Gilbert J. Burckart

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.

Nuclear factor kappa B : Important transcription factor and therapeutic target

Nuclear factor kappa B (NF‐κB) is an ubiquitous rapid response transcription factor in cells involved in immune and inflammatory reactions, and exerts its effect by expressing cytokines, chemokines, cell adhesion molecules, growth factors, and immunoreceptors. In this manner, NF‐κB contributes to immunologically mediated diseases such as allograft rejection, rheumatoid arthritis, and bronchial asthma. The prototypic inducible form of NF‐κB is a heterodimer composed of NF‐kB1 and RelA, which both belong to the NF‐κB/Rel family of proteins. Inactive NF‐κB is present in the cytoplasm complexed with an inhibitory protein, IκB. NF‐κB is activated by a number of incoming signals from the cell surface. Released from IκB inhibition, NF‐κB translocates into the nucleus and binds to the κB motif of the target gene. The NF‐κB activation process can be inhibited by pharmacologic agents at each activation step. Glucocorticoids inhibit NF‐κB by directly associating with NF‐κB or by upregulating IκB expression. Cyclosporin and tacrolimus prevent NF‐κB activation by inhibiting the action of calcineurin, a phosphatase that indirectly induces IκB degradation. Deoxyspergualin inhibits NF‐κB by blocking its nuclear translocation. Aspirin and salicylates inhibit upstream events inducing IκB phosphorylation. Tepoxalin and antioxidants inhibit NF‐κB activation by influencing the redox state of the cell. Further research is required to develop more specific inhibitors to treat diseases mediated by NF‐κB.

Tacrolimus Dosing in Pediatric Heart Transplant Patients is Related to CYP3A5 and MDR1 Gene Polymorphisms

Tacrolimus is a substrate for P‐glycoprotein (P‐gp) and cytochrome (CYP) P4503A. P‐gp is encoded by the multiple drug resistance gene MDR1 and CYP3A is the major enzyme responsible for tacrolimus metabolism. Both MDR1 and CYP3A5 genes have multiple single nucleotide polymorphisms. The objective of this study was to evaluate whether the MDR1 exon21 and exon26 polymorphisms and the CYP3A5 polymorphism are associated with tacrolimus disposition in pediatric heart transplant patients. At 3, 6 and 12 months post transplantation, a significant difference in tacrolimus blood level per dose/kg/day was found between the CYP3A5 *1/*3 (CYP3A5 expressor) vs. *3/*3 (nonexpressor) genotypes with the *1/*3 patients requiring a larger tacrolimus dose to maintain the same blood concentration. There were no significant differences in tacrolimus blood level per dose/kg/day between MDR1 exon21 G2677T and exon 26 C3435T at 3 months, but both were found to have a significant association with tacrolimus blood level per dose/kg/day at 6 and 12 months. We conclude that specific genotypes of MDR1 and CYP3A5 in pediatric heart transplant patients require larger tacrolimus doses to maintain their tacrolimus blood concentration, and that this information could be used prospectively to manage patients immunosuppressive therapy.

Pharmacogenomic Biomarker Information in Drug Labels Approved by the United States Food and Drug Administration: Prevalence of Related Drug Use

Study Objectives. To review the labels of United States Food and Drug Administration (FDA)‐approved drugs to identify those that contain pharmacogenomic biomarker information, and to collect prevalence information on the use of those drugs for which pharmacogenomic information is included in the drug labeling.

New Era in Drug Interaction Evaluation: US Food and Drug Administration Update on CYP Enzymes, Transporters, and the Guidance Process

Predicting clinically significant drug interactions during drug development is a challenge for the pharmaceutical industry and regulatory agencies. Since the publication of the US Food and Drug Administrations (FDAs) first in vitro and in vivo drug interaction guidance documents in 1997 and 1999, researchers and clinicians have gained a better understanding of drug interactions. This knowledge has enabled the FDA and the industry to progress and begin to overcome these challenges. The FDA has continued its efforts to evaluate methodologies to study drug interactions and communicate recommendations regarding the conduct of drug interaction studies, particularly for CYP‐based and transporter‐based drug interactions, to the pharmaceutical industry. A drug interaction Web site was established to document the FDAs current understanding of drug interactions (http:www.fda.govcderdrugdrugInteractionsdefault.htm). This report provides an overview of the evolution of the drug interaction guidances, includes a synopsis of the steps taken by the FDA to revise the original drug interaction guidance documents, and summarizes and highlights updated sections in the current guidance document, Drug Interaction Studies—Study Design, Data Analysis, and Implications for Dosing and Labeling.

