U. Christians

Metabolism and Drug Interactions of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors in Transplant Patients: Are the Statins Mechanistically Similar?

3-Hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.88) inhibitors are the most effective drugs to lower cholesterol in transplant patients. However, immunosuppressants and several other drugs used after organ transplantation are cytochrome P4503A (CYP3A, EC 1.14.14.1) substrates. Pharmacokinetic interaction with some of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, specifically lovastatin and simvastatin, leads to an increased incidence of muscle skeletal toxicity in transplant patients. It is our objective to review the role of drug metabolism and drug interactions of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, and cerivastatin. In the treatment of transplant patients, from a drug interaction perspective, pravastatin, which is not significantly metabolized by CYP enzymes, and fluvastatin, presumably a CYP2C9 substrate, compare favorably with the other statins for which the major metabolic pathways are catalyzed by CYP3A.

Grapefruit juice activates P-glycoprotein-mediated drug transport.

AbstractPurpose. Grapefruit juice (GJ) is known to increase the oral bioavailability of many CYP3A-substrates by inhibiting intestinal phase-I metabolism. However, the magnitude of AUC increase is often insignificant and highly variable. Since we earlier suggested that CYP3A and P-glycoprotein (P-gp) form a concerted barrier to drug absorption, we investigated the role of P-gp in GJ-drug interactions. Methods. The transcellular bidirectional flux of drugs that are (i) CYP3A-and/or P-gp substrates (Vinblastine, Cyclosporine, Digoxin, Fexofenadine, Losartan) or that are (ii) primary CYP3A-substrates (Felodipine, Nifedipine) was evaluated across MDCK-MDR1 cell monolayers with or without GJ, verifying monolayer integrity at all times. Results. While both apical-to-basal (A-B) and basal-to-apical (B-A) fluxes of all CYP3A/P-gp substrates tested were increased in the presence of GJ, the resulting net efflux (B-A/A-B) was in all cases significantly greater with GJ than control (Vin, 28.0 vs. 5.1; CsA, 9.9 vs. 2.8; Dig, 22. 9 vs. 14.7, Fex, 22.3 vs. 11.1, Los, 39.6 vs. 26). In contrast, no such GJ flux effect was observed with Pel and Nif, substrates of CYP3A only (2 vs. 1.7 and 1.2 vs. 1.3). Conclusions. GJ significantly activates P-gp-mediated efflux of drugs that are substrates of P-gp, potentially partially counteracting the CYP3A-inhibitory effects of GJ.

The pharmacokinetics and metabolic disposition of tacrolimus: a comparison across ethnic groups.

Our objective was to compare the intravenous and oral pharmacokinetics of tacrolimus among subjects of three different ethnic backgrounds, African American, white, and Latin American.

Automated, fast and sensitive quantification of drugs in blood by liquid chromatography–mass spectrometry with on-line extraction: immunosuppressants

We developed a universal LC-mass spectrometry assay with automated online extraction (LC/LC-MS) to quantify the immunosuppressants cyclosporine, tacrolimus, sirolimus and SDZ-RAD alone or in combination in whole blood. After protein precipitation, samples were loaded on a C18 extraction column, were washed and, after activation of the column-switching valve, were backflushed onto the C8 analytical column. [M+Na]+ ions were detected in the selected ion mode. For tacrolimus, sirolimus and SDZ-RAD, the assay was linear from 0.25 to 100 microg/l and for cyclosporine from 7.5 to 1250 microg/l (all r2>0.99). Analytical recovery was >85% and, in general, inter-day, intra-day variability for precision and accuracy were <10%.

Active transport of the angiotensin‐II antagonist losartan and its main metabolite EXP 3174 across MDCK‐MDR1 and Caco‐2 cell monolayers

We studied the functional interaction between transport and metabolism by comparing the transport of losartan and its active metabolite EXP 3174 (EXP) across cell monolayers. Epithelial layers of Caco‐2 cells as well as MDR1, MRP‐1 and MRP‐2 overexpressing cells, in comparison to the respective wildtypes, were used to characterize the transcellular transport of losartan and EXP. Losartan transport in MDCK‐MDR1 and Caco‐2 cells was saturable and energy‐dependent with a significantly greater basolateral‐to‐apical (B/A) than apical‐to‐basolateral (A/B) flux (ratio=31±1 in MDCK‐MDR1 and ratio 4±1 in Caco‐2 cells). The B/A flux of losartan was inhibited by cyclosporine and vinblastine, inhibitors of P‐glycoprotein and MRP. In contrast, no active losartan transport was observed in MRP‐1 or MRP‐2 overexpressing cells. The metabolite was only transported in Caco‐2 cells with a B/A‐to‐A/B ratio of 5±1, while lacking active transport in the MDR1, MRP‐1 or MRP‐2 overexpressing cells. The B/A flux of EXP was significantly inhibited by cyclosporine and vinblastine. In conclusion, losartan is transported by P‐glycoprotein and other intestinal transporters, that do not include MRP‐1 and MRP‐2. In contrast, the carboxylic acid metabolite is not a P‐glycoprotein substrate, but displays considerably higher affinity for other transporters than losartan, that again most probably do not include MRP‐1 and MRP‐2.

