Immunosuppressive drugs are used to dampen the immune response in organ transplantation and autoimmune disease. In transplantation, the major classes of immunosuppressive drugs used today are: (1) glucocorticoids, (2) calcineurin inhibitors, (3) antiproliferative/antimetabolic agents, and (4) biologics (antibodies).
Immunosuppressive drugs are used to dampen the immune response in organ transplantation and autoimmune disease. In transplantation, the major classes of immunosuppressive drugs used today are: (1) glucocorticoids, (2) calcineurin inhibitors, (3) antiproliferative/antimetabolic agents, and (4) biologics (antibodies). These drugs have met with a high degree of clinical success in treating conditions such as acute immune rejection of organ transplants and severe autoimmune diseases. However, such therapies require lifelong use and nonspecifically suppress the entire immune system, exposing patients to considerably higher risks of infection and cancer. The calcineurin inhibitors and glucocorticoids, in particular, are nephrotoxic and diabetogenic, respectively, thus restricting their usefulness in a variety of clinical settings.
Monoclonal and polyclonal antibody preparations directed at reactive T cells are important adjunct therapies and provide a unique opportunity to target specifically immune-reactive cells. Finally, newer small molecules and antibodies have expanded the arsenal of immunosuppressives. In particular, mTOR (mammalian target of rapamycin) inhibitors (sirolimus, everolimus) and anti-CD25 [interleukin (IL)-2 receptor] antibodies (basiliximab, daclizumab) target growth factor pathways, substantially limiting clonal expansion and thus potentially promoting tolerance. Immunosuppressive drugs used more commonly today are described below. Many more selective therapeutic agents under development are expected to revolutionize immunotherapy in the next decade.
General Approach to Organ Transplantation Therapy
Organ transplant therapy is organized around five general principles. The first principle is careful patient preparation and selection of the best available ABO blood type-compatible HLA match for organ donation. Second, a multitiered approach to immunosuppressive drug therapy, similar to that in cancer chemotherapy, is employed. Several agents are used simultaneously, each of which is directed at a different molecular target within the allograft response (Table 52-1; Hong and Kahan, 2000a). Synergistic effects permit use of the various agents at relatively low doses, thereby limiting specific toxicities while maximizing the immunosuppressive effect. The third principle is that greater immunosuppression is required to gain early engraftment and/or to treat established rejection than to maintain long-term immunosuppression. Therefore, intensive induction and lower-dose maintenance drug protocols are employed. Fourth, careful investigation of each episode of transplant dysfunction is required, including evaluation for rejection, drug toxicity, and infection, keeping in mind that these various problems can and often do coexist. Organ-specific problems (e.g., obstruction in the case of kidney transplants) must also be considered. The fifth principle, which is common to all drugs, is that a drug should be reduced or withdrawn if its toxicity exceeds its benefit.
Biologic Induction Therapy. Induction therapy with polyclonal and monoclonal antibodies (mAbs) has been an important component of immunosuppression dating back to the 1960s, when Starzl and colleagues demonstrated the beneficial effect of antilymphocyte globulin (ALG) in the prophylaxis of rejection in renal transplant recipients. Over the past 40 years, several polyclonal antilymphocyte preparations have been used in renal transplantation; however, only 2 preparations are currently FDA approved: lymphocyte immune globulin (ATGAM) and antithymocyte globulin (THYMOGLOBULIN). Another important milestone in biologic therapy was the development of mAbs and the introduction of the murine anti-CD3 mAb (muromonab-CD3 or OKT3).
In many transplant centers, induction therapy with biologic agents is used to delay the use of the nephrotoxic calcineurin inhibitors or to intensify the initial immunosuppressive therapy in patients at high risk of rejection (i.e., repeat transplants, broadly presensitized patients, African-American patients, or pediatric patients). Most of the limitations of murine-based mAbs generally were overcome by the introduction of chimeric or humanized mAbs that lack antigenicity and have prolonged serum half-lives. The anti-interleukin-2 receptor (IL-2R) mAbs (frequently referred to as anti-CD25) were the first biologics proven to be effective as induction agents in randomized double-blind prospective trials.
Biologic agents for induction therapy in the prophylaxis of rejection currently are used in approximately 70% of de novo transplant patients and have been propelled by several factors, including the introduction of the safe anti-IL-2R antibodies and the emergence of antithymocyte globulin as a safer and more effective alternative to lymphocyte immune globulin or muromonab-CD3 mAb. Biologics for induction can be divided into 2 groups: the depleting agents and the immune modulators. The depleting agents consist of lymphocyte immune globulin, antithymocyte globulin, and muromonab-CD3 mAb (the latter also produces immune modulation); their efficacy derives from their ability to deplete the recipient's CD3-positive cells at the time of transplantation and antigen presentation. The second group of biologic agents, the anti-IL-2R mAbs, do not deplete T lymphocytes, but rather block IL-2-mediated T-cell activation by binding to the chain of IL-2R.
For patients with high levels of anti-HLA antibodies, humoral rejection mediated by B cells can be modified by plasmapheresis, usually given every other day for 4 to 5 treatments followed by intravenous immunoglobulin to suppress antibody production.
Maintenance Immunotherapy. The basic immunosuppressive protocols in most transplant centers use multiple drugs simultaneously. Therapy typically involves a calcineurin inhibitor, glucocorticoids, and mycophenolate mofetil (a purine metabolism inhibitor; see below), each directed at a discrete site in T-cell activation). Glucocorticoids, azathioprine, cyclosporine, tacrolimus, mycophenolate mofetil, sirolimus, and various monoclonal and polyclonal antibodies are all approved for use in transplantation. Glucocorticoid-free regimens have achieved special prominence in recent successes in using pancreatic islet transplants to treat patients with type I diabetes mellitus. Protocols employing steroid withdrawal or steroid avoidance are being evaluated in many transplant centers. Short-term results are good, but the effects on long-term graft function are unknown. Recent data suggest that calcineurin inhibitors may shorten graft half-life by their nephrotoxic effects. Protocols under evaluation include calcineurin dose reduction or switching from calcineurin to sirolimus-based immunosuppressive therapy at 3 to 4 months.
Therapy for Established Rejection. Although low doses of prednisone, calcineurin inhibitors, purine metabolism inhibitors, or sirolimus are effective in preventing acute cellular rejection, they are less effective in blocking activated T lymphocytes, and thus are not very effective against established, acute rejection or for the total prevention of chronic rejection. Therefore, treatment of established rejection requires the use of agents directed against activated T cells. These include glucocorticoids in high doses (pulse therapy), polyclonal antilymphocyte antibodies, or muromonab-CD3 mAb.
The introduction of glucocorticoids as immunosuppressive drugs in the 1960s played a key role in making organ transplantation possible. Their chemistry, pharmacokinetics, and drug interactions are described in. Prednisone, prednisolone, and other glucocorticoids are used alone and in combination with other immunosuppressive agents for treatment of transplant rejection and autoimmune disorders.
Mechanism of Action. The immunosuppressive effects of glucocorticoids have long been known, but the specific mechanism(s) of their immunosuppressive action remains somewhat elusive. Glucocorticoids lyse (in some species) and induce the redistribution of lymphocytes, causing a rapid, transient decrease in peripheral blood lymphocyte counts. To effect longer-term responses, steroids bind to receptors inside cells; either these receptors, glucocorticoid-induced proteins, or interacting proteins regulate the transcription of numerous other genes. Additionally, glucocorticoid-receptor complexes increase IB expression, thereby curtailing activation of NF-B, which increases apoptosis of activated cells. Of central importance, key proinflammatory cytokines such as IL-1 and IL-6 are downregulated. T cells are inhibited from making IL-2 and proliferating. The activation of cytotoxic T lymphocytes is inhibited. Neutrophils and monocytes display poor chemotaxis and decreased lysosomal enzyme release. Therefore, glucocorticoids have broad antiinflammatory effects on multiple components of cellular immunity. In contrast, they have relatively little effect on humoral immunity.
