Immunosuppressive drugs are used to dampen the immune response in organ transplantation and auto-immune disease. In transplantation, the major classes of immunosuppressive drugs used today are:
These drugs have met with a high degree of clinical success in treating conditions such as acute immune rejection of organ transplants and severe auto-immune 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, mammalian target of rapamycin (mTOR) inhibitors (sirolimus, everolimus) and anti-CD25 (interleukin-2 receptor [IL-2R]) antibodies (basiliximab, daclizumab) target growth-factor pathways, substantially limiting clonal expansion and thus potentially promoting tolerance. Immunosuppressive drugs used more commonly today are described in the rest of this section. Many more selective therapeutic agents under development are expected to revolutionize immunotherapy in the next decade.
General Approach to Organ Transplantation Therapy
Organ transplantation 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, each of which is directed at a different molecular target within the allograft response (Table 35–1; Hong and Kahan, 2000), are used simultaneously. 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 co-exist. Organ-specific problems (e.g., obstruction in the case of kidney transplants) also must 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.
Biological 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 two preparations currently are approved by the FDA for use in transplantation: lymphocyte immune globulin (ATGAM) and antithymocyte globulin (ATG; thymoglobulin) (Howard et al., 1997). Another important milestone in biological therapy was the development of mAbs and the introduction of the murine anti-CD3 mAb (muromonab-CD3 or OKT3) (Ortho Multicenter Transplant Study Group, 1985). ATG is the most frequently used depleting agent. Lymphocyte immune globulin and OKT3 are rarely used because of poorer efficacy and acute side effects, respectively. Alemtuzumab, a humanized anti-CD52 monoclonal antibody that produces prolonged lymphocyte depletion, is approved for use in chronic lymphocytic leukemia but is increasingly used off label as induction therapy in transplantation.
In many transplant centers, induction therapy with biological 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 a prolonged serum t1/2. Antibodies derived from transgenic mice carrying human antibody genes are labeled "humanized" (90-95% human) or "fully human" (100% human); antibodies derived from human cells are labeled "human." However, all three types of antibodies probably are of equal efficacy and safety. Chimeric antibodies generally contain ∼33% mouse protein and 67% human protein and can still produce an antibody response, resulting in reduced efficacy and t1/2 compared to humanized antibodies. The anti–IL-2R mAbs (frequently referred to as anti-CD25) were the first biologicals proven to be effective as induction agents in randomized double-blind prospective trials (Vincenti et al., 1998).
Biological agents for induction therapy in the prophylaxis of rejection currently are used in ∼70% of de novo transplant patients and have been propelled by several factors, including the introduction of the relatively safe anti–IL-2R antibodies and the emergence of ATG as a safer and more effective alternative to lymphocyte immune globulin or muromonab-CD3. Biologicals for induction can be divided into two groups: the depleting agents and the immune modulators. The depleting agents consist of lymphocyte immune globulin, ATG, 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 biological agents, the anti–IL-2R mAbs, do not deplete T lymphocytes, with the possible exception of T regulatory cells, but rather block IL-2–mediated T-cell activation by binding to the α chain of IL-2R.
More aggressive approaches have been recently utilized in patients with high levels of anti-HLA antibodies, donor-specific antibodies detected by cytotoxicity cross-match, or flow cytometry and humoral rejection. These therapies include plasmapheresis, intravenous immunoglobulin, and rituximab, a chimeric anti-CD20 monoclonal antibody (Akalin et al., 2003; Zachary et al., 2003; Vo et al., 2008).
Maintenance Immunotherapy. The basic immunosuppressive protocols use multiple drugs simultaneously. Therapy typically involves a calcineurin inhibitor, glucocorticoids, and mycophenolate (a purine metabolism inhibitor; see "Mycophenolate Mofetil"), each directed at a discrete site in T-cell activation (Suthanthiran et al., 1996). Glucocorticoids, azathioprine, cyclosporine, tacrolimus, mycophenolate, sirolimus, and various monoclonal and polyclonal antibodies all are 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 rapid steroid withdrawal (within 1 week after transplantation) are being utilized in more than a third of renal transplant recipients. Short-term results are good, but the effects on long-term graft function are unknown (Vincenti et al., 2008). Recent data suggest that calcineurin inhibitors may shorten graft t1/2 by their nephrotoxic effects (Nankivell et al., 2003). Protocols under evaluation include calcineurin dose reduction or switching from calcineurin to sirolimus-based immunosuppressive therapy at 3-4 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 (Monaco et al., 1999). 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.
