Protopic
Localization of calcineurin/NFAT in human skin and psoriasis and inhibition of calcineurin/NFAT activation in human keratinocytes by cyclosporin A.

Al-Daraji WI, Grant KR, Ryan K, Saxton A, Reynolds NJ.

Department of Dermatology, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, UK.

Systemic cyclosporin A and tacrolimus are effective treatments for psoriasis. Cyclosporin A and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor nuclear factor of activated T cells (NFAT). Inhibition of T cell activation is thought to account for their therapeutic action in psoriasis. We investigated whether nonimmune cells in human skin express calcineurin and NFAT1 and whether cyclosporin A and tacrolimus block activation of calcineurin/NFAT in epidermal keratinocytes. The expression patterns of the principal components of calcineurin/NFAT signaling pathway in normal human skin and psoriasis were determined by immunohistochemistry. We assessed calcineurin/NFAT activation in cultured keratinocytes by measuring the degree of nuclear localization of calcineurin and NFAT1 using immunofluorescence/confocal microscopy and assessed if cyclosporin A and tacrolimus blocked nuclear translocation of these proteins. A variety of cell types in normal and psoriatic skin expressed calcineurin and NFAT1, but expression was particularly prominent in keratinocytes. The principal cyclosporin A and tacrolimus binding proteins cyclophilin A and FKBP12 were also expressed by keratinocytes and nonimmune cells in skin. NFAT1 was predominantly nuclear in normal basal epidermal keratinocytes. Increased nuclear localization of NFAT1 was observed in suprabasal keratinocytes within lesional and to a lesser extent nonlesional psoriatic epidermis compared to normal skin (p = 0.001 and p = 0.03, respectively), suggesting increased activation of calcineurin in psoriatic epidermal keratinocytes. Agonists that induce keratinocyte differentiation, specifically 12-0-tetradecanoyl-phorbol-13-acetate (TPA) plus ionomycin, TPA, and raised extracellular calcium, induced nuclear translocation of NFAT1 and calcineurin in keratinocytes that was inhibited by pretreatment with cyclosporin A or tacrolimus. In contrast in human dermal fibroblasts, TPA plus ionomycin or TPA did not significantly alter the proportion of nuclear-associated NFAT1. These data provide the first evidence that calcineurin is functionally active in human keratinocytes inducing nuclear translocation of NFAT1 and also indicate that regulation of NFAT1 nuclear translocation in skin is cell type specific. Inhibition of this pathway in epidermal keratinocytes may account, in part, for the therapeutic effect of cyclosporin A and tacrolimus in skin diseases such as psoriasis.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11982754&dopt=Abstract tacrolimus Protopic



Protopic
Decreased lung ischemia-reperfusion injury in rats after preoperative administration of cyclosporine and tacrolimus.

Krishnadasan B, Naidu B, Rosengart M, Farr AL, Barnes A, Verrier ED, Mulligan MS.

Division of Cardiothoracic Surgery, University of Washington, Seattle 98195, USA.

OBJECTIVES: Calcineurin inhibitors reduce experimental reperfusion injury in the liver, brain, heart, kidney, and small bowel. These studies were undertaken to determine whether these agents are similarly protective against lung ischemia-reperfusion injury. METHODS: Left lungs of male rats were rendered ischemic for 90 minutes and reperfused for as long as 4 hours. Treated animals received cyclosporine A (INN: ciclosporin; 1 or 5 mg/kg) or tacrolimus (0.2 mg/kg) 6 hours before ischemia, at reperfusion, or 2 hours after reperfusion. Injury was quantitated in terms of tissue polymorphonuclear leukocyte accumulation (myeloperoxidase content), vascular permeability (iodine 125-labeled bovine serum albumin extravasation), and bronchoalveolar lavage leukocyte content. Separate tissue samples were processed for nuclear protein and cytokine messenger RNA. RESULTS: Treatment with cyclosporine (5 mg/kg) or tacrolimus (0.2 mg/kg) 6 hours before reperfusion reduced lung vascular permeability by 54% and 56% relative to control animals (P <.03). The protective effects of cyclosporine and tacrolimus treatment before reperfusion correlated with 42% and 43% reductions in tissue polymorphonuclear leukocyte (myeloperoxidase) content (P <.008) and marked reductions in bronchoalveolar lavage leukocyte accumulation (P <.01). Administration of cyclosporine or tacrolimus at the time of reperfusion or 2 hours into the reperfusion period offered little or no protection. Animals treated before reperfusion also demonstrated marked reductions in nuclear factor kappaB activation and expression of proinflammatory cytokine messenger RNA. CONCLUSION: Cyclosporine and tacrolimus treatment before reperfusion was protective against lung ischemia-reperfusion injury in rats. The mechanism of these protective effects may involve the inhibition of nuclear factor kappaB, a central transcription factor mediating inflammatory injury. The decreased expression of cytokine messenger RNA indicates that both cyclosporine and tacrolimus may exert their protective effects at the pretranscriptional level.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11986604&dopt=Abstract tacrolimus Protopic



