Cardiac transplant experience with cyclosporine

Cardiac Transplant Experience With Cyclosporine J.K. Patel and J.A. Kobashigawa ABSTRACT The advent of cyclosporine 20 years ago was a major advance in the field of solid organ transplantation. Its use enabled directed immunosuppression with a consequent decrease in the incidence of graft failure, acute rejection, and systemic infection. The early oil-based preparation, however, was difficult to administer and had limited bioavailability and unpredictable pharmacokinetics. The drug also has a fairly narrow therapeutic window with major long-term side effects, which include nephrotoxicity, malignancy, hyperlipidemia, and hypertension. The introduction of a microemulsion preparation (Neoral) with improved bioavailability has been associated with lower rates of rejection and comparable tolerability, therefore allowing the use of lower doses. Traditionally cyclosporine toxicity has been minimized by monitoring trough levels. Monitoring of levels 2 hours after dosing may provide a more accurate determination of cyclosporine exposure. The next phase in cardiac transplantation immunosuppression will most likely see a significantly diminished role for cyclosporine with the introduction of newer, more potent immunosuppressive agents with more favorable side-effect profiles. These agents, which include mycophenolate mofetil, sirolimus, and everolimus, also hold the promise of having a major impact on the development of transplant vasculopathy, which up to now has been an important determinant of limiting long-term allograft survival.

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N THE LAST 20 YEARS, cardiac transplantation has become an established and acceptable therapeutic option for patients with end-stage heart disease. Surgical techniques, however, were developed as early as 1966 by Shumway and colleagues and the first clinical human cardiac transplant was performed in 1967 by Barnard. In the ensuing years, initial enthusiasm was tempered by fairly poor outcomes with 1-year survival rates as low as 56%.1 Survival was limited by high rates of rejection and infection. Early immunosuppression protocols consisted of antithymocyte globulin (ATG), prednisone, and azathioprine. The low specificity of this regimen resulted in suppression of a broad base of host immune responses. This led to an increase in the incidence of opportunistic infections including fungi, protozoa, and viruses. The use of ATG led to an almost universal occurrence of cytomegalovirus infection. Limiting the use of ATG resulted in fairly poor allograft rejection prophylaxis with prednisone and azathioprine combination unless higher doses were used. Progress in cardiac transplantation was therefore limited, awaiting development in improved immunosuppressive regimens. While clinicians struggled with the challenges of this new therapeutic option for heart failure, Borel at Sandoz was © 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 36 (Suppl 2S), 323S⫺330S (2004)

exploring immunosuppressive properties of cyclosporine (CsA), a lipophilic endecapeptide derived from a soil fungus in 1969.2 The agent was found to suppress delayedtype hypersensitivity skin reaction to tuberculin in guinea pigs but seemed to have no effect on antibody synthesis, suggesting a mechanism of immunosuppression specific to T cells. Clinical use for cardiac transplantation started in 1981 with some encouraging results.3 A subsequent randomized controlled trial in cardiac transplantation at Stanford confirmed improved patient survival with CsA over conventional therapy.4 One-year survival improved to 80% and there was a significant associated reduction in the length of hospital stay. Other centers confirmed this benefit.5,6 Significant improvements in morbidity and mortality led to a remarkable expansion in cardiac transplantation. Up to 1978, 378 cardiac transplants had been performed worldFrom the Division of Cardiology, David Geffen School of Medicine at UCLA, Los Angeles, Calif, USA. Address reprint requests to Jon A. Kobashigawa, MD, Division of Cardiology, David Geffen School of Medicine at UCLA, 47-123 CHS, 10833 Le Conte Ave, Los Angeles, CA 90045. E-mail: [email protected] 0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.01.039 323S

