Protopic
Comparative studies of the enhancing effects of cyclodextrins on the solubility and oral bioavailability of tacrolimus in rats.

Arima H, Yunomae K, Miyake K, Irie T, Hirayama F, Uekama K.

Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan.

The enhancing effects of cyclodextrins (CyDs) on the solubility, the dissolution rate, and the bioavailability of tacrolimus after oral administration to rats were examined and compared with those after administration of a PROGRAF capsule containing the solid dispersion formulation of tacrolimus. Here we used natural CyDs and the hydrophilic beta-CyD derivatives; that is, randomly methylated-beta-cyclodextrin (RM-beta-CyD), heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CyD), 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD), and sulfobutyl ether beta-cyclodextrins (SBE-beta-CyDs). Of the natural CyDs, the solubility of tacrolimus increased in the addition of beta-CyD, indicating that the cavity of beta-CyD comfortably fits the drug. Of the beta-CyD derivatives, DM-beta-CyD had the greatest solubilizing activity and gave the A(p) type phase solubility curve as defined by Higuchi and Connors, suggesting the formation of higher-order complexes. The result of van't Hoff plot suggests that the enthalpy is dominant for the complexation of tacrolimus with DM-beta-CyD. The dissolution rate of tacrolimus was markedly augmented by the complexation with DM-beta-CyD, reflecting its solubilizing activity. An in vivo study revealed that DM-beta-CyD increased the bioavailability of tacrolimus with low variability in the absorption after oral administration of the tacrolimus suspension to rats. The present results suggest that DM-beta-CyD is particularly useful in designing oral preparations of tacrolimus with an enhanced bioavailability and a reduced variability in absorption. Copyright 2001 Wiley-Liss, Inc.

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Protopic
Therapeutic monitoring of tacrolimus concentrations in blood: semi-automated extraction and liquid chromatography-electrospray ionization mass spectrometry.

Lensmeyer GL, Poquette MA.

University of Wisconsin Hospitals & Clinics, Division of Laboratory Medicine, Toxicology Laboratory, Madison, Wisconsin, USA. gl.lensmeyer hosp.wisc.edu

The authors describe a highly selective liquid chromatographic/mass spectrometric (LC/MS) method for measurement of the immunosuppressive drug tacrolimus. A protein-free supernatant of a patient's whole-blood sample (500 microL) is applied to an Empore styrene-divinylbenzene (SDB-XC) disk cartridge (Varian Sample Preparation Products; Harbor City, CA) to isolate tacrolimus and the internal standard, ascomycin. The entire extraction is automated on the Gilson ASPEC XL4 (Gilson; Middleton, WI). Separation takes place on an octyldecyl (C18) high-performance liquid chromatographic (HPLC) column maintained at 75 degrees C with a simple mobile phase of acetonitrile/water (90/10, v/v). An atmospheric pressure ionization (API)-electrospray ionization(ESI)-mass spectrometer (MS) is used for analysis. Detection is by selected ion monitoring (SIM) of the positively charged sodium adduct of tacrolimus and ascomycin, m/z 826.5 and 814.5, respectively. Collision-induced dissociation (CID) fragment ions of tacrolimus and ascomycin (m/z 616.4 and 604.4, respectively) are also monitored to enhance overall selectivity. Total run time is 1 minute per injection. A plot of chromatographic peak height ratio of m/z 826.5 to m/z 814.5 vs tacrolimus blood concentration is linear from the lowest limit of quantitation (0.3 microg/L) to at least 120.0 microg/L. Metabolites of tacrolimus and other immunosuppressive and commonly prescribed drugs do not interfere. Analytical recovery is 94% to 113% over a range of 4.4 to 41.0 microg/L. Between-run precision coefficients of variation (CV) are 3.6% to 4.2% over a range of 8.1 to 32.4 microg/L. Comparison data (n = 156) of the LC/MS method versus a commercial immunoassay (Abbott Tacrolimus IMx MEIA II) (Abbott; Chicago, IL) demonstrated that the tacrolimus concentration as measured by LC/MS = (0.912 x MEIA concentration) - 0.049; r2 = 0.968. This validated LC/MS method demonstrates significant advantages over conventional immunoassays and improves on the lower limit of detection, sensitivity, accuracy, and range of linearity. In the authors' laboratory, this method is approximately 60% less costly to perform than the most commonly implemented immunoassay. Together, the authors' daily clinical experience during 1 year and participation in a proficiency testing program has demonstrated that the method is robust and suitable for routine therapeutic monitoring of tacrolimus.