Protopic
Evaluating mechanisms of post-transplant diabetes mellitus.

van Hooff JP, Christiaans MH, van Duijnhoven EM.

Department of Nephrology, University Hospital Maastricht, PO Box 5800, NL-6202 AZ Maastricht, The Netherlands. jho sint.azm.nl

Post-transplant diabetes mellitus (PTDM) is a frequent complication in renal transplantation. While both tacrolimus and ciclosporin are known to be associated with PTDM, the mechanisms underlying this metabolic disturbance and the relative contribution of concomitant corticosteroids have been unclear. At the University Hospital Maastricht, a series of studies have been conducted to investigate these issues. Administering tacrolimus to non-diabetic, dialysis patients was shown to result in a dose-related reduction in insulin secretion without altering insulin resistance. The patients who developed diabetes after transplantation already had impaired glucose metabolism pre-transplant. In a second study, corticosteroid withdrawal from tacrolimus-based immunosuppression reduced insulin resistance without changing insulin secretion. Moreover, reducing tacrolimus blood levels by 30% within the therapeutic window increased both insulin and C-peptide secretion by 24 and 36%, respectively. Accordingly, the effects of tacrolimus on insulin secretion are both dose dependent and reversible. A comparison of the effects of tacrolimus and ciclosporin on glucose metabolism revealed reduced insulin release with tacrolimus at week 3 post-transplant, but for the remainder of the 3 year follow-up there were no significant differences between the two treatment arms. Also, no difference was reported in glucose metabolism following conversion of stable renal recipients from ciclosporin to tacrolimus. Therefore, replacing tacrolimus with ciclosporin in patients experiencing glucose metabolism disturbances is unlikely to be helpful. In a recent study, early corticosteroid withdrawal from tacrolimus-based therapy resulted in a significantly lower incidence of new-onset diabetes mellitus than that achieved with a corticosteroid dose-tapering regimen. In conclusion, corticosteroid minimization plus dose-optimized tacrolimus immunosuppression is likely to be the best option for patients at risk of developing PTDM.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15575024&dopt=Abstract tacrolimus Protopic



Protopic
Markers of oxidative stress in cyclosporine-treated and tacrolimus-treated children after liver transplantation.

Granot E, Elinav H, Kohen R.

Department of Pediatrics, Hadassah University Hospital, Jerusalem, Israel. etgranot md2.huji.ac.il

Oxidative stress is presumed to have a major role in cyclosporine A (CsA)- and tacrolimus-induced tissue toxicity. The present study was performed to elucidate the degree of oxidative stress after liver transplantation in CsA- and tacrolimus-treated patients. Twenty-three patients (14 patients, CsA; 9 patients, tacrolimus) aged 2.5 to 18 years (mean, 9.8 years) who had undergone liver transplantation 1.5 to 12 years (mean, 5.4 years) before were studied. Eighteen healthy children aged 2 to 16.5 years (mean, 9.4 years) served as a control group. The following parameters were assessed: plasma lipoprotein levels; plasma carbonyl levels, as markers of oxidative damage to proteins; total plasma oxidizability, which evaluates plasma antioxidant capacity (lag phase) and lipoprotein susceptibility to oxidation; and plasma antioxidant capacity by cyclic voltammetry (CV), which measures antioxidant capacity stemming from hydrophilic low-molecular-weight antioxidant components. Carbonyl levels and rates of plasma oxidation did not differ between groups. The lag phase of plasma oxidation was significantly longer in CsA-treated children compared with tacrolimus-treated children or controls (mean, 54.4 +/- 4.8 [SE] v 40.2 +/- 2.2 v 46.5 +/- 2.8 minutes, respectively; P < 0.05). Antioxidant capacity, assessed by CV, did not differ among CsA-treated patients, tacrolimus-treated patients, and healthy controls. Plasma alpha-tocopherol and beta-carotene levels did not differ between CsA-treated and tacrolimus-treated patients. In children post-liver transplantation, oxidative damage assessed by markers of lipid and protein oxidation is not increased, and plasma antioxidant capacity is not diminished.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12004347&dopt=Abstract tacrolimus Protopic