Therapeutic Uses. There are numerous indications for glucocorticoids. They commonly are combined with other immunosuppressive agents to prevent and treat transplant rejection. High dose pulses of intravenous methylprednisolone sodium succinate (SOLU-MEDROL, A-METHAPRED) are used to reverse acute transplant rejection and acute exacerbations of selected autoimmune disorders. Glucocorticoids also are efficacious for treatment of graft-versus-host disease in bone-marrow transplantation. Glucocorticoids are used routinely to treat autoimmune disorders such as rheumatoid and other arthritides, systemic lupus erythematosus, systemic dermatomyositis, psoriasis and other skin conditions, asthma and other allergic disorders, inflammatory bowel disease, inflammatory ophthalmic diseases, autoimmune hematologic disorders, and acute exacerbations of multiple sclerosis (see below). In addition, glucocorticoids limit allergic reactions that occur with other immunosuppressive agents and are used in transplant recipients to block first-dose cytokine storm caused by treatment with muromonad-CD3 and to a lesser extent thymoglobulin (see below).
Toxicity. Unfortunately, the extensive use of steroids often results in disabling and life-threatening adverse effects. These effects include growth retardation in children, avascular necrosis of bone, osteopenia, increased risk of infection, poor wound healing, cataracts, hyperglycemia, and hypertension. The advent of combined glucocorticoid/cyclosporine regimens has allowed reduced doses of steroids, but steroid-induced morbidity remains a major problem in many transplant patients.
Perhaps the most effective immunosuppressive drugs in routine use are the calcineurin inhibitors, cyclosporine and tacrolimus, which target intracellular signaling pathways induced as a consequence of T-cell-receptor activation. Although they are structurally unrelated and bind to distinct, albeit related molecular targets, they inhibit normal T-cell signal transduction essentially by the same mechanism. Cyclosporine and tacrolimus do not act per se as immunosuppressive agents. Instead, these drugs bind to an immunophilin (cyclophilin for cyclosporine or FKBP-12 for tacrolimus), resulting in subsequent interaction with calcineurin to block its phosphatase activity. Calcineurin-catalyzed dephosphorylation is required for movement of a component of the nuclear factor of activated T lymphocytes (NFAT) into the nucleus (Figure 52-2). NFAT, in turn, is required to induce a number of cytokine genes, including that for interleukin-2 (IL-2), a prototypic T-cell growth and differentiation factor.
Cyclosporine. Chemistry. Cyclosporine (cyclosporin A), a cyclic polypeptide consisting of 11 amino acids, is produced by the fungus species Beauveria nivea. Of note, all amide nitrogens are either hydrogen bonded or methylated, the single D-amino acid is at position 8, the methyl amide between residues 9 and 10 is in the cis configuration, and all other methyl amide moieties are in the trans form (Figure 52-1). Because cyclosporine is lipophilic and highly hydrophobic, it is formulated for clinical administration using castor oil or other strategies to ensure solubilization.
Mechanism of Action. Cyclosporine suppresses some humoral immunity, but is more effective against T-cell-dependent immune mechanisms such as those underlying transplant rejection and some forms of autoimmunity (Kahan, 1989). It preferentially inhibits antigen-triggered signal transduction in T lymphocytes, blunting expression of many lymphokines including IL-2, and the expression of antiapoptotic proteins. Cyclosporine forms a complex with cyclophilin, a cytoplasmic receptor protein present in target cells. This complex binds to calcineurin, inhibiting Ca2+-stimulated dephosphorylation of the cytosolic component of NFAT (Schreiber and Crabtree, 1992). When cytoplasmic NFAT is dephosphorylated, it translocates to the nucleus and complexes with nuclear components required for complete T-cell activation, including transactivation of IL-2 and other lymphokine genes. Calcineurin phosphatase activity is inhibited after physical interaction with the cyclosporine/cyclophilin complex. This prevents NFAT dephosphorylation such that NFAT does not enter the nucleus, gene transcription is not activated, and the T lymphocyte fails to respond to specific antigenic stimulation. Cyclosporine also increases expression of transforming growth factor- (TGF-), a potent inhibitor of IL-2-stimulated T-cell proliferation and generation of cytotoxic T lymphocytes (CTL)
Disposition and Pharmacokinetics. Cyclosporine can be administered intravenously or orally. The intravenous preparation (SANDIMMUNE Injection) is provided as a solution in an ethanol-polyoxyethylated castor oil vehicle that must be further diluted in 0.9% sodium chloride solution or 5% dextrose solution before injection. The oral dosage forms include soft gelatin capsules and oral solutions. Cyclosporine supplied in the original soft gelatin capsule (SANDIMMUNE) is absorbed slowly with 20% to 50% bioavailability. A modified microemulsion formulation (NEORAL) is available. It has more uniform and slightly increased bioavailability compared to SANDIMMUNE and is provided as 25-mg and 100-mg soft gelatin capsules and a 100-mg/ml oral solution. Since SANDIMMUNE and NEORAL are not bioequivalent, they cannot be used interchangeably without supervision by a physician and monitoring of drug concentrations in plasma. Comparison of blood concentrations in published literature and in clinical practice must be performed with a detailed knowledge of the assay system employed. Generic preparations of both NEORAL and SANDIMMUNE are available that are bioequivalent by FDA criteria. The generic preparations for NEORAL have been shown to be bioequivalent in normal volunteers, and, in some studies, also in transplant recipients. A consensus conference held under the auspices of the American Society of Transplantation recommended that generic preparations of cyclosporine could be used de novo in transplantation to substitute for NEORAL. However, when switching between generic and NEORAL formulations, increased surveillance is recommended to ensure that drug levels remain in the therapeutic range. This need for increased monitoring is based on anecdotal experience rather than validated differences. In fact the generic preparations were comparable to NEORAL for immunosuppressive purposes in most studies. Since SANDIMMUNE and NEORAL differ in terms of their pharmacokinetics and are definitely not bioequivalent, their generic versions cannot be used interchangeably. This has been a source of confusion to pharmacists and patients. Transplant units need to educate patients that SANDIMMUNE and its generics are not the same as NEORAL and its generics, such that one preparation cannot be substituted for another without risk of inadequate immunosuppression or increased toxicity.
Both radioimmunoassays and high-performance liquid chromatography assays for cyclosporine and tacrolimus are available. Because these methods differ, the prescribing physician should ensure that the methods are consistent when monitoring an individual patient. Blood is most conveniently sampled before the next dose, namely a C0 or trough level. While this is convenient, it has been shown repeatedly that C0 concentrations do not reflect the area under the curve (AUC) for cyclosporine exposure in individual patients. As a practical solution to this problem and to better measure the overall exposure of a patient to the drug, it has been proposed that levels be taken 2 hours after a dose administration, so-called C2 levels. Some studies have shown a better correlation of C2 with the AUC, but no single time point can simulate the exposure as measured by more frequent drug sampling. In complex patients with delayed absorption, such as diabetics, the C2 level may underestimate the peak cyclosporine level obtained, and in others who are rapid absorbers the C2 level may have peaked before the blood sample is drawn. In practice if a patient has clinical signs or symptoms of toxicity, or there is unexplained rejection or renal dysfunction, a pharmacokinetic profile can be used to estimate that person's exposure to the drug. Many clinicians, particularly those caring for transplant patients some time after the transplant, monitor cyclosporine blood levels only when a clinical event (e.g., renal dysfunction or rejection) occurs. In that setting, either a C0 or C2 level helps to ascertain whether inadequate immunosuppression or drug toxicity is present. As described above, cyclosporine absorption is incomplete following oral administration and varies with the individual patient and the formulation used. The elimination of cyclosporine from the blood is generally biphasic, with a terminal half-life of 5 to 18 hours. After intravenous infusion, clearance is approximately 5 to 7 ml/min per kg in adult recipients of renal transplants, but results differ by age and patient populations. For example, clearance is slower in cardiac transplant patients and more rapid in children. Thus, the intersubject variability is so large that individual monitoring is required.