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Fig 1. Survival rates of first orthotopic heart transplants according to immunosuppressive regimen. CyA, cyclosporine; AZA, azathioprine; STE, steroids. Adapted from Opelz.8

wide but only 78 had survived.7 By 1998, more than 2800 transplants were being performed annually, with acturial survival rates at 1 and 5 years of 85% and 75%, respectively.7 In a multicenter study of different immunosuppressive regimens involving over 16000 transplant patients, CsAbased therapies were associated with significantly improved 5 year survival compared with non-CsA-based regimens8 (Fig 1). Therapy with this new class of immunosuppressive agent, however, required an appreciation of its potency. CsA, used with the previous immunosuppressive regimens, resulted in an increased risk of lymphomas. Outcomes improved with adjustment of immunosuppressive regimens and the use of lower doses.9 CsA treatment was also associated with an abrogation in the clinical signs and symptoms of acute allograft rejection, increasing the importance for the need for surveillance endomyocardial biopsies. MECHANISM OF ACTION

Early experimental studies revealed that CsA inhibits T-cell activation by blocking the transcription of cytokine genes, including those of interleukin (IL)-2 and IL-4.10 –12 Upon T-cell entry, CsA binds with high affinity to cyclophilins. The cyclophilin–CsA complex can associate with another cytosolic protein, calcineurin. Calcineurin belongs to a superfamily of protein serine/threonine phosphatases, and its activity is tightly regulated by Ca2⫹/calmodulin. Binding of the T-cell receptor with its ligand induces the elevation of intracellular calcium concentration and results in activation of calmodulin. Activated calmodulin, then, interacts with calcineurin and releases the autoinhibitory domain from its active site, leading to the activation of its phosphatase activity. Calcineurin dephosphorylates NFAT (the nuclear

factor of activated T cells) family members, allowing them to translocate into the nucleus and activate gene expression through the cis-element named NFAT. Activated calcineurin also translocates into the nucleus together with NFAT family members, where it may maintain the sustained activation of NFAT proteins.13 The cyclophilin–CsA complex directly binds to calcineurin and inhibits the phosphatase activity, thereby preventing translocation of NFATs into the nucleus and subsequent gene expression (Fig 2). Among NFAT family members, NFAT1, NFAT2, and NFAT4 are involved in the transcriptional activation of genes encoding cytokines including IL-2 and IL-4, and CD40L.14 Transcriptional activation of the IL-2 gene requires cooperative interaction of several transcription factors, including AP-1, NF-␬B, and NFAT. It has been shown that CsA also affects the activities of AP-1 and NF-␬B in addition to NFAT, implying the presence of another target of CsA as well as the calcineurin/NFAT pathway.15 It has also been shown that CsA can inhibit an antigen-specific and Ca2⫹-independent response.16 There is evidence to suggest that CsA blocks both JNK and p38 signaling pathways in addition to the calcineurin/NFAT pathway.17 PREPARATIONS

Early preparations consisted of a crystalline powder that was insoluble in water but readily dissolved in alcohol, and more slowly in lipids such as olive oil. Commercially available oral preparation (Sandimmun) had a variable and somewhat unpredictable bioavailability ranging from 1% to 67% but averaging around 30%. This variability has been attributed to suboptimal gastrointestinal absorption and a significant first-pass effect through the enterohepatic circu-

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Fig 2. Mechanism of action of cyclosporine. TcR, T-cell receptor; CpN, cyclophilin; CsA, cyclosporine; CaN, calcineurin, CalM, calmodulin.

lation with extensive hepatic metabolism of the drug. Intravenous dosing is therefore about a third of the total daily oral dose, usually administered as a continuous infusion over a 24-hour period. Further advances were made with the introduction of a new microemulsion formulation of CsA known as Neoral. This agent demonstrated greater bioavailability and more predictable pharmacokinetics than Sandimmun.18 In a prospective, randomized, multicenter, double-blind study, Neoral demonstrated equivalent graft and patient survival compared with Sandimmun at 24 months.19 Fewer Neoraltreated patients, however, required antilymphocyte antibody therapy for rejection. Neoral was also better tolerated with fewer discontinuations of the study drug and the average corticosteroid dose was lower in the Neoral group. Other studies confirmed clinical benefit or equivalence of Neoral with Sandimmun with the main benefit pertaining to a reduction in the number of rejection episodes.20 –22 THERAPEUTIC DRUG MONITORING