Extrapolation of Adult Data and Other Data in Pediatric Drug-Development Programs

OBJECTIVES: In 1994, the US Food and Drug Administration (FDA) proposed an approach, based on extrapolation of efficacy findings from adults to the pediatric population, to maximize the use of adult data and other data when designing pediatric drug-development programs. We examined the experience of the FDA in using extrapolation to evaluate how and when it was used and any changes in scientific assumptions over time. METHODS: We reviewed 370 pediatric studies submitted to the FDA between 1998 and 2008 in response to 159 written requests (166 products) issued under the Pediatric Exclusivity Provision. We identified cases in which efficacy was extrapolated from adult data or other data, we categorized the type of pediatric data required to support extrapolation, and we determined whether the data resulted in new pediatric labeling. RESULTS: Extrapolation of efficacy from adult data occurred for 82.5% of the drug products (137 of 166). Extrapolation was defined as complete for 14.5% of the products (24 of 166) and partial for 68% of them (113 of 166). Approaches to extrapolation changed over time for 19% of the therapeutic indications studied (13 of 67). When extrapolation was used, 61% of the drug products (84 of 137) obtained a new pediatric indication or extension into a new age group; this number decreased to 34% (10 of 29) when there was no extrapolation. CONCLUSIONS: Extrapolating efficacy from adult data or other data to the pediatric population can streamline pediatric drug development and help to increase the number of approvals for pediatric use.

Cyclosporine A inhibits the expression of costimulatory molecules on in vitro-generated dendritic cells: association with reduced nuclear translocation of nuclear factor kappa B.

BACKGROUND The maturation of dendritic cells (DC) is influenced by various factors, in particular cytokine-mediated signaling events. These include modulation of the activation of nuclear factor kappa B (NF-kappaB), which controls the transcription of genes encoding major histocompatibility complex (MHC) antigens, and costimulatory/accessory molecules for T-cell activation. Here, we investigated the influence of cyclosporine A (CsA) on the in vitro maturation of DC, and on the nuclear translocation and DNA binding of NF-kappaB. METHODS DC progenitors were propagated from mouse bone marrow in granulocyte-macrophage colony-stimulating factor (GM-CSF) or in GM-CSF plus either transforming growth factor (TGF)-beta or interleukin (IL)-4, in the presence or absence of CsA (1 microg/ml). After 5 days of culture, cell surface expression of MHC class I/II, CD40, CD80, and CD86 was analyzed by flow cytometry, and nuclear NF-kappaB proteins by electrophoretic mobility shift, antibody supershift, and Western blot assays. The antigen-presenting function of DC was determined in one-way mixed leukocyte reactions. RESULTS Exposure of replicating DC progenitors propagated in GM-CSF or GM-CSF+TGF-beta to CsA reduced costimulatory molecule expression, without affecting MHC antigen expression. Nuclear extracts from the CsA-treated DC revealed a decrease in nuclear translocation of NF-kappaB (p50). Mixed leukocyte reaction data were consistent with the flow cytometry and gel shift assay results, and showed reduced allostimulatory ability of the CsA-treated cells compared with untreated controls. Addition of IL-4 from the start of DC cultures conferred resistance to CsA-induced inhibition of NF-kappaB nuclear translocation and DC maturation. CONCLUSIONS CsA differentially inhibits the expression of key cell surface costimulatory molecules by in vitro-generated DC. This effect can be overcome, at least in part, by IL-4 and augmented by TGF-beta. The inhibition is linked to a decrease in nuclear translocation/DNA binding of NF-kappaB. Thus, CsA can alter the antigen-presenting function of DC for T-cell activation.

