Raman Venkataramanan

Clinical Pharmacokinetics of Tacrolimus

SummaryTacrolimus, a novel macrocyclic lactone with potent immunosuppressive properties, is currently available as an intravenous formulation and as a capsule for oral use, although other formulations are under investigation.Tacrolimus concentrations in biological fluids have been measured using a number of methods, which are reviewed and compared in the present article. The development of a simple, specific and sensitive assay method for measuring concentrations of tacrolimus is limited by the low absorptivity of the drug, low plasma and blood concentrations, and the presence of metabolites and other drugs which may interfere with the determination of tacrolimus concentrations. Currently, most of the pharmacokinetic data available for tacrolimus are based on an enzyme-linked immunosorbent assay method, which does not distinguish tacrolimus from its metabolites.The rate of absorption of tacrolimus is variable with peak blood or plasma concentrations being reached in 0.5 to 6 hours; approximately 25% of the oral dose is bioavailable. Tacrolimus is extensively bound to red blood cells, with a mean blood to plasma ratio of about 15; albumin and α1-acid glycoprotein appear to primarily bind tacrolimus in plasma. Tacrolimus is completely metabolised prior to elimination. The mean disposition half-life is 12 hours and the total body clearance based on blood concentration is approximately 0.06 L/h/kg. The elimination of tacrolimus is decreased in the presence of liver impairment and in the presence of several drugs.Various factors that contribute to the large inter- and interindividual variability in the pharmacokinetics of tacrolimus are reviewed here. Because of this variability, the narrow therapeutic index of tacrolimus, and the potential for several drug interactions, monitoring of tacrolimus blood concentrations is useful for optimisation of therapy and dosage regimen design.

Clinical Pharmacokinetics of Cyclosporin

SummaryCyclosporin (cyclosporin A) is a unique immunosuppressant used to prevent the rejection of transplanted organs and to treat diseases of autoimmune origin. Therapeutic drug monitoring of cyclosporin is essential for several reasons: (a) wide variability in cyclosporin pharmacokinetics has been observed after the oral or intravenous administration of the drug. The variability in the kinetics of cyclosporin is related to a patient’s disease state, the type of organ transplant, the age of the patient and therapy with other drugs that interact with cyclosporin; (b) maintaining a blood concentration of cyclosporin required to prevent rejection of the transplanted organ; (c) minimising drug toxicity by maintaining trough concentrations below that which toxicity is most likely to occur; and (d) monitoring for compliance since patient non-compliance with drug regimens is a significant reason for graft loss after 60 days. Clinical monitoring and pharmacokinetic studies of cyclosporin can be performed using different biological fluids (plasma, serum or whole blood) and different analytical techniques (radioimmunoassay or high pressure liquid chromatography). The available analytical methods provide different results when using blood, plasma, or serum. Comparison of therapeutic ranges and pharmacokinetic parameters should be made with careful attention given to the method of cyclosporin analysis.Following oral administration, the absorption of cyclosporin is slow and incomplete. Peak concentrations in blood or plasma are reached in 1 to 8 hours after dosing. The bioavailability of cyclosporin ranges from less than 5% to 89% in transplant patients; poor absorption has frequently been observed in liver and kidney transplant patients and in bone marrow recipients. Factors that affect the oral absorption of cyclosporin include the elapsed time after surgery, the dose administered, gastrointestinal dysfunction, external bile drainage, liver disease, and food.Cyclosporin is widely distributed throughout the body. Following intravenous administration, the drug exhibits multicompartmental behaviour. The volume of distribution (whole blood; HPLC) ranges from 0.9 to 4.8 L/kg. Cyclosporin is highly bound to erythrocytes and plasma proteins and has a blood to plasma ratio of approximately 2. In plasma, approximately 80% of the drug is bound to lipoproteins. The distribution of cyclosporin in blood can be affected by a patient’s haematocrit and lipoprotein profile.Cyclosporin is extensively metabolised, primarily by mono- and dihydroxylation as well as N-demethylation, and is considered a low-to-intermediate clearance drug. The clearance of cyclosporin (whole blood; HPLC) ranges from 2.0 ml/min/kg in children with congestive heart failure to 11.8 ml/min/kg in paediatric kidney transplant patients. The terminal elimination half-life is highly variable and ranges from 6.3 hours in healthy volunteers to 20.4 hours in patients with severe liver disease (blood; H PLC). Factors affecting the metabolism of cyclosporin include liver disease, age, and concurrent drug therapy.The major route of elimination of cyclosporin is via the bile, primarily as metabolites of the drug. Renal excretion is a minor elimination pathway. Renal failure and haemodialysis do not alter the pharmacokinetics of cyclosporin.Several drugs are known to interact with cyclosporin, including microsomal enzyme inducing and inhibiting agents. Several drugs including amphotericin B, aminoglycoside antibiotics and co-irimoxazole may potentiate the nephrotoxicity of cyclosporin.The dose of cyclosporin used in a patient should be adjusted after considering factors such as the initial response to therapy, the patient’s age, transplant type, disease state and concurrent drug therapy. Initial doses are usually in the range of 10 to 20 mg/kg/day orally or 2.5 to 5 mg/kg/day as an intravenous infusion, and should be adjusted based on the clinical status of the patient and cyclosporin blood concentrations. Long term oral maintenance doses of less than 3 mg/kg/day have resulted in adequate immunosuppression in some patients.The therapeutic range for cyclosporin is poorly defined and depends on the biological fluid being analysed, the analytical technique and the time after transplant. Cyclosporin concentration monitoring should be used in conjunction with other assessment criteria such as serum biochemical parameters, radiological studies, biopsy results and the clinical status of the patient.Even though our understanding of cyclosporin is incomplete, a thorough knowledge of different factors that affect its kinetics will aid the clinician in optimising immunosuppression with this promising new agent.

