Corticosteroids act by altering transcriptional regulation of multiple genes that affect leukocytes (T and B lymphocytes, granulocytes, macrophages, and monocytes) as well as endothelial cell function [4]. The major effect on lymphocytes is mediated by inhibition of the transcription factor activator protein 1 and nuclear factor kappa B (NF-kB), which negatively affect expression of several genes, including those controlling cytokine production, growth factors and adhesion molecules. Furthermore, steroids cause a decrease in the production of vasoactive/chemoattractant factors and lipolytic/proteolytic enzymes in non-lymphoid cells. Downstream, this results in inhibition of neutrophil adhesion to endothelial cells, prevention of macrophage differentiation, and down-regulation of endothelial function.

Glucocorticoids also exert their anti-inflammatory effects through inducing the release of lipocortin, which acts by inhibiting phospholipase A2, in turn suppressing the production of prostaglandins and leukotrienes [5, 6].

While effective at preventing rejection, steroids are associated with a significant number of long-term adverse effects. Hypertension, poor wound healing, gastric ulcers, emotional lability, cataracts, and proximal myopathy are all associated with corticosteroid therapy. Furthermore, cosmetic side-effects such as hirsutism, acne, moon facies, easy bruising, skin fragility, “buffalo hump”, and truncal obesity may also occur. From a metabolic point of view, hyperlipidemia, salt and water retention, diabetes mellitus, osteopenia, and growth retardation in children may result [6, 7]. If high-dose steroids are administered long-term, chronic adrenal suppression may result (via negative feedback mechanisms). Adrenal insufficiency may also follow a steroid taper or physiologic “stress” (illness, surgical procedures, infections).

Cyclosporine and tacrolimus both function by blocking calcium-activated calcineurin (see Fig. 10.1) [8, 9]. The agents are able to enter cells through diffusion and bind to different immunophilins: cyclosporine binds to cyclophilin and tacrolimus to FK binding protein-12 (FKBP-12). This drug-immunophilin complex proceeds to bind to calcineurin, a phosphatase that dephosphorylates multiple molecules, including nuclear factor of activated T cells (NF-AT). In turn, dephosphorylated NF-AT translocates to the nucleus, where it binds to specific DNA sites in the promoter regions of several cytokine genes, including interleukin (IL)-2. Through this series of actions, cyclosporine and tacrolimus inhibit transcription of IL-2 and other cytokines, tumor necrosis factor alpha (TNF-a), granulocyte-macrophage colony-stimulating factor, and interferon-gamma [10]. In a mechanism specific to cyclosporine, transforming growth factor-ß (TGFß) production is also stimulated, augmenting its immunosuppressive activity [11]. Furthermore, cyclosporine has been found to suppress delayed-type hypersensitivity skin reactions to tuberculin in guinea-pigs but appeared have no effects on antibody synthesis, suggesting a mechanism of immunosuppression specific to T cells.

Cyclosporine is available as oil-based or microemulsion formulations, as well as intravenous solution (for post-operative administration). Due to an improved pharmacokinetic profile and clinical data, microemulsion preparations are generally preferred over the older oil-based formulations [12]. Indeed, randomized studies comparing the two demonstrated similar survival at 2 years, but lower rates of treated rejection in the microemulsion group [13–15]. Furthermore, the microemulsion formulation exhibited better tolerance and fewer discontinuations, and allowed lower average doses of corticosteroids compared to the oil-based formulation.

Tacrolimus has become the most widely used CNI in recent years, preferred over cyclosporine. There is evidence from uncontrolled studies that tacrolimus results in lower rates of rejection and fewer adverse effects as compared to cyclosporine [16–18]. While there is no demonstrated difference in post-transplant survival between tacrolimus and oil-based cyclosporine [19, 20], randomized controlled trials show patients on tacrolimus display lower moderate-severe cellular rejection rates at 6 months compared to those on microemulsion cyclosporine [21]. Despite this, tacrolimus patients have been noted to display a higher incidence of de novo diabetes mellitus compared to microemulsion ciclosplorin.

While not an adverse effect per se, cyclosporine treatment has been previously noted to mask the clinical signs and symptoms of acute allograft rejection, making endomyocardial biopsy essential for rejection surveillance (Chap. 12).