After oral administration of cyclosporine (as NEORAL), the time to peak blood concentrations is 1.5 to 2 hours (Faulds et al., 1993; Noble and Markham, 1995). Administration with food delays and decreases absorption. High- and low-fat meals consumed within 30 minutes of administration decrease the AUC by approximately 13% and the maximum concentration by 33%. This makes it imperative to individualize dosage regimens for outpatients.
Cyclosporine is distributed extensively outside the vascular compartment. After intravenous dosing, the steady-state volume of distribution is reportedly as high as 3 to 5 L/kg in solid-organ transplant recipients.
Only 0.1% of cyclosporine is excreted unchanged in urine (Faulds et al., 1993). Cyclosporine is extensively metabolized in the liver by CYP3A and to a lesser degree by the gastrointestinal tract and kidneys (Fahr, 1993). At least 25 metabolites have been identified in human bile, feces, blood, and urine (Christians and Sewing, 1993). Although the cyclic peptide structure of cyclosporine is relatively resistant to metabolism, the side chains are extensively metabolized. All of the metabolites have reduced biological activity and toxicity compared to the parent drug. Cyclosporine and its metabolites are excreted principally through the bile into the feces, with only about 6% being excreted in the urine. Cyclosporine also is excreted in human milk. In the presence of hepatic dysfunction, dosage adjustments are required. No adjustments generally are necessary for dialysis or renal failure patients.
Therapeutic Uses. Clinical indications for cyclosporine are kidney, liver, heart, and other organ transplantation; rheumatoid arthritis; and psoriasis (Faulds et al., 1993). Its use in dermatology is discussed in Chapter 62. Cyclosporine generally is recognized as the agent that ushered in the modern era of organ transplantation, increasing the rates of early engraftment, extending kidney graft survival, and making cardiac and liver transplantation possible. Cyclosporine usually is combined with other agents, especially glucocorticoids and either azathioprine or mycophenolate mofetil, and most recently, sirolimus. The dose of cyclosporine varies, depending on the organ transplanted and the other drugs used in the specific treatment protocol(s). The initial dose generally is not given before the transplant because of the concern about nephrotoxicity. Especially for renal transplant patients, therapeutic algorithms have been developed to delay cyclosporine introduction until a threshold renal function has been attained. The amount of the initial dose and reduction to maintenance dosing is sufficiently variable that no specific recommendation is provided here. Dosage is guided by signs of rejection (too low a dose), renal or other toxicity (too high a dose), and close monitoring of blood levels. Great care must be taken to differentiate renal toxicity from rejection in kidney transplant patients. Ultrasound-guided allograft biopsy is the best way to assess the reason for renal dysfunction. Because adverse reactions have been ascribed more frequently to the intravenous formulation, this route of administration is discontinued as soon as the patient is able to take the drug orally.
In rheumatoid arthritis, cyclosporine is used in severe cases that have not responded to methotrexate. Cyclosporine can be combined with methotrexate, but the levels of both drugs must be monitored closely (Baraldo et al., 1999). In psoriasis, cyclosporine is indicated for treatment of adult immunocompetent patients with severe and disabling disease for whom other systemic therapies have failed (Linden and Weinstein, 1999). Because of its mechanism of action, there is a theoretical basis for the use of cyclosporine in a variety of other T-cell-mediated diseases (Faulds et al., 1993). Cyclosporine reportedly is effective in Behcet's acute ocular syndrome, endogenous uveitis, atopic dermatitis, inflammatory bowel disease, and nephrotic syndrome, even when standard therapies have failed.
Toxicity. The principal adverse reactions to cyclosporine therapy are renal dysfunction, tremor, hirsutism, hypertension, hyperlipidemia, and gum hyperplasia (Burke et al., 1994). Hyperuricemia may lead to worsening of gout, increased P-glycoprotein activity, and hypercholesterolemia. Nephrotoxicity occurs in the majority of patients treated and is the major indication for cessation or modification of therapy. Hypertension occurs in approximately 50% of renal transplant and almost all cardiac transplant patients. Combined use of calcineurin inhibitors and glucocorticoids is particularly diabetogenic, although this apparently is more problematic in patients treated with tacrolimus (see below). Especially at risk are obese patients, African-American or Hispanic recipients, or those with family history of type II diabetes or obesity. Cyclosporine, as opposed to tacrolimus, is more likely to produce elevations in LDL cholesterol (Artz et al., 2003; Kramer et al., 2003; Tanabe, 2003).
Drug Interactions. Cyclosporine interacts with a wide variety of commonly used drugs, and close attention must be paid to drug interactions. Any drug that affects microsomal enzymes, especially the CYP3A system, may impact cyclosporine blood concentrations (Faulds et al., 1993). Substances that inhibit this enzyme can decrease cyclosporine metabolism and increase blood concentrations. These include Ca2+ channel blockers (e.g., verapamil, nicardipine), antifungal agents (e.g., fluconazole, ketoconazole), antibiotics (e.g., erythromycin), glucocorticoids (e.g., methylprednisolone), HIV-protease inhibitors (e.g., indinavir), and other drugs (e.g., allopurinol, metoclopramide). Grapefruit and grapefruit juice block CYP3A and the multidrug efflux pump and should be avoided by patients taking cyclosporine; these effects can increase cyclosporine blood concentrations. In contrast, drugs that induce CYP3A activity can increase cyclosporine metabolism and decrease blood concentrations. Such drugs include antibiotics (e.g., nafcillin, rifampin), anticonvulsants (e.g., phenobarbital, phenytoin), and other drugs (e.g., octreotide, ticlopidine). In general, close monitoring of cyclosporine blood levels and the levels of other drugs is required when such combinations are used.
Interactions between cyclosporine and sirolimus (see below) have led to the recommendation that administration of the two drugs be separated by time. Sirolimus aggravates cyclosporine-induced renal dysfunction, while cyclosporine increases sirolimus-induced hyperlipidemia and myelosuppression. Other drug interactions of concern include additive nephrotoxicity when cyclosporine is coadministered with nonsteroidal antiinflammatory drugs and other drugs that cause renal dysfunction; elevation of methotrexate levels when the two drugs are coadministered; and reduced clearance of other drugs, including prednisolone, digoxin, and statins.
Tacrolimus. Tacrolimus (PROGRAF, FK506) is a macrolide antibiotic produced by Streptomyces tsukubaensis (Goto et al., 1987). Its formula is shown in Figure 52-1.