Disrupted Bile Acid Homeostasis Reveals an Unexpected Interaction among Nuclear Hormone Receptors, Transporters, and Cytochrome P450

Sister of P-glycoprotein (SPGP) is the major hepatic bile salt export pump (BSEP). BSEP/SPGP expression varies dramatically among human livers. The potency and hierarchy of bile acids as ligands for the farnesyl/bile acid receptor (FXR/BAR) paralleled their ability to induce BSEP in human hepatocyte cultures. FXR:RXR heterodimers bound to IR1 elements and enhanced bile acid transcriptional activation of the mouse and human BSEP/SPGP promoters. In FXR/BAR nullizygous mice, which have dramatically reduced BSEP/SPGP levels, hepatic CYP3A11 and CYP2B10 were strongly but unexpectedly induced. Notably, the rank order of bile acids as CYP3A4 inducers and activators of pregnane X receptor/steroid and xenobiotic receptor (PXR/SXR) closely paralleled each other but was markedly different from their hierarchy and potency as inducers of BSEP in human hepatocytes. Moreover, the hepatoprotective bile acid ursodeoxycholic acid, which reverses hydrophobic bile acid hepatotoxicity, activates PXR and efficaciously induces CYP3A4 (a bile-metabolizing enzyme) in primary human hepatocytes thus providing one mechanism for its hepatoprotection. Because serum and urinary bile acids increased in FXR/BAR −/− mice, we evaluated hepatic transporters for compensatory changes that might circumvent the profound decrease in BSEP/SPGP. We found weak MRP3 up-regulation. In contrast, MRP4 was substantially increased in the FXR/BAR nullizygous mice and was further elevated by cholic acid. Thus, enhanced hepatocellular concentrations of bile acids, due to the down-regulation of BSEP/SPGP-mediated efflux in FXR nullizygous mice, result in an alternate but apparent compensatory up-regulation of CYP3A, CYP2B, and some ABC transporters that is consistent with activation of PXR/SXR by bile acids.

Mycophenolic acid pharmacodynamics and pharmacokinetics provide a basis for rational monitoring strategies.

Mycophenolic acid (MPA), the active immunosuppressant form of the pro-drug mycophenolate mofetil (MMF), is a widely used component of immunosuppressive regimens in organ transplantation. This immunosuppressant is commonly administered in combination with a calcineurin inhibitor (CIN)+ corticosteroid with or without induction therapy in the form of a polyclonal anti-T lymphocyte preparation or one of the available humanized monoclonal interleukin 2 inhibitors. There is strong evidence that maintenance CIN-MMFsteroid-based triple therapy, initiated in the early posttransplant period significantly reduces the risk of acute rejection in the first post-transplant year, when compared to double therapy regimens comprising CIN and steroids alone. After the initial phase of graft stabilization, there are several therapeuticapproachescurrentlyemployed in transplantpractice, including: (1) maintenance of a three-drug regimen, at reduced doses compared to the early post-transplant period; (2) elimination of corticosteroid; (3) reduction or elimination of CIN; (4) replacement of CIN with sirolimus in patients who are especially sensitive to the nephrotoxic effects of these agents; or (5) reduction or discontinuation of MMF, while retaining full-dose CIN or sirolimus and maintenance corticosteroids are among the other therapeutic options under investigation. Dose individualization of CIN is facilitated by target drug concentration monitoring, whereas for MMF, empiric dosing is most commonly practiced.