Sirolimus, but not the structurally related RAD (everolimus), enhances the negative effects of cyclosporine on mitochondrial metabolism in the rat brain

Clinical studies have shown enhancement of cyclosporine toxicity when co‐administered with the immunosuppressant sirolimus. We evaluated the biochemical mechanisms underlying the sirolimus/cyclosporine interaction on rat brain metabolism using magnetic resonance spectroscopy (MRS) and compared the effects of sirolimus with those of the structurally related RAD. Two‐week‐old rats (25 g) were allocated to the following treatment groups (all n=6): I. control, II. cyclosporine (10 mg kg−1 d−1), III. sirolimus (3 mg kg−1 d−1), IV. RAD (3 mg kg−1 d−1), V. cyclosporine+sirolimus and VI. cyclosporine+RAD. Drugs were administered by oral gavage for 6 days. Twelve hours after the last dose, metabolic changes were assessed in brain tissue extracts using multinuclear MRS. Cyclosporine significantly inhibited mitochondrial glucose metabolism (glutamate: 78±6% of control; GABA: 67±12%; NAD+: 76±3%; P<0.05), but increased lactate production. Sirolimus and RAD inhibited cytosolic glucose metabolism via lactate production (sirolimus: 81±3% of control, RAD: 69±2%; P<0.02). Sirolimus enhanced cyclosporine‐induced inhibition of mitochondrial glucose metabolism (glutamate: 60±4%; GABA: 59±8%; NAD+: 45±5%; P<0.02 versus cyclosporine alone). Lactate production was significantly reduced. In contrast, RAD antagonized the effects of cyclosporine (glutamate, GABA, and NAD+, not significantly different from controls). The results can partially be explained by pharmacokinetic interactions: co‐administration increased the distribution of cyclosporine and sirolimus into brain tissue, while co‐administration with RAD decreased cyclosporine brain tissue concentrations. In addition RAD, but not sirolimus, distributed into brain mitochondria. The combination of cyclosporine/RAD compares favourably to cyclosporine/sirolimus in regards to their effects on brain high‐energy metabolism and tissue distribution in the rat.

Suppression of acute rejection in allogeneic rat lung transplantation: a study of the efficacy and pharmacokinetics of rapamycin derivative (SDZ RAD) used alone and in combination with a microemulsion formulation of cyclosporine.

BACKGROUND The novel immunosuppressant SDZ RAD, 40-0 (2-hydroxyethyl)rapamycin, is an orally active rapamycin analogue developed for use in combination with cyclosporine (Neoral). The present study was designed to evaluate the efficacy of SDZ RAD, Neoral, or a combination of both drugs for suppression of acute rejection in an allogeneic, unilateral rat lung transplant model. METHODS Brown-Norway (RT1n) donor lungs were implanted into Lewis (RT1l) recipients that were observed for 21 days. Postoperative evaluation included daily weights, serial chest radiographs, drug trough levels, and histology scores of the transplanted lung on the day of sacrifice. Treatment groups were comprised of rats treated orally with the RAD vehicle as controls (n = 6); SDZ RAD 2.5 mg/kg/day (n = 9); Neoral 7.5 mg/kg/day (n = 8); Neoral 2.5 mg/kg/day (n = 6); SDZ RAD 2.5 mg/kg/day plus Neoral 7.5 mg/kg/day (n = 7); and Neoral 2.5 mg/kg/day plus SDZ RAD 2.5 mg/kg/day (n = 6). RESULTS The results of this study showed that neither monotherapy with 2.5 mg/kg/day of Neoral, nor 2.5 mg/kg/day of SDZ RAD prevented severe acute rejection in unilateral lung transplant recipients. Furthermore, despite high dose (7.5 mg/kg/day) Neoral treatment, graft histology showed moderate rejection. However, addition of 2.5 mg/kg/day of SDZ RAD to 7.5 mg/kg/day of Neoral completely prevented histologic rejection in four of seven grafts, although the remaining 3 grafts showed minimal rejection. This combination resulted in significantly higher RAD trough levels when compared to SDZ RAD treatment alone. Combining a lower dose of Neoral (2.5 mg/ kg/day) with 2.5 mg/kg/day of SDZ RAD resulted in less weight loss and improved animal health; however, the histology of lung grafts in these rats showed mild rejection. CONCLUSIONS This is the first study on the efficacy of the novel rapamycin derivative SDZ RAD for the control of acute lung allograft rejection. Results showed that acute unilateral rat lung allograft rejection is refractory to monotherapy with either high dose Neoral or SDZ RAD. The two regimens of combined treatment with Neoral plus SDZ RAD used in these studies produced either minimal rejection and reduced tolerability or mild rejection and better tolerability and showed potentiation of immunosuppression when both drugs were used together. Additional investigation of these two drugs is needed, however, to devise regimens that produce both high immunosuppressive efficacy and good tolerability.