Mechanism of Action. Like cyclosporine, tacrolimus inhibits T-cell activation by inhibiting calcineurin (Schreiber and Crabtree, 1992). Tacrolimus binds to an intracellular protein, FK506-binding protein-12 (FKBP-12), an immunophilin structurally related to cyclophilin. A complex of tacrolimus-FKBP-12, Ca2+, calmodulin, and calcineurin then forms, and calcineurin phosphatase activity is inhibited. As described for cyclosporine and depicted in Figure 52-2, the inhibition of phosphatase activity prevents dephosphorylation and nuclear translocation of NFAT and inhibits T-cell activation. Thus, although the intracellular receptors differ, cyclosporine and tacrolimus target the same pathway for immunosuppression (Plosker and Foster, 2000).
Disposition and Pharmacokinetics. Tacrolimus is available for oral administration as capsules (0.5, 1, and 5 mg) and as a sterile solution for injection (5 mg/ml). Immunosuppressive activity resides primarily in the parent drug. Because of intersubject variability in pharmacokinetics, individualized dosing is required for optimal therapy (Fung and Starzl, 1995). Whole blood, rather than plasma, is the most appropriate sampling compartment to describe tacrolimus pharmacokinetics. For tacrolimus, the C0 level seems to correlate better with clinical events than it does for cyclosporine. Target concentrations in many centers are 200 to 400 ng/ml in the early preoperative period and 100 to 200 ng/ml 3 months after transplantation. Unlike cyclosporine, more frequent tacrolimus dosing has not been formally evaluated. Gastrointestinal absorption is incomplete and variable. Food decreases the rate and extent of absorption. Plasma protein binding of tacrolimus is 75% to 99%, involving primarily albumin and 1-acid glycoprotein. Its half-life is about 12 hours. Tacrolimus is extensively metabolized in the liver by CYP3A, with a half-life of ~12 hours; at least some of the metabolites are active. The bulk of excretion of the parent drug and metabolites is in the feces. Less than 1% of administered tacrolimus is excreted unchanged in the urine.
Therapeutic Uses. Tacrolimus is indicated for the prophylaxis of solid-organ allograft rejection in a manner similar to cyclosporine and as rescue therapy in patients with rejection episodes despite "therapeutic" levels of cyclosporine (Mayer et al., 1997; The U.S. Multicenter FK506 Liver Study Group, 1994). The recommended starting dose for tacrolimus injection is 0.03 to 0.05 mg/kg per day as a continuous infusion. Recommended initial oral doses are 0.15 to 0.2 mg/kg per day for adult kidney transplant patients, 0.1 to 0.15 mg/kg per day for adult liver transplant patients, and 0.15 to 0.2 mg/kg per day for pediatric liver transplant patients in two divided doses 12 hours apart. These dosages are intended to achieve typical blood trough levels in the 5- to 15-ng/ml range. Pediatric patients generally require higher doses than do adults (Shapiro, 1998).
Toxicity. Nephrotoxicity, neurotoxicity (tremor, headache, motor disturbances, seizures), GI complaints, hypertension, hyperkalemia, hyperglycemia, and diabetes are all associated with tacrolimus use (Plosker and Foster, 2000). As with cyclosporine, nephrotoxicity is limiting (Mihatsch et al., 1998; Henry, 1999). Tacrolimus has a negative effect on pancreatic islet beta cells, and glucose intolerance and diabetes mellitus are well-recognized complications of tacrolimus-based immunosuppression. As with other immunosuppressive agents, there is an increased risk of secondary tumors and opportunistic infections. Notably, tacrolimus does not adversely affect uric acid or LDL cholesterol.
Drug Interactions. Because of its potential for nephrotoxicity, tacrolimus blood levels and renal function should be monitored closely, especially when tacrolimus is used with other potentially nephrotoxic drugs. Coadministration with cyclosporine results in additive or synergistic nephrotoxicity; therefore a delay of at least 24 hours is required when switching a patient from cyclosporine to tacrolimus. Since tacrolimus is metabolized mainly by CYP3A, the potential interactions described above for cyclosporine also apply for tacrolimus (Venkataramanan et al., 1995; Yoshimura et al., 1999).
Antiproliferative and Antimetabolic Drugs
Sirolimus. Sirolimus (rapamycin; RAPAMUNE) is a macrocyclic lactone produced by Streptomyces hygroscopicus (Vezina et al., 1975). Its structure is shown in Figure 52-1.
Mechanism of Action. Sirolimus inhibits T-lymphocyte activation and proliferation downstream of the IL-2 and other T-cell growth factor receptors (Figure 52-2) (Kuo et al., 1992). Like cyclosporine and tacrolimus, therapeutic action of sirolimus requires formation of a complex with an immunophilin, in this case FKBP-12. However, the sirolimus-FKBP-12 complex does not affect calcineurin activity. It binds to and inhibits a protein kinase, designated mammalian target of rapamycin (mTOR), which is a key enzyme in cell-cycle progression (Brown et al., 1994). Inhibition of mTOR blocks cell-cycle progression at the G1 S phase transition. In animal models, sirolimus not only inhibits transplant rejection, graft-versus-host disease, and a variety of autoimmune diseases, but its effect also lasts several months after discontinuing therapy, suggesting a tolerizing effect (see Tolerance, below; Groth et al., 1999). A newer indication for sirolimus is the avoidance of calcineurin inhibitors, even when patients are stable, to protect kidney function (Stegall et al., 2003).
Disposition and Pharmacokinetics. After oral administration, sirolimus is absorbed rapidly and reaches a peak blood concentration within about 1 hour after a single dose in healthy subjects and within about 2 hours after multiple oral doses in renal transplant patients (Napoli and Kahan, 1996; Zimmerman and Kahan, 1997). Systemic availability is approximately 15%, and blood concentrations are proportional to doses between 3 and 12 mg/m2. A high-fat meal decreases peak blood concentration by 34%; sirolimus therefore should be taken consistently either with or without food, and blood levels should be monitored closely. About 40% of sirolimus in plasma is protein bound, especially to albumin. The drug partitions into formed elements of blood, with a blood-to-plasma ratio of 38 in renal transplant patients. Sirolimus is extensively metabolized by CYP3A4 and is transported by P-glycoprotein. Seven major metabolites have been identified in whole blood (Salm et al., 1999). Metabolites also are detectable in feces and urine, with the bulk of total excretion being in feces. Although some of its metabolites are active, sirolimus itself is the major active component in whole blood and contributes more than 90% of the immunosuppressive effect. The blood half-life after multiple doses in stable renal transplant patients is 62 hours (Napoli and Kahan, 1996; Zimmerman and Kahan, 1997). A loading dose of three times the maintenance dose will provide nearly steady-state concentrations within 1 day in most patients.
Therapeutic Uses. Sirolimus is indicated for prophylaxis of organ transplant rejection in combination with a calcineurin inhibitor and glucocorticoids (Kahan et al., 1999a). In patients experiencing or at high risk for calcineurin inhibitor-associated nephrotoxicity, sirolimus has been used with glucocorticoids and mycophenolate mofetil to avoid permanent renal damage. The initial dosage in patients 13 years or older who weigh less than 40 kg should be adjusted based on body surface area (1 mg/m2 per day) with a loading dose of 3 mg/m2. Data regarding doses for pediatric and geriatric patients are lacking at this time (Kahan, 1999). It is recommended that the maintenance dose be reduced by approximately one-third in patients with hepatic impairment (Watson et al., 1999). Sirolimus also has been incorporated into stents to inhibit local cell proliferation and blood vessel occlusion.