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Protopic
Reasons for long-term use of steroid in primary adult liver transplantation under tacrolimus.

Jain A, Kashyap R, Marsh W, Rohal S, Khanna A, Fung JJ.

Department of Surgery, Thomas E. Starzl Transplantation Institute, Pittsburgh, Pennsylvania, USA.

BACKGROUND: Tacrolimus is a potent immunosuppressive agent that provides higher freedom from acute and chronic rejection than cyclosporine after liver transplantation (LTx). Initially, a steroid-free state was observed in about 70% of patients at 1 year; this did not change over the next 5 years. The present study identifies the various reasons why the remaining 30% of adult patients still require steroids even after 5 years after successful LTx. METHOD: Eight hundred thirty-four consecutive patients who underwent LTx between August 1989 and December 1992 were included in this study. Four hundred ninety-nine patients were alive in January 1999 and were available for this study. The dose of steroid and the reason for steroid use were retrospectively determined from the clinical records. RESULTS: Three hundred sixty-five patients (73.1%) were off steroid, whereas 134 patients (26.9%) were receiving prednisone (mean dose was 6.4+/-3.7 mg/day) at the time of the study. Four hundred and eight-four patients (97%) were off prednisone at some time after LTx; however, in 119 (23.8%) patients, steroids were reintroduced. Fifteen patients (3%) continued to receive prednisone; eight receive prednisone due to reluctance of the local physician to withdraw the medication; in five patients, the prednisone was not withdrawn because these patients were on cyclosporine; in the remaining two patients, repeated attempts to withdraw steroid resulted in a rise in liver function test. In the 49 (36.6%) of 119 patients in whom the steroid was reintroduced, it was restarted secondary to pathologically proven or clinically suspected rejection (group I). In five patients steroid was reintroduced for abnormal liver function after being off immunosuppression for treatment of a posttransplantation lymphoproliferative disorder. Six patients were noncompliant with their immunosuppressive medication, and the steroid was reintroduced to control rejection. Steroids were reintroduced in 30 patients (22.4%) for recurrence of original disease: primary biliary cirrhosis (n= 19), sclerosing cholangitis (n=6), and autoimmune hepatitis (n=5) (group II). In 24 patients (20.2%), steroids were reintroduced to lower the dose of tacrolimus secondary to nephrotoxicity. Six of these patients received kidney transplantation (group III). In 16 patients (13.4%) the steroid was reintroduced for concomitant medical problems, consisting of ulcerative/Crohn's colitis (n=6), adrenal insufficiency (n=5), hematological disorders (n=3), dermatitis (n=1), and rheumatoid arthritis (n=1) (group IV). CONCLUSION: Ninety-seven percent of patients under tacrolimus were weaned off steroid; however, 23.8% required steroid reintroduction for late rejection, recurrence of autoimmune process(es), renal impairment, or the concomitant presence of other medical conditions. Although the use of other immunosuppressive agents may reduce the rate of reintroduction of steroid, long-term sustained freedom from steroid may not be possible in all patients under tacrolimus secondary to these conditions.

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Protopic
Tacrolimus based immunosuppression.

First MR.