Cyclosporine is also noted to cause acute or chronic dose-related nephrotoxicity, with the possible sequelae of arteriolar sclerosis and tubulo-interstitial fibrosis (see Table 10.2). In most patients, hypertension and hyperlipidemia tend to occur [22] and the development of de novo diabetes mellitus is fairly common. Electrolyte abnormalities are common, especially hyperkalemia, but are rarely life-threatening if renal function remains intact. Hypertrichosis, which occurs in at least 50% of patients, and gingival hyperplasia are side-effects seen with cyclosporine. Neurotoxic symptoms may also occur; such manifestations include tremor, paresthesias, headache, seizures, mental status changes, visual symptoms, and insomnia. Other possible side effects include nausea, vomiting, cholestasis/cholelithiasis, and long-term, may accelerate the development of osteoporosis (especially in combination with corticosteroids).

Tacrolimus has been noted to exhibit a similar side effect profile to cyclosporine, although the incidence of hyperlipidemia and hypertension are reduced (see Table 10.2) [19], while the incidence of hyperglycemia and neurotoxicity is relatively increased. There is some evidence to suggest that the onset of diabetes may be more common when tacrolimus is given with azathioprine compared to with mycophenolate mofetil [23]. Care must be taken in specific demographic groups, such as African-Americans and females, with regards to high tacrolimus doses and hyperglycemia [24]. In contrast to the side-effect profile seen with cyclosporine, hirsutism and gingival hypertrophy do not occur with tacrolimus. Indeed, alopecia may be a side effect of tacrolimus.

Tacrolimus is frequently used as a substitute for cyclosporine when cyclosporine-related toxic effects occur; the converse is also applicable to tacrolimus-related toxic effects [24].

The calcineurin inhibitors and proliferation signal inhibitors are extensively metabolized by the cytochrome P-450 3A4 enzyme pathway in the liver; as a result, their blood levels are affected by drugs that induce or inhibit this pathway. As a result, the nephrotoxic effects of CNIs may be enhanced. The interactions may occur with very commonly used drugs; as such, constant attention is required and vigilance as to potential interactions, and utmost care should be taken when introducing new drugs. Table 10.3 summarizes potential interactions of CNIs with common, everyday medications.

Azathioprine is a prodrug that is hydrolyzed rapidly in the blood to 6-mercaptopurine, which is subsequently converted to thioinosine monophosphate, a purine analog which is its active metabolite (see Fig. 10.1). This purine analog is incorporated into DNA, thereby inhibiting its synthesis and the consequent proliferation of both T and B lymphocytes.

The major side-effects of azathioprine are hematologic, and hence complete blood counts should be regularly monitored. Myelosuppressive adverse effects, including leukopenia, anemia, and thrombocytopenia (see Table 10.2) may occur. Generally, dose-dependent, these events typically resolve after 7–10 days with dose reduction. More rarely, pancreatitis, hepatitis, and hepatic veno-occlusive disease may also occur.

MMF is a reversible inhibitor of inosine monophosphate dehydrogenase, a crucial enzyme in the de novo synthesis of guanine nucleotides. Proliferating lymphocytes are dependent on this pathway because it is their only pathway for the purine synthesis and DNA replication; in contrast, other cells use both de novo and salvage pathways for purine synthesis. Therefore, MMF is a more selective inhibitor of lymphocyte proliferation than azathioprine. In vivo and in vitro mycophenolic acid blocks the proliferation of T and B cells, inhibits antibody formation and inhibits the generation of cytotoxic T cells [27]. Furthermore, MMF down-regulates the expression of adhesion molecules on lymphocytes.

MMF is largely preferred over azathioprine due to its reduced adverse-effect profile combined with superior efficacy in maintaining survival and preventing rejection. In a multi-center, active-controlled, randomized trial [28], MMF was compared with azathioprine when used in conjunction with cyclosporine and corticosteroids in 650 de novo heart transplant recipients. Because an intravenous form of the study drug was not available at the time of the trial, 11 percent of the patients withdrew before receiving the drug. Survival and rejection were similar in both groups when analyzed in an intention-to-treat manner. However, among treated patients, MMF was associated with a significant reduction in both mortality (6 vs. 11 percent, p = 0.031) and in the incidence of treatable rejection (66 vs. 74 percent, p = 0.026) at 1 year. These findings are supported by retrospective data from the International Society for Heart and Lung Transplantation (ISHLT) Thoracic Registry [29], which find significantly superior actuarial 1 and 3-year survival in MMF patients compared to azathioprine patients. (1 year, 96% vs. 93%; 3 year, 91% vs. 86%; p = 0.0012). MMF has also been demonstrated to be effective in reversing recurrent rejection when used in place of azathioprine [30, 31]. In patients with chronic renal dysfunction, switching from azathioprine to MMF in combination with cyclosporine reduction or withdrawal to improve renal function has also been employed as an effective strategy [32].