Protopic
In vitro evaluation of motilin agonism by macrolide immunosuppressive drugs.

Van Vlem B, Schoonjans R, Vanholder R, De Vos M, Depoortere I, Peeters TL, Lefebvre R.

Renal Division, Department of Internal Medicine, Ghent University Hospital, Ghent, Belgium. bruno.vanvlem rug.ac.be

BACKGROUND: The immunosuppressive drugs tacrolimus and sirolimus may have a stimulatory influence on gastric emptying, in view of their macrolide structure. The aim of this study was to investigate in vitro the possible interaction of tacrolimus and sirolimus with motilin receptors in the rabbit antrum and duodenum. METHODS: Rabbit duodenum strips were mounted under a load of 1 g in 10 ml organ baths containing Krebs solution. Erythromycin, tacrolimus, cyclosporin, and sirolimus were added to the bathing solution in a cumulative way; in a second series, after incubation with cyclosporin (10(-7) mol/l), tacrolimus (10(-8) mol/l), or sirolimus (3 x 10(-8) mol/l), a cumulative concentration-response curve to erythromycin was obtained. The effect of cumulatively added tacrolimus and nle(13)-porcine motilin on the contractile response to electrical field stimulation was tested in strips from the rabbit gastric antrum. Displacement by tacrolimus of (125)I-nle(13)-porcine motilin bound to its receptor was tested with crude homogenates of the smooth-muscle layer of the rabbit antrum. RESULTS: In rabbit duodenum strips, carbachol (10(-5) mol/l) induced a stable and reproducible contraction. Erythromycin induced concentration-dependent contractions. Expressed as a percentage of the response to carbachol, the maximal attained effect was 78%; the EC(50) was 4.8 x 10(-7) mol/l. Tacrolimus (10(-8) to 10(-5) mol/l), cyclosporin (10(-8) to 10(-5) mol/l), and sirolimus (10(-8) to 3 x 10(-5) mol/l) had no influence. The response to erythromycin (10(-8) to 3 x 10(-5) mol/l) in the presence of cyclosporin (10(-7) mol/l), tacrolimus (10(-8) mol/l), or sirolimus (3 x 10(-8) mol/l) did not differ from that obtained with erythromycin alone, except for a decrease of the EC(50) in the presence of tacrolimus (2.2 x 10(-7) mol/l) (P<0.05 vs erythromycin alone). Motilin enhanced the response to electrical field stimulation of rabbit antral strips, but tacrolimus had no stimulatory effect. Tacrolimus weakly displaced motilin bound to its receptor. The pIC(50) was 4.97. CONCLUSIONS: As opposed to erythromycin, neither tacrolimus nor sirolimus showed a contractile effect in the rabbit duodenum. Tacrolimus did not activate the neural motilin receptor of the rabbit gastric antrum and had low affinity for the smooth-muscle motilin receptor. It is thus unlikely that these macrolide immunosuppressive drugs possess gastroprokinetic effects via motilin agonism.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12032184&dopt=Abstract tacrolimus Protopic



Protopic
Pharmacokinetic interaction between tacrolimus and diltiazem: dose-response relationship in kidney and liver transplant recipients.

Jones TE, Morris RG.