Research and Development, Fujisawa Healthcare, Inc., Deerfield, IL 60015-2548, USA. roy_first fujisawa.com

The safety and efficacy of tacrolimus (Prograf) in renal transplantation is well established. Achieving longterm patient and graft survival are the ultimate goals following transplantation. Many factors negatively impact long-term transplant outcomes, including graft rejection, renal dysfunction and cardiovascular risk factors (hypertension, hyperlipidaemia, and post-transplant diabetes mellitus (PTDM)). Accordingly, careful consideration of the immunosuppressive strategy and its impact on these factors is critical to optimising outcomes. Clinical trials and registry studies conducted over the past decade have demonstrated tacrolimus to be a cornerstone immunosuppressant in renal transplantation. Compared with cyclosporine treatment, tacrolimus has been shown to be associated with decreased acute and chronic rejection, improved renal function over the long-term post-transplant, and a lower incidence of hyperlipidaemia and hypertension. In early studies, the incidence of PTDM was significantly higher in patients receiving tacrolimus; however, recent large clinical trials have revealed no significant between-group differences in the incidence of PTDM with tacrolimus treatment and cyclosporine microemulsion treatment. Together, these findings may translate into improved long-term transplant outcomes with tacrolimus-based immunosuppression. Although approved only for kidney and liver transplantation in the US, Prograf was the calcineurin inhibitor used in the majority of patients transplanted in 2003: kidney (67%), liver (89%), kidney/pancreas (81%), pancreas (77%), lung (65%), heart/lung (48%), and heart (42%).

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Protopic
Induction of transplantation tolerance with a short course of tacrolimus (FK506): I. Rapid and stable tolerance to two-haplotype fully mhc-mismatched kidney allografts in miniature swine.

Utsugi R, Barth RN, Lee RS, Kitamura H, LaMattina JC, Ambroz J, Sachs DH, Yamada K.

Department of Pathology, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02129, USA.

BACKGROUND: Inbred miniature swine provide a large animal model in which the effects of selective major histocompatibility complex (MHC) matching can be reproducibly studied. We have previously demonstrated that although a 12-day course of cyclosporine uniformly induces tolerance to class I-mismatched renal allografts, it does not induce tolerance across full MHC barriers. In this study, we assessed whether and at what dose tacrolimus might permit allografts to induce tolerance across different MHC barriers. METHODS: Recipients of MHC disparate renal allografts were treated with a 12-day course of tacrolimus by continuous intravenous infusion. Groups were divided as follows: (1) class I-mismatched kidneys with 0.3 mg/kg/day tacrolimus (n=3); (2) fully MHC-mismatched kidneys with 0.3 mg/kg/day tacrolimus (n=2); and (3) fully MHC-mismatched kidneys with 0.12-0.16 mg/kg/day tacrolimus (n=4). RESULTS: In groups 1 and 2, recipients with tacrolimus levels of 45-80 ng/ml accepted renal allografts long-term with stable renal function. Donor-specific hyporesponsiveness was demonstrated by cell-mediated lymphocytotoxicity and mixed lymphocyte response, and subsequent donor-matched grafts were also accepted, without further immunosuppression (n=4), confirming systemic tolerance. In group 3, recipients that achieved tacrolimus levels of 35 ng/ml (n=2) accepted their grafts without chronic changes, whereas recipients with levels of 20-26 ng/ml (n=2) developed chronic allograft glomerulopathy, suggesting 35 ng/ml as the threshold blood level for tolerance induction. In vitro assays demonstrated that peripheral blood lymphocytes from tolerant animals produced inhibitory cytokines, suggesting the involvement of regulatory mechanisms. CONCLUSIONS: To our knowledge, this study represents the first demonstration of the induction of transplant tolerance across a two-haplotype full MHC barrier with a short course of immunosuppression in a large animal model. These studies may also have clinical applicability, because the time course required to induce tolerance was sufficiently short that the high drug levels required might be expected to be tolerated clinically with only transient toxicity.

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Protopic
The effect of gut metabolism on tacrolimus bioavailability in renal transplant recipients.

Tuteja S, Alloway RR, Johnson JA, Gaber AO.

Department of Pharmacy, University of Kentucky, Lexington, USA.