Changes in the pharmacokinetics of the low-molecular-weight heparin enoxaparin sodium during pregnancy

OBJECTIVE We sought to evaluate the pharmacokinetics of subcutaneously administered enoxaparin sodium during and after pregnancy. STUDY DESIGN Daily subcutaneous injections of enoxaparin sodium (40 mg) were administered to 13 pregnant women. On 3 separate occasions, once early in pregnancy (12-15 weeks), once late in pregnancy (30-33 weeks), and once in the nonpregnant state (6-8 weeks post partum), serial blood samples were collected, and plasma was analyzed for antifactor Xa activity. Analysis of variance was used for statistical analysis. P <.05 was significant. RESULTS The time to maximum concentration and the mean residence time in pregnancy compared with the postpartum state were not significantly different. During early and late pregnancy, maximum concentration and the last measurable anti-factor Xa activity level were lower than in the nonpregnant state (P <.05). The area under the plasma activity-versus-time curve was significantly lower in pregnancy than in the postpartum state (P <.05). CONCLUSION The pharmacokinetics of enoxaparin sodium are significantly different during pregnancy than in the same women when nonpregnant. The observed difference is likely because of increased renal clearance of enoxaparin during pregnancy. This finding has significant implications for appropriate dosing of enoxaparin in pregnancy.

Traditional Chinese Medicines Wu Wei Zi (Schisandra chinensis Baill) and Gan Cao (Glycyrrhiza uralensis Fisch) Activate Pregnane X Receptor and Increase Warfarin Clearance in Rats

The traditional Chinese medicines (TCMs) are essential components of alternative medicines. Many TCMs are known to alter the expression of hepatic drug-metabolizing enzymes and transporters. The molecular mechanism by which TCMs and/or their constituents regulate enzyme and transporter expression, however, has remained largely unknown. In this report, we show that two TCMs, Wu Wei Zi (Schisandra chinensis Baill) and Gan Cao (Glycyrrhiza uralensis Fisch), and their selected constituents activate the xenobiotic orphan nuclear receptor pregnane X receptor (PXR). Treatment with TCM extracts and the Schisandrol and Schisandrin constituents of Wu Wei Zi induced the expression of drug-metabolizing enzymes and transporters in reporter gene assays and in primary hepatocyte cultures. The affected enzymes and transporters include CYP3A and 2C isozymes and the multidrug resistance-associated protein 2. In transient transfection and reporter gene assays, the Schisandrin constituents of Wu Wei Zi had an estimated EC50 of 2 and 1.25 μM on hPXR and mPXR, respectively. Interestingly, mutations that were intended to alter the pore of the ligand-binding cavity of PXR had species-specific effects on the activities of the individual Schisandrols and Schisandrins. In rats, the administration of Wu Wei Zi and Gan Cao increased the metabolism of the coadministered warfarin, reinforcing concerns involving the safe use of herbal medicines and other nutraceuticals to avoid PXR-mediated drug-drug interactions. Meanwhile, the activation of PXR and induction of detoxifying enzymes provide a molecular mechanism for the hepatoprotective effects of certain TCMs.

Therapeutic monitoring of mycophenolic acid: A consensus panel report

Biochem 1998;5:317–22. 4. Oellerich M, Shipkova M, Schütz E, Weber L, Tönshoff B, Armstrong VW, et al. Pharmacokinetic and metabolic investigations of mycophenolic acid in pediatric patients after renal transplantation: implications for therapeutic drug monitoring. Ther Drug Monit 2000;22:20–6. 5. Pescovitz MD, Conti D, Dunn J, Gonwa T, Halloran P, Sollinger H, et al. Intravenous mycophenolate mofetil: safety, tolerability, and pharmacokinetics. Clin Transplant 2000:14;179–88. 6. Tsina I, Kaloostian M, Lee R, Tarnowski T, Wong B. High-performance liquid chromatographic method for the determination of mycophenolate mofetil in human plasma. J Chromatogr B 1996;681:347–53. 7. McBride JH, Kim S, Reyes A, Rodgerson DO. Measurement of plasma mycophenolic acid in pediatric renal transplant recipients. Clin Chem 1998;44(Suppl 6):A93. 8. Shipkova M, Niedmann PD, Armstrong VW, Schütz E, Wieland E, Oellerich M. Simultaneous determination of mycophenolic acid and its glucuronide in human plasma using a simple high-performance liquid chromatographic procedure. Clin Chem 1998;44:1481–8. 9. Shipkova M, Schütz E, Armstrong VW, Niedmann PD, Oellerich M, Wieland, E. Determination of the acyl glucuronide metabolite of mycophenolic acid in human plasma by HPLC and Emit. Clin Chem 2000;46:365–72. 10. Stamm D. A new concept for quality control of clinical laboratory investigations in the light of clinical requirements and based on reference method values. J Clin Chem Clin Biochem 1982;20:817–24. 11. Hyneck ML, Munafo A, Benet LZ. Effect of pH on acyl migration and hydrolysis of tolmetin glucuronide. Drug Metab Dispos 1988;16:322–4.