Due to the complex pharmacokinetics and pharmacodynamics of CsA, monitoring of serum levels is essential to minimize both allograft rejection and the risk of adverse effects. Over the years, a number of chromatographic and immunologic techniques for measuring CsA have become available with differing sensitivity and specificity. Highperformance liquid chromatography (HPLC) remains the gold standard. It can be used to measure the parent drug or its metabolites with high sensitivity and specificity. However, the technique is both time-consuming and laborintensive. Tandem mass spectroscopy offers high volume

rapid throughput with great accuracy.23 Immunoassay techniques are commonly used in clinical practice. They have the advantage of simplicity, fairly good accuracy, and good sensitivity. Fluorescence polarization immunoassay is a popular technique, but in addition to measuring the parent compound, it also detects metabolites and therefore may provide falsely elevated CsA levels. The radioimmunoassay utilizes a monoclonal antibody with little affinity for metabolites and is fairly comparable to HPLC. The enzymemultiplication immunoassay likely has the greatest specificity for the parent compound of all the immunologic methods.24 The ratio of parent drug to metabolite is significantly greater at peak blood concentration compared to the trough concentration of cyclosporine.25 Tests that are more likely to detect metabolites therefore may perform better when measuring CsA levels within the first few hours of dosing in contrast to when measuring trough concentrations (Table 1). In clinical practice, CsA exposure has usually been determined by measurement of trough levels taken prior to dosing. The improved bioavailability of Neoral compared to the conventional formulation revealed a significantly higher peak value and greater area under the concentration time curve (AUC) for Neoral. Trough levels however were not significantly different26 (Fig 3). Therefore equivalent trough levels are associated with greater CsA exposure with Neoral compared with the conventional formulation. In clinical practice, a change from conventional formulation to Neoral has correlated with decreased allograft rejection but also decreased renal function.27 To assess the molecular effects of CsA levels, investiga-

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Table 1. Comparison of Available Assays for Detection of Cyclosporine in Blood METHOD

HPLC TMS EMIT Monoclonal RIA FPIA

ACCURACY

Highest (Specific for parent molecule)

2 Lowest (also detects metabolites)

SPEED

Slow Rapid; Rapid; Rapid; Rapid;

high high high high

volume volume volume volume

HPLC ⫽ High-performance liquid chromatography; TMS ⫽ Tandem Mass Spectroscopy; EMIT ⫽ Enzyme-multiplication immunoassay; RIA ⫽ Radioimmunoassay; FPIA ⫽ Fluorescence Polarization Immuno-assay.

tors have determined blood CsA levels and correlated these to calcineurin inhibition and IL-2 production in circulating leukocytes.28,29 CsA was shown to produce only partial inhibition of calcineurin; this inhibition varied with CsA blood levels. Greatest inhibition of calcineurin and IL-2 production occurred within the first 2 hours after dose administration, with this effect waning totally by 24 hours. These studies suggest that immunosuppression with CsA monotherapy may be incomplete even at therapeutic levels and that for a significant time period between dosing, there may be no effective immunosuppression. Furthermore, monitoring of CsA levels at 2 hours after dosing (C2) may be a better indictor of immunosuppression efficacy than trough levels. Prospective studies in renal transplant patients have confirmed lower incidence of both acute rejection and renal dysfunction with early postdose monitoring compared to monitoring of trough levels.30 These authors also demonstrated that C2 monitoring provided a good estimate of peak levels and AUC within the first 4 hours. Experience with C2 monitoring in cardiac transplant patients is more limited. In a longitudinal study of 114 stable cardiac allograft recipients more than 1 year after transplantation, patients were initially followed for about a year with C2 monitoring (target range 300 to 600 mcg/L).31 The patients were then switched to trough level (C0)

Fig 3. Comparison of available assays for detection of cyclosporine in blood. HPCL, high-performance liquid chromatography; TMS, tandem mass spectroscopy; EMIT, enzyme-multiplication immunoassay; RIA, radioimmunoassay; FPIA, fluorescence polarization immunoassay.