Toxicity. The use of sirolimus in renal transplant patients is associated with a dose-dependent increase in serum cholesterol and triglycerides that may require treatment (Murgia et al., 1996). While immunotherapy with sirolimus per se is not nephrotoxic, patients treated with cyclosporine plus sirolimus have impaired renal function compared to patients treated with cyclosporine and either azathioprine or placebo. Sirolimus also may prolong delayed graft function in deceased donor kidney transplants, presumably because of its antiproliferative action (Smith et al., 2003; McTaggart et al., 2003). Renal function therefore must be monitored closely in such patients. Lymphocele, a known surgical complication associated with renal transplantation, is increased in a dose-dependent fashion by sirolimus, requiring close postoperative follow-up. Other adverse effects include anemia, leukopenia, thrombocytopenia (Hong and Kahan, 2000b), hypokalemia or hyperkalemia, fever, and gastrointestinal effects. Delayed wound healing may occur with sirolimus use. As with other immunosuppressive agents, there is an increased risk of neoplasms, especially lymphomas, and infections. Prophylaxis for Pneumocystis carinii pneumonia and cytomegalovirus is recommended (Groth et al., 1999).
Drug Interactions. Since sirolimus is a substrate for CYP3A4 and is transported by P-glycoprotein, close attention to interactions with other drugs that are metabolized or transported by these proteins is required (Yoshimura et al., 1999). As noted above, cyclosporine and sirolimus interact, and their administration should be separated by time. Dose adjustment may be required when sirolimus is coadministered with diltiazem or rifampin. The combination of sirolimus plus tacrolimus probably is more nephrotoxic than cyclosporine plus sirolimus. Dose adjustment apparently is not required when sirolimus is coadministered with acyclovir, digoxin, glyburide, nifedipine, norgestrel/ethinyl estradiol, prednisolone, or trimethoprim-sulfamethoxazole. This list is incomplete, and blood levels and potential drug interactions must be monitored closely.
Everolimus. Everolimus (40-0-[2-hydroxy] ethyl-rapamycin) is closely related chemically and clinically to sirolimus but has distinct pharmacokinetics. The main difference is a shorter half-life and thus a shorter time to achieve steady-state concentrations of the drug. Dosage on a milligram per kilogram basis is similar to sirolimus. Aside from the shorter half-life, no studies have compared everolimus with sirolimus in standard immunosuppressive regimens (Eisen et al., 2003). As with sirolimus, the combination of a calcineurin inhibitor and an mTOR inhibitor produces worse renal function at 1 year than does calcineurin inhibitor therapy alone, suggesting a drug interaction between the mTOR inhibitors and the calcineurin inhibitors to enhance toxicity and to reduce rejection. The toxicity of everolimus and the drug interactions reported to date seem to be the same as with sirolimus.
Azathioprine. Azathioprine (IMURAN) is a purine antimetabolite. It is an imidazolyl derivative of 6-mercaptopurine (Figure 52-1).
Mechanism of Action. Following exposure to nucleophiles such as glutathione, azathioprine is cleaved to 6-mercaptopurine, which in turn is converted to additional metabolites that inhibit de novo purine synthesis (see Chapter 51). 6-Thio-IMP, a fraudulent nucleotide, is converted to 6-thio-GMP and finally to 6-thio-GTP, which is incorporated into DNA. Cell proliferation is thereby inhibited, impairing a variety of lymphocyte functions. Azathioprine appears to be a more potent immunosuppressive agent than 6-mercaptopurine, which may reflect differences in drug uptake or pharmacokinetic differences in the resulting metabolites.
Disposition and Pharmacokinetics. Azathioprine is well absorbed orally and reaches maximum blood levels within 1 to 2 hours after administration. The half-life of azathioprine is about 10 minutes, while that of its metabolite 6-mercaptopurine is about an hour. Other metabolites have half-lives of up to 5 hours. Blood levels have limited predictive value because of extensive metabolism, significant activity of many different metabolites, and high tissue levels attained. Azathioprine and mercaptopurine are moderately bound to plasma proteins and are partially dialyzable. Both are rapidly removed from the blood by oxidation or methylation in the liver and/or erythrocytes. Renal clearance has little impact on biological effectiveness or toxicity, but the dose should be reduced in patients with renal failure.
Therapeutic Uses. Azathioprine was first introduced as an immunosuppressive agent in 1961, helping to make allogeneic kidney transplantation possible. It is indicated as an adjunct for prevention of organ transplant rejection and in severe rheumatoid arthritis (Hong and Kahan, 2000a; Gaffney and Scott, 1998). Although the dose of azathioprine required to prevent organ rejection and minimize toxicity varies, 3 to 5 mg/kg per day is the usual starting dose. Lower initial doses (1 mg/kg per day) are used in treating rheumatoid arthritis. Complete blood count and liver function tests should be monitored.
Toxicity. The major side effect of azathioprine is bone marrow suppression, including leukopenia (common), thrombocytopenia (less common), and/or anemia (uncommon). Other important adverse effects include increased susceptibility to infections (especially varicella and herpes simplex viruses), hepatotoxicity, alopecia, GI toxicity, pancreatitis, and increased risk of neoplasia.
Drug Interactions. Xanthine oxidase, an enzyme of major importance in the catabolism of azathioprine metabolites, is blocked by allopurinol. If azathioprine and allopurinol are used concurrently, the azathioprine dose must be decreased to 25% to 33% of the usual dose; it is best not to use these two drugs together. Adverse effects resulting from coadministration of azathioprine with other myelosuppressive agents or angiotensin-converting enzyme inhibitors include leukopenia, thrombocytopenia, and anemia as a result of myelosuppression.
Mycophenolate Mofetil. Mycophenolate mofetil (CELLCEPT) is the 2-morpholinoethyl ester of mycophenolic acid (MPA) (Allison and Eugui, 1993). Its structure is shown in Figure 52-1.
Mechanism of Action. Mycophenolate mofetil is a prodrug that is rapidly hydrolyzed to the active drug, mycophenolic acid (MPA), a selective, noncompetitive, and reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH) (Natsumeda and Carr, 1993), an important enzyme in the de novo pathway of guanine nucleotide synthesis. B and T lymphocytes are highly dependent on this pathway for cell proliferation, while other cell types can use salvage pathways; MPA therefore selectively inhibits lymphocyte proliferation and functions, including antibody formation, cellular adhesion, and migration.
Disposition and Pharmacokinetics. Mycophenolate mofetil undergoes rapid and complete metabolism to MPA after oral or intravenous administration. MPA, in turn, is metabolized to the inactive phenolic glucuronide MPAG. The parent drug is cleared from the blood within a few minutes. The half-life of MPA is about 16 hours. Negligible (1%) amounts of MPA are excreted in the urine (Bardsley-Elliot et al., 1999). Most (87%) is excreted in the urine as MPAG. Plasma concentrations of MPA and MPAG are increased in patients with renal insufficiency. In early renal transplant patients (40 days posttransplant), plasma concentrations of MPA after a single dose of mycophenolate mofetil are about half of those found in healthy volunteers or stable renal transplant patients. Studies in children are limited, and safety and effectiveness have not been established (Butani et al., 1999).
Therapeutic Uses. Mycophenolate mofetil is indicated for prophylaxis of transplant rejection, and it typically is used in combination with glucocorticoids and a calcineurin inhibitor, but not with azathioprine (Kimball et al., 1995; Ahsan et al., 1999; Kreis et al., 2000). Combined treatment with sirolimus is possible, although potential drug interactions necessitate careful monitoring of drug levels. For renal transplants, 1 g is administered orally or intravenously (over 2 hours) twice daily (2 g per day). A higher dose, 1.5 g twice daily (3 g per day), is recommended for African-American renal transplant patients and all cardiac transplant patients. Use of mycophenolate mofetil in other clinical settings is under investigation.