Use of organotypical cultures of primary hepatocytes to analyse drug biotransformation in man and animals

1. In conventional single-gel culture systems for primary hepatocytes, rapid loss of drug metabolizing capacities is a common feature and parallels general loss of function. An organotypical (double gel) culture technique for primary hepatocytes is established by enclosing the cells within two layers of extra cellular matrix. This serves to imitate the in vivo microenvironment within the space of Dissé. Using rat hepatocytes, this technique has been shown previously to maintain protein synthetic functions in vitro and to allow more efficient P450A-dependent biotransformation of drugs than a standard single-gel culture system. 2. The aim was to test the capacity of this organotypical culture model for primary rat and human hepatocytes to generate drug metabolites in a typical species-dependent pattern. 3. Urapidil, an antihypertensive drug, was used as a test compound, since it is metabolized in vivo in a species-dependent manner in rat and man. 4. Primary rat and human hepatocytes were cultured within two layers of collagen and exposed to 2.25 micrograms/ml urapidil for periods of 1-24 h at 3 days in culture. Urapidil metabolites were measured using hplc. 5. Metabolite M1 (hydroxylated product) was produced preferentially in human hepatocyte cultures, and metabolites M2/M3 (O-demethylated, N-demethylated product) were preferentially generated in rat cultures. This corresponded to the in vivo pattern found in man and rat, respectively. 6. Since in vitro urapidil metabolism by human and rat hepatocytes cultured in a double-gel system reflects that in vivo, it is suggested that information from such a system may be useful to predict the metabolic pathway of novel xenobiotics and to direct further toxicological evaluation.

Combined immunosuppression with cyclosporine (neoral) and SDZ RAD in non-human primate lung transplantation: systematic pharmacokinetic-based trials to improve efficacy and tolerability.

BACKGROUND We studied the efficacy and tolerability of combined immunosuppressive therapy with cyclosporine A microemulsion (Neoral) plus the macrolide SDZ RAD 40-0 (2-hydroxyethyl) rapamycin (RAD) in a stringent cynomolgus monkey lung graft model in comparison with cyclosporine or SDZ RAD monotherapy. METHODS Thirty-nine cynomolgus monkeys received mixed lymphocyte reaction (MLR) mismatched unilateral lung transplants. Immunosuppressants were administered orally as single daily doses. The observation period was 28 days and follow-up included serial trough blood drug concentrations measured by high performance liquid chromatography/mass spectrometry, blood analyses, chest radiographs, open lung biopsies, as well as tissue drug concentrations and graft histology at necropsy. RESULTS Graft biopsies in monkeys treated with vehicle (n=4), Neoral (day 1-7: 150 mg/kg/day; day 8-28: 100 mg/kg/day; n=6; mean +/- SE trough level (MTL): 292+/-17 ng/ml) or SDZ RAD monotherapy (1.5 mg/kg/day; n=6; MTL: 15+/-1 ng/ml) showed severe rejection. Coadministration in two transplant monkeys of Neoral (150/100 mg/kg/day) and SDZ RAD (1.5 mg/kg/day) caused their early death. In both animals, SDZ RAD blood levels were more than 5-fold higher than under monotherapy (MTL: 82+/-18 ng/ml). Simultaneous administration (n=6) of Neoral (150/100 mg/kg/day; MTL: 217+/-16 ng/ml) and SDZ RAD (0.3 mg/kg/day; MTL: 24+/-2 ng/ml) improved graft outcome (mild rejection). Side effects included renal failure (n=2) and seizures (n=1). Three monkeys survived to day 28. In this group the MTL for cyclosporin was 143+/-13 and for RAD 38+/-3. Staggered treatment completely prevented rejection in four of six grafts. However, five of six monkeys had moderate to severe diarrhea. In a concentration-controlled trial of simultaneously administered Neoral and SDZ RAD in transplant monkeys (target SDZ RAD MTL: 20-40 ng/ml; cyclosporine MTL: 100-200 ng/ml) all six monkeys survived with improved drug tolerability and an average biopsy score of mild rejection. CONCLUSION Combination of orally administered SDZ RAD and Neoral showed excellent immunosuppressive efficacy in a stringent lung transplant model. The drug interaction and the narrow therapeutic index of this drug combination required careful dose adjustments to optimize tolerability and efficacy.

Simultaneous on-line extraction and analysis of sirolimus (rapamycin) and ciclosporin in blood by liquid chromatography–electrospray mass spectrometry

We developed a sensitive and specific semi-automated liquid chromatography-electrospray mass spectrometric (HPLC-ESI-MS) assay for the simultaneous quantification of sirolimus and ciclosporin in blood. Following a simple protein precipitation step, the supernatants were injected into the HPLC system and extracted on-line. After column switching, the analytes were backflushed from the extraction column onto the analytical narrow-bore column and eluted into the ESI-MS system. The assay was linear from 0.4 to 100 microg/l sirolimus and from 2 to 1500 microg/l ciclosporin. The mean recoveries of sirolimus and ciclosporin were 98 and 96%, respectively. The mean interday precision/accuracy was 8.6%/-4.8% for sirolimus and 9.3%/-2.9% for ciclosporin.