Tacrolimus Dosing in Adult Lung Transplant Patients Is Related to Cytochrome P4503A5 Gene Polymorphism

Tacrolimus is a potent immunosuppressive agent used in lung transplantation and is a substrate for both P‐glycoprotein (P‐gp, encoded by the gene MDR1) and cytochrome (CYP) P4503A. A previous study by the authors identified a correlation between the tacrolimus blood level per dose with CYP3A5 and MDR1 gene polymorphisms in pediatric heart transplant patients. The objective of this study was to confirm the influence of these polymorphisms on tacrolimus dosing in adult lung transplant patients. Adult lung transplant patients who had been followed for at least 1 year after lung transplantation were studied. Tacrolimus blood level (ng/mL) per dose (mg/day) at 1, 3, 6, 9, and 12 months after transplantation was calculated as [L/D]. DNA was extracted from blood. MDR1 3435 CC, CT, and TT; MDR1 2677 GG, GT, and TT; and CYP3A5*1 (expressor) and *3 (nonexpressor) genotypes were determined by PCR amplification, direct sequencing, and sequence evaluation. Eightythree patients were studied. At 1, 3, 6, 9, and 12 months after the transplant, a significant difference in [L/D] was found between the CYP3A5 expressor versus nonexpressor genotypes (mean ± SD of 1.49 ± 0.88 vs. 3.11 ± 4.27, p = 0.01; 1.23 ± 0.82 vs. 3.44 ± 8.97, p = 0.05; 1.32 ± 0.96 vs. 3.81 ± 6.66, p = 0.005; 0.95 ±1.19 vs. 3.74 ±5.98, p = 0.0015; and 0.45 ± 0.2 vs. 3.76 ± 6.75, p = 0.0001, respectively). MDR1 G2677T and C3435T genotypes had only minimal effects on [L/D] at 1 and 3 months after transplantation. This study confirms the relationship of CYP3A5 polymorphisms to tacrolimus dosing in organ transplant patients. CYP3A5 expressor genotypes required a larger tacrolimus dose to achieve the same blood levels than the CYP3A5 nonexpressors at all time points during the first posttransplant year. This was not uniformly true for MDR1. The authors therefore conclude that tacrolimus dosing in adult lung transplant patients is associated with CYP3A5 gene polymorphisms.

Increased serum nitrite and nitrate concentrations in children with the sepsis syndrome

OBJECTIVES To measure total serum nitrite and nitrate concentrations in children with the sepsis syndrome as an indicator of endogenous nitric oxide production. To determine if there is an association between total serum nitrite and nitrate concentrations and vascular responsiveness to norepinephrine. DESIGN A prospective, clinical study. SETTING Tertiary, multidisciplinary, pediatric intensive care unit. PATIENTS Thirty-one children with the sepsis syndrome, 18 of whom were also hypotensive. Sixteen critically ill children without signs of the sepsis syndrome served as controls. INTERVENTIONS Blood samples were obtained from indwelling catheters. The norepinephrine dose to reach the age appropriate, 50th percentile mean arterial blood pressure was determined in patients receiving norepinephrine. MEASUREMENTS AND MAIN RESULTS Total serum nitrite and nitrate concentrations were measured on the first three days after the recognition of the sepsis syndrome. Patients with the sepsis syndrome had increased mean total serum nitrite and nitrate concentrations (day 1, 118 +/- 93 microM; day 2, 112 +/- 94 microM; day 3, 112 +/- 93 microM) vs. controls (43 +/- 24 microM, p < .05) on all 3 days. When sepsis syndrome patients were separated into nonhypotensive and hypotensive groups, only the patients with hypotension had increased concentrations vs. controls on all three days (p < .05). Sepsis syndrome patients with hypotension also had higher total serum nitrite and nitrate concentrations (145 +/- 97 microM) than sepsis syndrome patients without hypotension (82 +/- 76 microM, p < .05) on day 1. In five patients receiving norepinephrine infusions, increased total serum nitrite and nitrate concentrations were associated with higher norepinephrine requirements to maintain an age-appropriate, 50th percentile mean arterial blood pressure on each of the three study days (day 1, rs = 0.821, p < .05; day 2, rs = 0.900, p < .05; day 3, rs = 0.872, p < .05). CONCLUSIONS Children with the sepsis syndrome, particularly those patients with hypotension, have increased total serum nitrite and nitrate concentrations that likely reflect increased endogenous production of nitric oxide. Vascular hyporesponsiveness to norepinephrine during the sepsis syndrome may be, in part, a nitric oxide-mediated process.