Toxicity. The principal toxicities of mycophenolate mofetil are gastrointestinal and hematologic (Fulton and Markham, 1996; Bardsley-Elliot et al., 1999). These include leukopenia, diarrhea, and vomiting. There also is an increased incidence of some infections, especially sepsis associated with cytomegalovirus. Tacrolimus in combination with mycophenolate mofetil has been associated with devastating viral infections including polyoma nephritis (Zavos et al., 2004; Elli et al., 2002).
Drug Interactions. Potential drug interactions between mycophenolate mofetil and several other drugs commonly used by transplant patients have been studied (Bardsley-Elliot et al., 1999). There appear to be no untoward effects produced by combination therapy with cyclosporine, trimethoprim-sulfamethoxazole, or oral contraceptives. Unlike cyclosporine, tacrolimus delays elimination of mycophenolate mofetil by impairing the conversion of MPA to MPAG. This may enhance GI toxicity. Mycophenolate mofetil has not been tested with azathioprine. Coadministration with antacids containing aluminum or magnesium hydroxide leads to decreased absorption of mycophenolate mofetil; thus, these drugs should not be administered simultaneously. Mycophenolate mofetil should not be administered with cholestyramine or other drugs that affect enterohepatic circulation. Such agents decrease plasma MPA concentrations, probably by binding free MPA in the intestines. Acyclovir and ganciclovir may compete with MPAG for tubular secretion, possibly resulting in increased concentrations of both MPAG and the antiviral agents in the blood, an effect that may be compounded in patients with renal insufficiency.
Other Antiproliferative and Cytotoxic Agents. Many of the cytotoxic and antimetabolic agents used in cancer chemotherapy (see Chapter 51) are immunosuppressive due to their action on lymphocytes and other cells of the immune system. Other cytotoxic drugs that have been used as immunosuppressive agents include methotrexate, cyclophosphamide (CYTOXAN), thalidomide, and chlorambucil (LEUKERAN). Methotrexate is used for treatment of graft-versus-host disease, rheumatoid arthritis, and psoriasis, as well as in anticancer therapy (see Chapter 51) (Grosflam and Weinblatt, 1991). Cyclophosphamide and chlorambucil are used in treating childhood nephrotic syndrome (Neuhaus et al., 1994) and a variety of malignancies (see Chapter 51). Cyclophosphamide also is used widely for treatment of severe systemic lupus erythematosus (Valeri et al., 1994) and other vasculitides such as Wegener's granulomatosis. Leflunomide (ARAVA) is a pyrimidine-synthesis inhibitor indicated for the treatment of adults with rheumatoid arthritis (Prakash and Jarvis, 1999). This drug has found utility in the treatment of polyomavirus nephropathy seen in immunosuppressed renal transplant recipients and is increasingly being used for that purpose. There are no studies showing efficacy, however, compared with control patients treated with withdrawal or reduction of immunosuppression alone in BK virus nephropathy. The drug inhibits dihydroorotate dehydrogenase in the de novo pathway of pyrimidine synthesis. It is hepatotoxic and can cause fetal injury when administered to pregnant women.
FTY720 (see Figure 52-1), an S1P receptor prodrug, is the first agent in a new class of small molecules, sphingosine 1-phosphate receptor (S1P-R) agonists, which reduce recirculation of lymphocytes from the lymphatic system to the blood and peripheral tissues, including inflammatory lesions and organ grafts.
Therapeutic Uses. FTY720 may have an important role in combination immunosuppression therapy in the prevention of acute rejection. FTY720 is not effective as monotherapy; it has demonstrated efficacy in clinical trials with cyclosporine and prednisone or in combination with the mTOR inhibitor everolimus and steroids. In Phase II trials with cyclosporine, FTY720 at daily doses of 2.5 and 5 mg showed comparable efficacy in the prevention of acute rejection in de novo renal transplant patients when compared to immunosuppression with cyclosporine, mycophenolate mofetil, and prednisone. FTY720 now is in Phase III trials.
Mechanism of Action. Unlike other immunosuppressive agents, FTY720 acts via "lymphocyte homing." It specifically and reversibly sequesters host lymphocytes into the lymph nodes and Peyer's patches, and thus away from the circulation. This protects the graft from T-cell-mediated attack. FTY720 sequesters lymphocytes but does not impair either T or B cell functions. FTY720 is phosphorylated by sphingosine kinase-2 and the FTY720-phosphate product is a potent agonist of S1P receptors. Altered lymphocyte traffic induced by FTY720 clearly results from its effect on S1P receptors.
Toxicity. Lymphopenia, the most common side effect of FTY720, is predicted from its pharmacologic effect and is fully reversible upon drug discontinuation. Of greater concern is the negative chronotropic effect of FTY720 on the heart, which has been observed with the first dose of FTY720 in up to 30% of patients. In most patients, the heart rate returns to baseline within 48 hours after the administration of the first dose of FTY720, with the remainder returning to baseline thereafter. The negative chronotropic effect of FTY720 likely is related to the presence of S1P-R on human atrial myocytes, thus affecting S1P signaling pathways. This effect can be functionally antagonized by 1-receptor agonists or by the muscarinic receptor antagonist atropine. Importantly, cardiac rhythm was not affected in patients treated chronically with FTY720; however, the safety of the first-dose response of FTY720 on the heart rate in patients with underlying coronary artery disease remains to be determined.
Both polyclonal and monoclonal antibodies against lymphocyte cell-surface antigens are widely used for prevention and treatment of organ transplant rejection. Polyclonal antisera are generated by repeated injections of human thymocytes (antithymocyte globulin, ATG) or lymphocytes (antilymphocyte globulin, ALG) into animals such as horses, rabbits, sheep, or goats, and then purifying the serum immunoglobulin fraction. Although highly effective immunosuppressive agents, these preparations vary in efficacy and toxicity from batch to batch. The advent of hybridoma technology to produce monoclonal antibodies was a major advance in immunology (Kohler and Milstein, 1975). It is now possible to make essentially unlimited amounts of a single antibody of a defined specificity (Figure 52-3). These monoclonal reagents have overcome the problems of variability in efficacy and toxicity seen with the polyclonal products, but they are more limited in their target specificity. The first-generation murine monoclonal antibodies have been replaced by newer chimeric or humanized monoclonal antibodies that lack antigenicity, have prolonged half-lives, and can be mutagenized to alter their affinity to Fc receptors. Thus, both polyclonal and monoclonal products have a place in immunosuppressive therapy.
Antithymocyte Globulin. Antithymocyte globulin is a purified gamma globulin from the serum of rabbits immunized with human thymocytes (Regan et al., 1999). It is provided as a sterile, freeze-dried product for intravenous administration after reconstitution with sterile water.
Mechanism of Action. Antithymocyte globulin contains cytotoxic antibodies that bind to CD2, CD3, CD4, CD8, CD11a, CD18, CD25, CD44, CD45, and HLA class I and II molecules on the surface of human T lymphocytes (Bourdage and Hamlin, 1995). The antibodies deplete circulating lymphocytes by direct cytotoxicity (both complement and cell-mediated) and block lymphocyte function by binding to cell surface molecules involved in the regulation of cell function.