monitoring (target range 100 to 200 mcg/L). During C2 monitoring, cyclosporine dosage, C0 levels, C2, and creatinine decreased by 26%, 56%, 45% and 2.3%, respectively, compared to baseline. When changed to C0 monitoring, these variables increased by 24%, 56%, 38%, and 10%, respectively. Prospective randomized studies to assess the impact of C2 monitoring in cardiac transplantation are currently underway. COMPLICATIONS OF CYCLOSPORINE THERAPY Hypertension

Systemic hypertension is a common finding following cardiac transplantation and is generally associated with steroid and CsA immunosuppressive therapy. Mechanisms of CsAassociated hypertension are not completely understood but may include augmented production of endothelin, impairment of nitric oxide synthesis, neuroendocrine activation (specifically renin-angiotensin and sympathetic nervous systems), hypervolemia, and alteration of vascular reactivity.32 Hyperlipidemia

CsA and corticosteroids have both been implicated in the development of hyperlipidemia.33 They may therefore indirectly contribute to the development of transplant vasculopathy. It has been suggested that CsA may inhibit the enzyme 26-hydroxylase, which is important in the bile acid synthetic pathway. CsA would thereby decrease the synthesis of bile acids from cholesterol and subsequently the transport of cholesterol to the intestines. CsA is also reported to bind to the low-density lipoprotein (LDL) receptor, which results in increased serum levels of LDL cholesterol. It is also thought that CsA increases hepatic lipase activity and decreases lipoprotein lipase activity, enzymes important in clearance of very low-density lipoprotein and LDL. Infection

A consequence of CsA therapy is overimmunosuppression. In the short term, this may lead to an increase in opportunistic infections. As the drug mainly affects T-cell responses with no significant effect on antibody production, response to bacterial and fungal infection is relatively preserved. CsA does, however, have intrinsic antibiotic activity but this effect is limited; although it inhibits certain viruses, fungi, protozoa, and helminths, in practical terms these effects are insignificant. The use of CsA has little effect on reducing the incidence and severity of herpes infections and also likely predisposes to Pneumocystis carinii infection, necessitating the use of prophylaxis, especially within the first year following cardiac transplantation. Compared with immunosuppressive therapy with azathioprine and prednisone, the use of CsA has generally decreased the incidence of infection (especially cytomegalovirus), an effect most likely attributable to its specific inhibition of T-cell responses.34

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Malignancy

Chronic immunosuppression has been associated with an increased risk for malignancy. It has been widely held that this is a consequence of the failure of immune surveillance, whereby the immune system eliminates cancerous cells. However, recent studies suggest that CsA may have an independent pro-oncogenic effect. Hojo and colleagues35 demonstrated that the application of CsA in vitro and in vivo altered the characteristics of cancerous cell lines. In vitro CsA was shown to induce phenotypic changes in adenocarcinoma cells, resulting in striking morphological alterations, including membrane ruffling and numerous pseudopodial protrusions, increased cell motility, and anchorage-independent (invasive) growth. These changes were prevented by treatment with monoclonal antibodies directed at transforming growth factor-beta (TGF-beta). In vivo, CsA enhanced tumor growth in immunodeficient mice; anti-TGF-beta monoclonal antibodies but not control antibodies prevented the CsA induced increase in the number of metastases. These findings suggest that CsA can promote cancer progression by a direct cellular effect that is independent of its effect on the host’s immune cells, and that CsA-induced TGF-beta production is involved in this. Initial experience with CsA in transplantation resulted in over immunosuppression, leading to a high incidence of certain types of malignancies, especially those implicated with a viral etiology such as lymphomas36,37 and Kaposi’s sarcoma. The selective effect of CsA on T cells may impair resistance to Epstein-Barr virus–induced B-cell proliferation. This mechanism is supported by the observation that in many patients regression of lymphoma is seen after discontinuation or reduction in the dose of CsA.38,39 The incidence of lymphomas is also significantly higher in the first year following transplantation when the level of immunosuppression is generally higher than in subsequent years.40 While lymphomas only represent 5% of all cancers in the general population, they represent 22% of cancers in the transplant populations.41 Skin cancers are also much more common, especially squamous cell carcinoma. There is also a risk of recurrence of previously treated neoplasms following transplantation, and patients with a prior history of malignancy are generally excluded from transplantation unless the disease was successfully treated several years previously.

