA) had little effect on tropisetron hydroxylation (< 10%), whereas CsA and triacetyloleandomycin reduced ondansetron 7- and 8-hydroxylation up to 27%. Substrates for CYP1A (phenacetin and acetanilide), CYP2C (mephenytoin), and CYP2E (chlorzoxazone) had negligible inhibitory effects on the hydroxylation of either tropisetron or ondansetron. For the CYP2D6-dependent O-demethylation of dextromethorphan, tropisetron and ondansetron were competitive inhibitors with Ki values of 14 and 29 microM, respectively. The CYP3A specific metabolism of CsA was also competitively inhibited by tropisetron (Ki = 2.1 mM) and ondansetron (Ki = 31 microM). Other metabolites, which are only minor in vivo were also inhibited by CsA, 47-60% for tropisetron metabolism and 43% for ondansetron metabolism. To summarize, this study has identified the involvement of CYP2D6 in the formation of the hydroxylated metabolites of tropisetron and ondansetron and in addition of CYP3A in ondansetron hydroxylation. Because these are the major pathways in vivo, coadministration of drugs competing for CYP2D6 and possibly CYP3A4 could influence the human kinetics of tropisetron and ondansetron.

The biotransformation of the ergot derivative CQA 206-291 in human, dog, and rat liver slice cultures and prediction of in vivo plasma clearance.

Liver slice cultures from humans, dogs, and rats were used to investigate the biotransformation of the dopaminergic ergot agonist CQA 206-291 and to predict pharmacokinetic values for hepatic intrinsic clearance and plasma clearance. CQA 206-291 was extensively metabolized in the liver slice cultures and in vivo. The HPLC metabolite patterns from the liver slice cultures were similar for all three species, indicating the occurrence of the same metabolic pathways for CQA 206-291 biotransformation. The rate of formation of CQ 32-084, a pharmacologically active N-deethylated metabolite, exceeded that of metabolite d, a primary metabolite, by 1.4 fold in human liver slices, and by 1.7 fold in rat liver slices. In dog liver slice cultures, metabolite d formation exceeded CQ 32-084 formation by 1.3 fold and was formed at a statistically significantly greater rate (3 fold) than in either human or rat liver slices. The metabolism of ergots like CQA 206-291 by human fetal liver was also demonstrated in this study. However, the prominent metabolite from fetal and adult human liver microsomes was metabolite d with minor amounts of CQ 32-089 being formed. A major route of excretion for the metabolites of CQA 206-291 is the kidney, yet the kidney does not contribute to the metabolism of CQA 206-291. Kidney slices derived from humans, rats, and dogs did not metabolize CQA 206-291 within 24 hr. CQA 206-291 intrinsic clearance was derived from the half-life of parent drug disappearance in the liver slice and hepatocyte cultures, and from the ratio of Vmax/Km of human and rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclosporin A metabolism in human liver, kidney, and intestine slices. Comparison to rat and dog slices and human cell lines.

This study assesses the contribution of cyclosporin A (CsA) metabolism at sites of CsA-induced toxicity: kidney and liver, and a site of absorption, the intestine. With organ slice cultures (8 mm phi), it has been possible to demonstrate that the extrahepatic metabolism of CsA is significant. Both human kidney and colonic mucosal tissue metabolize CsA (1 microM, 24 hr) as analyzed by HPLC. The major metabolite M17 was formed in the kidney at an initial rate of 3 pmol/hr/mg slice protein, which was comparable to M17 formation in the liver slices (5 pmol/hr/mg slice protein). The rate of total CsA metabolism by human kidney slices represents about 42% the rate in liver slices. The metabolism of CsA to M17 was the same in the human kidney cell line 293; however, CsA metabolism was not detectable using human kidney microsomes, nor was metabolism clearly evident in either rat or dog kidney slice cultures. The metabolism of CsA by human colonic mucosal slices to at least three metabolites and the metabolism of CsA by the human intestinal cell line FHs74 Int indicates that the intestinal metabolism of CsA contributes to the first-pass effect of the drug. The liver proved to be the major site of CsA biotransformation in terms of the complexity of metabolites produced, whereas the human liver HepG2 cell line proved not to be a suitable model for CsA metabolism. A time course revealed that the first metabolites formed in the liver slice cultures were the monohydroxylated, M1 and M17, and N-demethylated, M21, followed by the secondary metabolites (including M8, M13, and M18).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the cytochrome P-450 gene family responsible for the N-dealkylation of the ergot alkaloid CQA 206-291 in humans.

The ergot alkaloid CQA 206-291 (CQA) was converted by human liver microsomes (n = 16) almost exclusively to the N-deethylated metabolite (I), as identified by the on-line coupling of liquid chromatography and mass spectroscopy. Metabolite I formation exhibited monophasic and linear enzyme kinetics (2.9-300 microM), and a 5.6-fold interindividual variability (7.2-40.2 nmol/mg/hr). Chemical inhibition experiments revealed that imidazole antimycotic agents (ketoconazole, miconazole, and clotrimazole) were potent inhibitors of this N-deethylation. Polymorphically metabolized substrates (sparteine and phenytoin), well-established cytochrome P-450 probe substrates (antipyrine and tolbutamide), and steroid hormones (estradiol and testosterone) were noninhibitory, indicating that their metabolism is catalyzed by forms of cytochrome P-450 that do not catalyze this route of CQA biotransformation. The ergot alkaloids--dihydroergotamine, bromocriptine, and SDZ 208-911--were competitive inhibitors of metabolite I formation, suggesting that these compounds are metabolized by similar enzymes. Cyclosporine A was a potent competitive inhibitor of CQA metabolism, providing initial evidence that formation of metabolite I was catalyzed by proteins of the CYP3 gene family. This was substantiated by the finding that CQA metabolism was completely inhibited by a polyclonal antibody directed against a pregnenolone 16 alpha-carbonitrile-inducible cytochrome P-450 of rat liver. The rate of CQA metabolism correlated significantly to the level of CYP3A4 expression, the rate of cyclosporine A metabolism to each of the primary metabolites (M-1, M-17, and M-21), and the rate of midazolam 4-hydroxylation. COS 1 cells transfected with human CYP3A4 and CYP3A5 provided direct evidence that these enzymes catalyze the metabolism of CQA.(ABSTRACT TRUNCATED AT 250 WORDS)