Department of Pharmacy, The Queen Elizabeth Hospital, Woodville South, South Australia, Australia. terryej hotmail.com

OBJECTIVE: To study the dose-response relationship of the pharmacokinetic interaction between diltiazem and tacrolimus in kidney and liver transplant recipients. DESIGN: Nonrandomised seven-period stepwise pharmacokinetic study. PATIENTS: Stable kidney (n = 2) and liver (n = 2) transplant recipients maintained on oral tacrolimus twice daily but not taking diltiazem. METHODS: Patients were treated with seven incremental dosages of diltiazem (0 to 180 mg/day) at > or = 2-weekly intervals. At the end of each interval, 13 blood samples were taken over a 24-hour period to allow determination of morning (AUC(12)), evening (AUC(12-24)) and 24-hour (AUC(24)) areas under the concentration-time curve for tacrolimus, as well as AUC(24) for diltiazem and three of its metabolites. RESULTS: There was considerable interpatient variability in tacrolimus-sparing effect. In the two kidney transplant recipients, an increase in tacrolimus AUC(24) occurred following the 20 mg/day dosage of diltiazem (26 and 67%). The maximum increase in tacrolimus AUC(24) occurred at the maximum diltiazem dosage used (180 mg/day), when the increase was 48 and 177%. In the two liver transplant recipients, an increase in tacrolimus AUC(24) did not occur until a higher diltiazem dosage (60 to 120 mg/day) was given. The increase at the maximum diltiazem dosages used (120 mg/day in one and 180 mg/day in the other) was also lower (18 and 22%) than that exhibited by the kidney transplant recipients. The increase in tacrolimus AUC(12) was similar to the increase in AUC(12-24) when diltiazem was given in the morning only (dosages < or = 60 mg/day). Hence, diltiazem affects blood tacrolimus concentrations for longer than would be predicted from the half-life of diltiazem in plasma. CONCLUSIONS: The mean tacrolimus-sparing effect of diltiazem was similar in magnitude to the cyclosporin-sparing effect previously reported. Whether the lesser tacrolimus-sparing effect with diltiazem seen in the liver transplant recipients was due to functional differences in the transplanted liver is not known, but it was not due to lower plasma diltiazem concentrations. Diltiazem makes a logical tacrolimus-sparing agent because of the potential financial savings and therapeutic benefits. Because of interpatient variability, the sparing effect should be demonstrated in each patient and not merely assumed.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12036394&dopt=Abstract tacrolimus Protopic



Protopic
Sirolimus/cyclosporine/tacrolimus interactions on bile flow and biliary excretion of immunosuppressants in a subchronic bile fistula rat model.

Deters M, Klabunde T, Kirchner G, Resch K, Kaever V.

Institute of Pharmacology, Medical School of Hannover, 30623 Hannover, Germany. Deters.Michael MH-Hannover.de

The new immunosuppressive agent sirolimus generally is combined in transplant patients with cyclosporine and tacrolimus which both exhibit cholestatic effects. Nothing is known about possible cholestatic effects of these combinations which might be important for biliary excretion of endogenous compounds as well as of immunosuppressants. Rats were daily treated with sirolimus (1 mg kg(-1) p.o.), cyclosporine (10 mg kg(-1) i.p.), tacrolimus (1 mg kg(-1) i.p.), or a combination of sirolimus with cyclosporine or tacrolimus. After 14 days a bile fistula was installed to investigate the effects of the immunosuppressants and their combinations on bile flow and on biliary excretion of bile salts, cholesterol, and immunosuppressants. Cyclosporine as well as tacrolimus reduced bile flow (-22%; -18%), biliary excretion of bile salts (-15%;-36%) and cholesterol (-15%; -47%). Sirolimus decreased bile flow by 10%, but had no effect on cholesterol or bile salt excretion. Combination of sirolimus/cyclosporine decreased bile flow and biliary bile salt excretion to the same extent as cyclosporine alone, but led to a 2 fold increase of biliary cholesterol excretion. Combination of sirolimus/tacrolimus reduced bile flow only by 7.5% and did not change biliary bile salt and cholesterol excretion. Sirolimus enhanced blood concentrations of cyclosporine (+40%) and tacrolimus (+57%). Sirolimus blood concentration was increased by cyclosporine (+400%), but was not affected by tacrolimus. We conclude that a combination of sirolimus/tacrolimus could be the better alternative to the cotreatment of sirolimus/cyclosporine in cholestatic patients and in those facing difficulties in reaching therapeutic ranges of sirolimus blood concentration.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12055139&dopt=Abstract tacrolimus Protopic