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careful monitoring and dose adjustment. Altered mental status is also rarely seen, particularly in patients with a low cholesterol level. Gingival Hyperplasia

CsA-associated gingival hyperplasia is generally related to poor dental hygiene with underlying chronic gingivitis. Dental health maintenance usually improves the condition but on occasion gingivectomy may be required. Many patients also respond to a change in immunosuppression from CsA to tacrolimus. Hypertrichosis

Steroids typically cause hirsuitism, which is hair growth in a male pattern. In contrast, CsA causes excessive growth of preexisting hair, or hypertrichosis. In the vast majority of cases, CsA-induced hypertrichosis does not require intervention. Some women and children may require the use of depilatory treatments. Hepatotoxicity

Early experience with CsA use was associated with a frequent occurance of hepatotoxicity as determined by elevated bilirubin and/or transaminases. In one series of renal allograft recipients, liver function abnormalities were seen in 19.7% of recipients. However, the dose of CsA was 20 mg/kg per day.42 In cardiac allograft recipients the incidence was even higher, up to 62.5% in one study.43 However, subsequent analysis revealed that almost half these patients had evidence of viral hepatitis at time of transplant. A small portion had hepatic dysfunction due to cardiac failure and in about 14% hepatic dysfunction was attributed to drugs, which included CsA. With a significantly lower incidence of allograft rejection and use of much lower doses of CsA in recent years, hepatic dysfunction is seen much less frequently, and when it occurs, it usually responds to dose adjustment. Bone Marrow Suppression

Unlike newer immunosuppressive agents such as mycophenolate mofetil and sirolimus (Rapamycin), bone marrow suppression is much less common with CsA and occurs in fewer than 1% of patients. Hemolytic anemia is also seen rarely.

Central Nervous System

Neurological side effects are sometimes seen with CsA use might be expected due to its high lipid solubility. The most common side effect is involuntary tremor. It is most common early following transplantation and generally reflects high blood levels. Tremor therefore usually responds to dose adjustment. Other less common side effects include seizure disorder, paraesthesias, and peripheral neuropathy. Seizures may occur in patients both with and without a prior history. Unfortunately many anticonvulsants, including phenylhydantoin, interfere with CsA metabolism, requiring

Nephrotoxicity

Some degree of renal dysfunction is seen in most patients treated with CsA, whether it is used to prevent allograft rejection44 or in the treatment of autoimmune diseases. In cardiac transplantation postoperative renal dysfunction is associated with an increased mortality particularly when dialysis is required.45,46 CsA nephrotoxicity manifests as a decrease in creatinine clearance with elevation of creatinine and blood urea nitrogen, potassium, hypertension, hyperuricemia, and hyperkalemic hyperchloremic renal tubular

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acidosis with preserved urine volume and sodium resorption. The greatest change in glomerular filtration rate occurs within the first few months of CsA therapy. However, serum creatinine is subsequently a relatively poor marker for progressive decline in renal function as this can conceal progressive injury. Proteinuria may be an indication of advanced renal dysfunction due to CsA.47 Lowering CsA dose may be helpful in slowing the progression of disease, especially with concomitant use of newer immunosuppressive regimens such as mycophenolate mofetil.48 Decreased erythropoietin production associated with CsA-induced renal dysfunction is also a cause for chronic anemia frequently seen in transplant recipients. A number of mechanisms may be responsible for CsAinduced renal injury. CsA-induced renal vasospasm may initially cause reversible kidney dysfunction.49 However, prolonged effects lead to chronic renal ischemia with irreversible changes, which include glomeulosclerosis and interstitial fibrosis.50 Renal vasospasm may lead to activation of the renin angiotensin aldosterone system, resulting in a hypervolemic state, which contributes to posttransplant hypertension.51 Another possible mechanism for CsA-induced renal hypoperfusion may involve decreased production of prostacyclin, an important regulator of renal blood flow. However, prostacyclin analogues do not seem to have a favorable impact on CsA-induced decrease in glomerular filtration.52 Recent studies suggest that CsA may promote renal production of factors that may contribute to interstitial fibrosis. In vitro studies demonstrate that CsA is able to stimulate production of insulin-like growth factor-1 by renal cultured fibroblasts.53 There is also a concomitant increase in production of the fibrogenic cytokines TGF-beta and platelet-derived growth factor.