BACKGROUND: Tacrolimus, a substrate of CYP3A, has low and variable bioavailability similar to cyclosporine. Co-administration of ketoconazole, potent inhibitor of gut and hepatic CYP3A, has been shown to increase tacrolimus bioavailability in healthy volunteers. The purpose of this study is to assess the role of gut metabolism in the overall bioavailability of tacrolimus in a renal transplant population. METHODS: We prospectively studied 19 adult renal transplant recipients who were receiving tacrolimus as part of a quadruple, sequential immunosuppression regimen. Each patient received tacrolimus (4-hr intravenous dose of 0.04 mg/kg between postoperative days 2 and 4). Whole blood samples were collected over 24 hr. After a 24-hr washout period, a single oral dose of ketoconazole (400 mg) was administered followed by the same intravenous dose of tacrolimus, and subsequent samples were obtained. Steady state oral pharmacokinetic profiles were obtained between 1 and 3 months after transplant while patients were receiving twice daily dosing of tacrolimus to maintain whole blood levels between 10 and 20 ng/ml. Two days later, 400 mg of ketoconazole was administered orally 2 hr before to the morning dose. Whole blood samples were collected over 12 hr. RESULTS: In the absence of ketoconazole, 8.0% of the tacrolimus dose underwent first pass metabolism (E(H)), whereas in the presence of ketoconazole, first pass metabolism was 6.2% (P=0.01). Based on this difference in first pass metabolism, an increase of 2% in bioavailability is expected, but an increase of 47% is observed (P=0.001). CONCLUSIONS: This indicates that the gut metabolism of tacrolimus is extensive and contributes significantly to its bioavailability.

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Protopic
Safety and efficacy of pravastatin therapy for the prevention of hyperlipidemia in pediatric and adolescent cardiac transplant recipients.

Penson MG, Fricker FJ, Thompson JR, Harker K, Williams BJ, Kahler DA, Schowengerdt KO.

Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida 32610, USA.

BACKGROUND: Hyperlipidemia is common after cardiac transplantation and it is a risk factor for post-transplantation coronary artery disease. Immunosuppression with corticosteroids and cyclosporine has been associated with hyperlipidemia. Pravastatin, a HMG-CoA reductase inhibitor, has been shown to be effective and safe for cholesterol reduction in adult heart transplant recipients. To our knowledge the safety and efficacy of pravastatin therapy in pediatric and adolescent heart transplant populations have not been previously analyzed. Therefore, we evaluated lipid profiles, liver transaminases, rejection data, and possible side effects in pediatric and adolescent cardiac transplant recipients treated with pravastatin. METHODS: The study group consisted of 40 cardiac transplant recipients 10 to 21 years old (mean age 16.9 years). Twenty-two patients received pravastatin in addition to an immunosuppressive regimen of either cyclosporine or tacrolimus, azathioprine or mycophenolate mofetil, and prednisone. Serial determinations of total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein, and triglycerides were available for all pravastatin-treated patients. Pre-treatment lipid values and hepatic transaminases were compared with those measured after therapy with pravastatin. Comparison of pravastatin-induced lipid reduction between groups treated with cyclosporine vs tacrolimus was also made. RESULTS: Patients receiving pravastatin experienced a mean 32 mg/dl decrease in TC (p < 0.005) and a mean 31 mg/dl decrease in LDL (p < 0.005), regardless of their immunosuppressive regimen. No statistical differences occurred in the magnitude of mean lipid reduction induced by pravastatin between the groups treated with cyclosporine vs tacrolimus. No significant changes in hepatic transaminase levels were noted, and no clinical evidence of pravastatin-induced myositis occurred in any subjects. CONCLUSION: Pravastatin therapy is effective and safe when used in pediatric and adolescent cardiac transplant recipients. Although the pravastatin-induced reduction in TC and LDL was more pronounced in patients receiving cyclosporine, the reduction was not statistically different from that in the tacrolimus group. No evidence of hepatic dysfunction or rhabdomyolysis in patients treated with pravastatin was noted. Long-term studies are required to evaluate the effect of pravastatin therapy on the incidence of accelerated coronary atherosclerosis in this population.

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