Therapeutic Uses. Antithymocyte globulin is used for induction immunosuppression, although the only approved indication is in the treatment of acute renal transplant rejection in combination with other immunosuppressive agents (Mariat et al., 1998). Antilymphocyte-depleting agents (THYMOGLOBULIN, ATGAM, and OKT3) have been neither rigorously tested in clinical trials nor registered for use as induction immunosuppression. However, a meta-analysis (Szczech et al., 1997) showed that antilymphocyte induction improves graft survival. A course of antithymocyte-globulin treatment often is given to renal transplant patients with delayed graft function to avoid early treatment with the nephrotoxic calcineurin inhibitors and thereby aid in recovery from ischemic reperfusion injury. The recommended dose for acute rejection of renal grafts is 1.5 mg/kg per day (over 4 to 6 hours) for 7 to 14 days. Mean T-cell counts fall by day 2 of therapy. Antithymocyte globulin also is used for acute rejection of other types of organ transplants and for prophylaxis of rejection (Wall, 1999).
Toxicity. Polyclonal antibodies are xenogeneic proteins that can elicit major side effects, including fever and chills with the potential for hypotension. Premedication with corticosteroids, acetaminophen, and/or an antihistamine and administration of the antiserum by slow infusion (over 4 to 6 hours) into a large-diameter vessel minimize such reactions. Serum sickness and glomerulonephritis can occur; anaphylaxis is a rare event. Hematologic complications include leukopenia and thrombocytopenia. As with other immunosuppressive agents, there is an increased risk of infection and malignancy, especially when multiple immunosuppressive agents are combined. No drug interactions have been described; anti-ATG antibodies develop, although they do not limit repeated use.
Monoclonal Antibodies. Anti-CD3 Monoclonal Antibodies. Antibodies directed at the chain of CD3, a trimeric molecule adjacent to the T-cell receptor on the surface of human T lymphocytes, have been used with considerable efficacy since the early 1980s in human transplantation. The original mouse IgG2a antihuman CD3 monoclonal antibody, muromonab-CD3 (OKT3, ORTHOCLONE OKT3), still is used to reverse glucocorticoid-resistant rejection episodes (Cosimi, et al., 1981).
Mechanism of Action. Muromonab-CD3 binds to the chain of CD3, a monomorphic component of the T-cell receptor complex involved in antigen recognition, cell signaling, and proliferation (Hooks et al., 1991). Antibody treatment induces rapid internalization of the T-cell receptor, thereby preventing subsequent antigen recognition. Administration of the antibody is followed rapidly by depletion and extravasation of a majority of T cells from the bloodstream and peripheral lymphoid organs such as lymph nodes and spleen. This absence of detectable T cells from the usual lymphoid regions is secondary both to cell death following complement activation and activation-induced cell death and to margination of T cells onto vascular endothelial walls and redistribution of T cells to nonlymphoid organs such as the lungs. Muromonab-CD3 also reduces function of the remaining T cells, as defined by lack of IL-2 production and great reduction in the production of multiple cytokines, perhaps with the exception of IL-4 and IL-10.
Therapeutic Uses Muromonab-CD3 is indicated for treatment of acute organ transplant rejection (Ortho Multicenter Transplant Study Group, 1985; Woodle et al., 1999; Rostaing et al., 1999). Muromonab-CD3 is provided as a sterile solution containing 5 mg per ampule. The recommended dose is 5 mg/day (in adults; less for children) in a single intravenous bolus (less than 1 minute) for 10 to 14 days. Antibody levels increase over the first 3 days and then plateau. Circulating T cells disappear from the blood within minutes of administration and return within approximately 1 week after termination of therapy. Repeated use of muromonab-CD3 results in the immunization of the patient against the mouse determinants of the antibody, which can neutralize and prevent its immunosuppressive efficacy (Jaffers et al., 1983). Thus, repeated treatment with the muromonab-CD3 or other mouse monoclonal antibodies generally is contraindicated. The use of muromonab-CD3 for induction and rejection therapy has diminished substantially in the past 5 years because of its toxicity and the availability of antithymocyte globulin.
Toxicity The major side effect of anti-CD3 therapy is the "cytokine release syndrome" (Wilde and Goa, 1996; Ortho Multicenter Transplant Study Group, 1985). The syndrome typically begins 30 minutes after infusion of the antibody (but can occur later) and may persist for hours. Antibody binding to the T-cell receptor complex combined with Fc receptor (FcR)-mediated crosslinking is the basis for the initial activating properties of this agent. The syndrome is associated with and attributed to increased serum levels of cytokines (including tumor necrosis factor [TNF]-, IL-2, IL-6, and interferon-), which are released by activated T cells and/or monocytes. In several studies, the production of TNF- has been shown to be the major cause of the toxicity (Herbelin et al., 1995). The symptoms usually are worst with the first dose; frequency and severity decrease with subsequent doses. Common clinical manifestations include high fever, chills/rigor, headache, tremor, nausea/vomiting, diarrhea, abdominal pain, malaise, myalgias, arthralgias, and generalized weakness. Less common complaints include skin reactions and cardiorespiratory and CNS disorders, including aseptic meningitis. Potentially fatal severe pulmonary edema, acute respiratory distress syndrome, cardiovascular collapse, cardiac arrest, and arrhythmias have been described.
Administration of glucocorticoids before the injection of muromonab-CD3 prevents the release of cytokines and reduces first-dose reactions considerably and is now a standard procedure. Volume status of patients also must be monitored carefully before therapy; steroids and other premedications should be given, and a fully competent resuscitation facility must be immediately available for patients receiving their first several doses of this therapy.
Other toxicities associated with anti-CD3 therapy include anaphylaxis and the usual infections and neoplasms associated with immunosuppressive therapy. "Rebound" rejection has been observed when muromonab-CD3 treatment is stopped (Wilde and Goa, 1996). Anti-CD3 therapies may be limited by anti-idiotypic or anti-murine antibodies in the recipient.
New-Generation Anti-CD3 Antibodies. Recently, genetically altered anti-CD3 monoclonal antibodies have been developed that are "humanized" to minimize the occurrence of antiantibody responses and mutated to prevent binding to FcRs (Friend et al., 1999). The rationale for developing this new generation of anti-CD3 monoclonal antibodies is that they could induce selective immunomodulation in the absence of toxicity associated with conventional anti-CD3 monoclonal antibody therapy. In initial clinical trials, a humanized anti-CD3 monoclonal antibody that does not bind to FcRs reversed acute renal allograft rejection without causing the first-dose cytokine-release syndrome (Woodle et al., 1999). Clinical efficacy of these agents in autoimmune diseases is being evaluated (Herold et al., 2002).
Anti-IL-2 Receptor (Anti-CD25) Antibodies. Daclizumab (ZENAPAX), a humanized murine complementarity-determining region (CDR)/human IgG1 chimeric monoclonal antibody, and basiliximab (SIMULECT), a murine-human chimeric monoclonal antibody, have been produced by recombinant DNA technology (Wiseman and Faulds, 1999). The composite daclizumab antibody consists of human (90%) constant domains of IgG1 and variable framework regions of the Eu myeloma antibody and murine (10%) CDR of the anti-Tac antibody.
Mechanism of Action. Daclizumab has a somewhat lower affinity than does basiliximab, but a longer half-life (20 days). The exact mechanism of action of the anti-CD25 mAbs is not completely understood, but likely results from the binding of the anti-CD25 mAbs to the IL-2 receptor on the surface of activated, but not resting, T cells (Vincenti et al., 1998; Amlot et al., 1995). Significant depletion of T cells does not appear to play a major role in the mechanism of action of these mAbs. However, other mechanisms of action may mediate the effect of these antibodies. In a study of daclizumab-treated patients, there was a moderate decrease in circulating lymphocytes staining with 7G7, a fluorescein-conjugated antibody that binds a different -chain epitope than that recognized and bound by daclizumab (Vincenti et al., 1998). Similar results were obtained in studies with basiliximab (Amlot et al., 1995). These findings indicate that therapy with the anti IL-2R mAbs results in a relative decrease of the expression of the chain, either from depletion of coated lymphocytes or modulation of the chain secondary to decreased expression or increased shedding. There is also recent evidence that the chain may be down-regulated by the anti-CD25 antibody.