NEWER IMMUNOSUPPRESSIVE REGIMENS

CsA has been the cornerstone of maintenance immunosuppressive therapy for 20 years. Newer agents, however, show great promise with even more effective reduction in acute rejection, improved tolerance, and decreased toxicity compared to CsA. These more effective adjunctive agents also allow a safe reduction in CsA dose. Mycophenolate mofetil is an antiproliferative agent that blocks clonal proliferation of activated T and B cells. Substitution of mycophenolate mofetil for azathioprine reduces the frequency and severity of acute rejection episodes, may also decrease the incidence of cardiac allograft vasculopathy, and has been shown to improve survival.54 Tacrolimus has a similar mode of action to CsA. Two multicenter randomized trials55,56 have compared tacrolimus to the oil-based CsA formulation (both combined with azathioprine and steroids) in heart transplant patients. The two drugs displayed similar patient survival rates and incidences of rejection, nephrotoxicity, diabetes, and infections. Tacrolimus treatment, however, was associated with a lower

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incidence of arterial hypertension and gingival hyperplasia (and, in one study, of dyslipidemia). Tacrolimus has also been effective for rescue from steroid-resistant rejection.57–59 Clinical trials are currently underway to compare tacrolimus with Neoral. Tacrolimus is currently used as primary therapy, conversion for acute rejection, or conversion for CsA toxicity.60 Sirolimus and the related agent everolimus block activation of T cells following autocrine stimulation by IL-2. Their action is therefore complementary to the calcineurin inhibitors. Sirolimus has been shown to effectively prevent acute graft rejection and inhibit refractory acute graft rejection in heart transplant recipients.61 Importantly, recent studies with both sirolimus and everolimus have shown that these agents may have a significant impact on reducing the development of transplant vasculopathy.62,63 Sirolimus has also recently been shown to allow safe withdrawal of CsA in renal transplant patients with resulting improvement in long-term renal function.64 SUMMARY

The advent of CsA 20 years ago was a major advance in the field of solid organ transplantation. Its use enabled directed immunosuppression with consequent decrease in the incidence of graft failure, acute rejection, and systemic infection. The early oil-based preparation, however, was difficult to administer and had limited bioavailability and unpredictable pharmacokinetics. The drug also has a fairly narrow therapeutic window with major long-term side effects, which include nephrotoxicity, malignancy, hyperlipidemia, and hypertension. The introduction of a microemulsion preparation (Neoral) improved bioavailability. It has been associated with lower rates of rejection with comparable tolerability and allows the use of lower doses. Traditionally CsA toxicity has been minimized with monitoring of trough levels. Recent research, however, suggests that monitoring of levels 2 hours after dosing may provide a more accurate determination of CsA exposure. The next phase in cardiac transplantation immunosuppression will most likely see a significantly diminished role for CsA with the introduction of newer, more potent immunosuppressive agents with more favorable side-effect profiles. These agents, which include mycophenolate mofetil, sirolimus, and everolimus, also hold the promise of having a major impact on the development of transplant vasculopathy, which up to now has been an important determinant of limiting long-term allograft survival. REFERENCES 1. Caves PK, Stinson EB, Griepp RB, et al: Results of 54 cardiac transplants. Surgery 74:307, 1973 2. Borel JF, Feurer C, Magnee C, et al: Effects of the new anti-lymphocytic peptide cyclosporin A in animals. Immunology 32:1017, 1977 3. Griffith BP, Hardesty RL, Deeb GM, et al: Cardiac transplantation with cyclosporin A and prednisone. Ann Surg 196:324, 1982