Voriconazole Inhibition of the Metabolism of Tacrolimus in a Liver Transplant Recipient and in Human Liver Microsomes

ABSTRACT The purpose of this study was to assess the effect of voriconazole on the blood tacrolimus concentration in a liver transplant recipient and to examine the interaction between voriconazole and tacrolimus by using human liver microsomes. Two subjects were enrolled in the clinical study: one received voriconazole, and the other received a placebo. Tacrolimus metabolism was evaluated in human liver microsomes at various concentrations in the absence and presence of various concentrations of voriconazole. Coadministration of voriconazole and tacrolimus resulted in elevated (nearly 10-fold-higher) trough tacrolimus blood concentrations in the liver transplant patient. In the in vitro study, voriconazole at a concentration of 10.4 ± 4.3 μg/ml inhibited the metabolism of tacrolimus by 50%. Clinically relevant concentrations of voriconazole inhibited the metabolism of tacrolimus in human liver microsomes. Close monitoring of the blood concentration and adjustment in the dose of tacrolimus are warranted in transplant recipients treated with voriconazole.

Clinical Utility of Monitoring Tacrolimus Blood Concentrations in Liver Transplant Patients

The relationship between the dose of tacrolimus, trough tacrolimus blood concentration, and selected clinical endpoints (acute rejection, nephrotoxicity, and other toxicities) were examined in a prospective, multicenter clinical trial to validate the use of an enzyme‐linked immunosorbent assay (ELISA) for monitoring whole‐blood concentrations of tacrolimus in liver transplant patients. A total of 111 subjects from six transplant centers were evaluated over 12 weeks posttransplantation. In addition to trough tacrolimus blood concentrations, hematocrit, ALT, AST, GGTP, alkaline phosphatase, total bilirubin, serum creatinine, BUN, serum potassium, serum magnesium, blood glucose, and serum albumin were also measured. The relationship between trough tacrolimus blood concentrations and clinical endpoints was analyzed using both a logistic regression model and a Cox proportional hazard model. By logistic regression analysis, a statistically significant (p = 0.0465) relationship between increasing trough tacrolimus blood concentrations and decreasing risk of acute rejection was demonstrated over a 7‐day time window. Nephrotoxicity and other toxicities also demonstrated statistically significant relationships with trough tacrolimus blood concentrations. The results of the Cox analysis were consistent with the logistic regression analysis. Using receiver operator characteristic curves, trough tacrolimus concentrations as measured by the ELISA method were able to differentiate the occurrence of nephrotoxicity and toxicity from nonevents. To minimize nephrotoxicity of tacrolimus, it is necessary to maintain trough blood concentrations below 15 ng/ml. This study demonstrates that the ELISA method used to measure tacrolimus blood concentrations in this study provides information of predictive value for managing the risk of nephrotoxicity, other toxicity, and rejection in liver transplant patients.

Novel Single Nucleotide Polymorphisms in the Promoter and Intron 1 of Human Pregnane X Receptor/NR1I2 and Their Association with CYP3A4 Expression

The hypothesis was tested that sequence diversity in pregnane X receptor (PXR) cis-regulatory regions is a significant determinant of variation in inducible and constitutive CYP3A4 expression. A combination of comparative genomics and computational algorithms was used to select regions of the human PXR promoter and intron 1 that were resequenced in the polymorphism discovery resource 24 DNA subset. PXR single nucleotide polymorphisms (SNP) were then genotyped in donor human livers phenotyped for CYP3A4 and multidrug resistance protein 1 mRNA and primary human hepatocytes phenotyped for basal and rifampin-inducible CYP3A4 activity. The human PXR promoter [16.9 kilobase (kb)] was significantly larger than in rodents (2.9 kb). Eighty-nine SNPs were identified in the promoter and intron 1 of PXR. The SNPs most consistently associated with CYP3A4 phenotypic measures were a 44477T>C(-1359) promoter SNP (in linkage disequilibrium with SNP 463970, a 6-base pair deletion in intron 1a, and SNP 46551, a C nucleotide insertion in intron 1b); SNP 63396C>T in intron 1 (in linkage disequilibrium with SNP 63704A>G, a 63813(CAAA)(CA) variable repeat, and SNP 65104T>C); and SNP 56348C>A, SNP 69789A>G, and SNP 66034T>C. Donor livers with the variant PXR alleles had altered hepatic expression of PXR targets compared with livers with PXR wild-type alleles. These results identified PXR promoter and intron 1 SNPs associated with PXR target gene expression (CYP3A4) in donor livers and cultured hepatocytes and that a striking number of the linked intron 1 SNPs will affect putative binding sites for hepatic nuclear factor 3β (FOXA2), a transcription factor linked with PXR expression.