Therapeutic Uses Anti-IL-2-receptor monoclonal antibodies are used for prophylaxis of acute organ rejection in adult patients. There are two anti-IL-2R preparations for use in clinical transplantation: daclizumab and basiliximab (Vincenti et al., 1998; Nashan et al., 1999). In Phase III trials, daclizumab was administered in five doses (1 mg/kg given intravenously over 15 minutes in 50 ml to 100 ml of normal saline) starting immediately preoperatively, and subsequently at biweekly intervals. The half-life of daclizumab was 20 days, resulting in saturation of the IL-2R on circulating lymphocytes for up to 120 days after transplantation. In these trials, daclizumab was used with maintenance immunosuppression regimens (cyclosporine, azathioprine, and steroids; cyclosporine and steroids). Subsequently, daclizumab was successfully used with a maintenance triple-therapy regimeneither with cyclosporine or tacrolimus, steroids, and mycophenolate mofetil (MMF) substituting for azathioprine (Ciancio et al., 2003; Pescovitz et al., 2003). In Phase III trials, basiliximab was administered in a fixed dose of 20 mg preoperatively and on days 0 and 4 after transplantation (Nashan et al., 1997; Kahan et al., 1999a). This regimen of basiliximab resulted in a concentration of 0.2 g/mL, sufficient to saturate IL-2R on circulating lymphocytes for 25 to 35 days after transplantation. The half-life of basiliximab was 7 days. In the Phase III trials, basiliximab was used with a maintenance regimen consisting of cyclosporine and prednisone. In one randomized trial, basiliximab was safe and effective when used in a maintenance regimen consisting of cyclosporine, MMF, and prednisone (Lawen et al., 2000).
There presently is no marker or test to monitor the effectiveness of anti-IL-2R therapy. Saturation of chain on circulating lymphocytes during anti-IL-2R mAb therapy does not predict rejection. The duration of IL-2R blockade by basiliximab was similar in patients with or without acute rejection episodes (34 14 days vs. 37 14 days, mean + SD) (Kovarik et al., 1999). In another daclizumab trial, patients with acute rejection were found to have circulating and intragraft lymphocytes with saturated IL-2R (Vincenti et al., 2001). A possible explanation is that those patients who reject despite anti-IL-2R blockade do so through a mechanism that bypasses the IL-2 pathway due to cytokine-cytokine receptor redundancy (i.e., IL-7, IL-15).
Toxicity No cytokine-release syndrome has been observed with these antibodies, but anaphylactic reactions can occur. Although lymphoproliferative disorders and opportunistic infections may occur, as with the depleting antilymphocyte agents, the incidence ascribed to anti-CD25 treatment appears remarkably low. No significant drug interactions with anti-IL-2-receptor antibodies have been described (Hong and Kahan, 1999).
Campath-1H. Campath-1H (ALEMTUZUMAB) is a humanized mAb that has been approved for use in chronic lymphocytic leukemia. The antibody targets CD52, a glycoprotein expressed on lymphocytes, monocytes, macrophages, and natural killer cells; thus, the drug causes extensive lympholysis by inducing apoptosis of targeted cells. It has achieved some use in renal transplantation because it produces prolonged T- and B-cell depletion and allows drug minimization. Large controlled studies of efficacy or safety are not available. Although short-term results are promising, further clinical experience is needed before Campath-1H is accepted into the clinical armamentarium for transplantation.
Infliximab Infliximab (REMICADE) is a chimeric anti-TNF- monoclonal antibody containing a human constant region and a murine variable region. It binds with high affinity to TNF- and prevents the cytokine from binding to its receptors.
Patients with rheumatoid arthritis have elevated levels of TNF- in their joints, while patients with Crohn's disease have elevated levels of TNF- in their stools. In one trial, infliximab plus methotrexate improved the signs and symptoms of rheumatoid arthritis more than methotrexate alone. Patients with active Crohn's disease who had not responded to other immunosuppressive therapies also improved when treated with infliximab, including those with Crohn's-related fistulae. Infliximab is approved in the United States for treating the symptoms of rheumatoid arthritis, and is used in combination with methotrexate in patients who do not respond to methotrexate alone. Infliximab also is approved for treatment of symptoms of moderate to severe Crohn's disease in patients who have failed to respond to conventional therapy, and in treatment to reduce the number of draining fistulae in Crohn's disease patients (see Chapter 38). About 1 of 6 patients receiving infliximab experiences an infusion reaction characterized by fever, urticaria, hypotension, and dyspnea within 1 to 2 hours after antibody administration. Serious infections also have occurred in infliximab-treated patients, most frequently in the upper respiratory and urinary tracts. The development of antinuclear antibodies, and rarely a lupuslike syndrome, have been reported after treatment with infliximab.
Although not a monoclonal antibody, etanercept (ENBREL) is mechanistically related to infliximab because it also targets TNF-. Etanercept contains the ligand-binding portion of a human TNF- receptor fused to the Fc portion of human IgG1, and binds to TNF- and prevents it from interacting with its receptors. It is approved in the United States for treatment of the symptoms of rheumatoid arthritis in patients who have not responded to other treatments. Etanercept can be used in combination with methotrexate in patients who have not responded adequately to methotrexate alone. As with infliximab, serious infections have occurred after treatment with etanercept. Injection-site reactions (erythema, itching, pain, or swelling) have occurred in more than one-third of etanercept-treated patients.
Adalimumab (HUMIRA) is another anti-TNF product for intravenous use. This recombinant human IgG1 monoclonal antibody was created by phage display technology and is approved for use in rheumatoid arthritis.
LFA-1 Inhibition. Efalizumab is a humanized IgG1 mAb targeting the CD11a chain of LFA-1 (lymphocyte function associated antigen). Efalizumab binds to LFA-1 and prevents the LFA-1-ICAM (intercellular adhesion molecule) interaction to block T-cell adhesion, trafficking, and activation (Arnaout, 1990). Pretransplant therapy with anti-CD11a prolonged survival of murine skin and heart allografts and monkey heart allografts (Nakakura et al., 1996). In a randomized, multicenter trial, a murine anti-ICAM-1 mAb (ENLIMOMAB) failed to reduce the rate of acute rejection or to improve delayed graft function of cadaveric renal transplants (Salmela et al., 1999). This may have been due to either the murine nature of the mAb or the redundancy of the ICAMs. Efalizumab also is approved for use in patients with psoriasis. In a Phase I/II open-label, dose-ranging, multidose, multicenter trial, efalizumab (dose 0.5 mg/kg or 2 mg/kg) was administered subcutaneously for 12 weeks after renal transplantation (Vincenti et al., 2001). Both doses of efalizumab decreased the incidence of acute rejection. Pharmacokinetic and pharmacodynamic studies showed that efalizumab produced saturation and 80% modulation of CD11a within 24 hours of therapy. In a subset of 10 patients who received the higher dose efalizumab (2 mg/kg) with full-dose cyclosporine, MMF, and steroids, 3 patients developed posttransplant lymphoproliferative diseases. While efalizumab appears to be an effective immunosuppressive agent, it may be best used in a lower dose and with an immunosuppressive regimen that spares calcineurin inhibitors.
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