CARDIAC TRANSPLANT AND CYCLOSPORINE 4. Oyer P, Stinson E, Jamieson S, et al: Cyclosporine in cardiac transplantation: a 2 1/2 year follow-up. Transplant Proc 15:2546, 1983 5. Cooley D, Frazier O, Painvin G, et al: Cardiac and cardiopulmonary transplantation using cyclosporine for immunosuppression: recent Texas Heart Institute experience. Transplant Proc 15:2567, 1983 6. Wallwork J, Cory-Pearce R, English T: Cyclosporine for cardiac transplantation. Transplant Proc 15:2559, 1983 7. Cheung A, Menkis AH: Cyclosporine heart transplantation. Transplant Proc 30:1881, 1998 8. Opelz G: Multicenter evaluation of immunosuppressive regimens in heart transplantation. The Collaborative Transplant Study. Transplant Proc 29:617, 1997 9. Grattan M, Moreno-Cabral C, Starnes V, et al: Eight-year results of cyclosporine-treated patients with cardiac transplants. J Thorac Cardiovasc Surg 99:500, 1990 10. Kronke M, Leonard WJ, Depper JM, et al: Cyclosporin A inhibits T-cell growth factor gene expression at the level of mRNA transcription. Proc Nati Acad Sci U S A 81:5214, 1984 11. Herold KC, Lancki DW, Moldwin RL, et al: Immunosuppressive effects of cyclosporin A on cloned T cells. J Immunol 136:1315, 1986 12. Granelli-Piperno A: In situ hybridization for interleukin 2 and interleukin 2 receptor mRNA in T cells activated in the presence or absence of cyclosporin A. J Exp Med 168:1649, 1988 13. Shibasaki F, Price ER, Milan D, et al: Role of kinases and the phosphatase calcineurin in the nuclear shuttling of transcription factor NF-AT4. Nature 382:370, 1996 14. Rao A, Luo C, Hogan PG: Transcription factors of the NFAT family: regulation and function. Ann Rev Immunol 15:707, 1997 15. Rincon M, Flavell RA: AP-1 transcriptional activity requires both T-cell receptor-mediated and co-stimulatory signals in primary T lymphocytes. EMBO J 13:4370, 1994 16. Metcalfe S, Alexander D, Turner J: FK506 and cyclosporin A each inhibit antigen-specific signaling in the T cell line 171 in the absence of a calcium signal. Cell Immunol 158:46, 1994 17. Matsuda S, Moriguchi T, Koyasu S, et al: T lymphocyte activation signals for interleukin-2 production involve activation of MKK6-p38 and MKK7-SAPK/JNK signaling pathways sensitive to cyclosporin A. J Biol Chem 273:12378, 1998 18. Cooney GF, Jeevanandam V, Choudhury S, et al: Comparative bioavailability of Neoral and Sandimmune in cardiac transplant recipients over 1 year. Transplant Proc 30:1892, 1998 19. Eisen HJ, Hobbs RE, Davis SF, et al: Safety, tolerability, and efficacy of cyclosporine microemulsion in heart transplant recipients: a randomized, multicenter, double-blind comparison with the oil-based formulation of cyclosporine—results at 24 months after transplantation. Transplantation 71:70, 2001 20. Carrier M, White M, Pellerin M, et al: Comparison of Neoral and Sandimmune cyclosporine for induction of immunosuppression after heart transplantation. Can J Cardiol 13:469, 1997 21. Maccherini M, Bernazzali S, Diciolla F, et al: Neoral versus Sandimmun: clinical impact and modification of immunosuppressive therapy in cardiac transplantation. Transplant Proc 30:1904, 1998 22. Yonan NA, Aziz T, el-Gamel A, et al: Long-term safety and efficacy of Neoral in heart transplantation. Transplant Proc 30: 1906, 1998 23. Keevil BG, Tierney DP, Cooper DP, et al: Rapid liquid chromatography-tandem mass spectrometry method for routine analysis of cyclosporin A over an extended concentration range. Clin Chem 48:69, 2002 24. Soldin SJ, Steele BW, Witte DL, et al: Lack of specificity of cyclosporine immunoassays. Results of a College of American Pathologists Study. Arch Pathol Lab Med 127:19, 2003

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