MMF is considerably less myelosuppressive than azathioprine and is usually well tolerated (see Table 10.2). The most common side-effects include nausea, vomiting, and diarrhea, which usually respond to dose adjustment. However, the toxicity of MMF may be more closely related to the mycophenolic acid levels than the dose itself. There is also data to suggest that the risk of opportunistic infections may be higher in patients on MMF compared with azathioprine [28].

Sirolimus and everolimus bind to the same family of immunophilins as tacrolimus, the FKBPs, but instead of blocking calcineurin-dependent T-cell activation, the resultant complex inhibits a key regulatory kinase; mammalian target of rapamycin (mTOR) (see Fig. 10.1). mTOR phosphorylates proteins that play a vital role in cell cycle regulation, in turn connecting signals from the growth factor receptors to the cell nucleus for stimulation of growth and proliferation of T and B lymphocytes [35, 36]. In this way, sirolimus/everolimus is able to specifically inhibit cell division. Notably, sirolimus has also been noted to inhibit arterial smooth muscle and endothelial cell growth via inhibition of mTOR; this has translated to reduced allograft atherosclerosis in animal models [37, 38].

Sirolimus, which was discovered before everolimus, has been shown to effectively inhibit acute graft rejection and treat refractory acute graft rejection in heart transplant recipients [39]. In randomized, open-label clinical trials, sirolimus has demonstrated reduced rejection compared to azathioprine, though with similar mortality [40]. Furthermore, sirolimus has been shown to decrease the development of cardiac allograft vasculopathy (CAV), as assessed by intravascular ultrasound (IVUS) at 6 months; the benefit was maintained at 2 years [40]. In existing patients with CAV, sirolimus was also demonstrated to slow the progression of CAV as per angiography [41]. Interestingly, sirolimus has also been noted for its antitumor effects; a useful quality in a field where a major cause of death after transplant is malignancy. In a recent study, the switch from cyclosporine to sirolimus in renal transplant recipients who subsequently developed Kaposi’s sarcoma was shown to reduce tumor burden significantly [42].

Clinical trials involving everolimus have also demonstrated largely positive results. In a randomized double-blind prospective 634-patient three-arm trial that compared everolimus (1.5 mg or 3 mg) to azathioprine [43], significantly fewer patients on everolimus reached the 6-month composite endpoint of death, graft loss or retransplantation, loss to follow-up, biopsy-proven severe acute rejection, or rejection with hemodynamic compromise (36.4% and 27.0%, compared to 46.0%). Furthermore, a decrease in the development of CAV, as assessed by intravascular ultrasound (IVUS) at 12 months, was observed in the everolimus groups compared to those on azathioprine. These study results are further supported by a recent 721-patient clinical trial in heart transplantation [44] which found no difference between everolimus and MMF in 2-year survival and rejection, and actually found a favorable effect of everolimus in reducing CAV compared to MMF [45]. Interestingly, the rates of cytomegalovirus (CMV) infection have been noted to be significantly lower in everolimus patients compared to azathioprine.

When administered alone, sirolimus/everolimus are not noted to adversely affect renal function; however, data from clinical trials shows that everolimus with low-dose cyclosporine has been shown not to worsen [46] and may even improve renal function when compared to standard-dose cyclosporine with MMF—a finding supported in multiple prospective studies [44, 46–49], including the more recent NOCTET study by Gullestad et al. and the recent SCHEDULE trial, which showed regular everolimus with no cyclosporine (with MMF) to be superior to cyclosporine with MMF for renal function.

Nevertheless, while potent effective immunosuppressive drugs, use of proliferation signal inhibitors following heart transplantation has remained limited because of evidence from clinical trials regarding worsening CNI nephrotoxicity, delayed wound healing and increased infection and dehiscence [50, 51]. Furthermore, data from the everolimus vs MMF trial showed an increased mortality from infection in the patient group with high-dose everolimus (3.0 mg) [44]. Other major adverse effects of the proliferation signal inhibitors include hyperlipidemia, hypertriglyceridemia with increased LDL cholesterol, mouth ulceration, deep venous thrombosis, proteinuria and more rarely, thrombocytopenia, neutropenia, and anemia (see Table 10.2) [52–56]. Rarely, cases of noninfectious pneumonitis have been reported with sirolimus [52].