Table 35-1Sites of Action of Selected Immunosuppressive Agents on T-Cell Activation ||Download (.pdf) Table 35-1 Sites of Action of Selected Immunosuppressive Agents on T-Cell Activation
|DRUG ||SITE OF ACTION |
|Glucocorticoids ||Glucocorticoid response elements in DNA (regulate gene transcription) |
|Muromonab-CD3 ||T-cell receptor complex (blocks antigen recognition) |
|Cyclosporine ||Calcineurin (inhibits phosphatase activity) |
|Tacrolimus ||Calcineurin (inhibits phosphatase activity) |
|Azathioprine ||DNA (false nucleotide incorporation) |
|Mycophenolate mofetil ||Inosine monophosphate dehydrogenase (inhibits activity) |
|Daclizumab, basiliximab ||IL-2 receptor (block IL-2-mediated T-cell activation) |
|Sirolimus ||Protein kinase involved in cell-cycle progression (mTOR) (inhibits activity) |
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 Chapter 42. Prednisone, prednisolone, and other glucocorticoids are used alone and in combination with other immunosuppressive agents for treatment of transplant rejection and auto-immune disorders.
Mechanism of Action. The immunosuppressive effects of glucocorticoids have long been known, but the specific mechanisms of their immunosuppressive actions 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 (Chapter 42). Additionally, glucocorticoids curtail activation of NF-κB, which increases apoptosis of activated cells (Auphan et al., 1995). Of central importance, key pro-inflammatory 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 anti-inflammatory 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, others) are used to reverse acute transplant rejection and acute exacerbations of selected auto-immune disorders. Glucocorticoids also are efficacious for treatment of graft-versus-host disease in bone-marrow transplantation. Glucocorticoids are routinely used to treat auto-immune 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, auto-immune hematological disorders, and acute exacerbations of MS (see "Multiple Sclerosis"). 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 muromonab-CD3 and to a lesser extent ATG (see "Antithymocyte Globulin").
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 (Chapter 42). The advent of combined glucocorticoid/calcineurin inhibitor regimens has allowed reduced doses or rapid withdrawal of steroids, resulting in lower steroid-induced morbidities.
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 (Figure 35–1) and bind to distinct (albeit related) molecular targets, they inhibit normal T-cell signal transduction essentially by the same mechanism (Figure 35–2). Cyclosporine and tacrolimus do not act per se as immunosuppressive agents. Instead, these drugs bind to an immunophilin (cyclophilin for cyclosporine [see "Cyclosporine"] or FKBP-12 for tacrolimus [see "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 35–2). 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.
Chemical structures of immunosuppressive drugs.
Mechanisms of action of cyclosporine, tacrolimus, and sirolimus on T lymphocytes. Both cyclosporine and tacrolimus bind to immunophilins (cyclophilin and FK506-binding protein [FKBP], respectively), forming a complex that binds the phosphatase calcineurin and inhibits the calcineurin-catalyzed dephosphorylation essential to permit movement of the nuclear factor of activated T cells (NFAT) into the nucleus. NFAT is required for transcription of interleukin-2 (IL-2) and other growth- and differentiation-associated cytokines (lymphokines). Sirolimus (rapamycin) works at a later stage in T-cell activation, downstream of the IL-2 receptor. Sirolimus also binds FKBP, but the FKBP-sirolimus complex binds to and inhibits the mammalian target of rapamycin (mTOR), a kinase involved in cell-cycle progression (proliferation). TCR, T-cell receptor. (From Pattison et al., 1997, with permission. Copyright © Lippincott Williams & Wilkins. http://lww.com.)
Tacrolimus. Tacrolimus (prograf, FK506) is a macrolide antibiotic produced by Streptomyces tsukubaensis (Goto et al., 1987). Because of perceived slightly greater efficacy and ease of blood level monitoring, tacrolimus has become the preferred calcineurin inhibitor in most transplant centers (Ekberg et al., 2008).
Mechanism of Action. Like cyclosporine, tacrolimus inhibits T-cell activation by inhibiting calcineurin. 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 35–2, 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.
Disposition and Pharmacokinetics. Tacrolimus is available for oral administration as capsules (0.5, 1, and 5 mg) and as a 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. Whole blood, rather than plasma, is the most appropriate sampling compartment to describe tacrolimus pharmacokinetics. For tacrolimus, the trough drug level seems to correlate better with clinical events than it does for cyclosporine. Target concentrations in most centers are 10-15 ng/mL in the early preoperative period and 100-200 ng/mL 3 months after transplantation. Gastrointestinal absorption is incomplete and variable. Food decreases the rate and extent of absorption. Plasma protein binding of tacrolimus is 75-99%, involving primarily albumin and α1-acid glycoprotein. The t1/2 of tacrolimus is ∼12 hours. Tacrolimus is extensively metabolized in the liver by CYP3A; 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 (see "Cyclosporine") and is used off label as rescue therapy in patients with rejection episodes despite "therapeutic" levels of cyclosporine.
Recommended initial oral doses are 0.2 mg/kg/day for adult kidney transplant patients, 0.1-0.15 mg/kg/day for adult liver transplant patients, 0.075 mg/kg/day for adult heart transplant patients, and 0.15-0.2 mg/kg/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 20-ng/mL range.
Toxicity. Nephrotoxicity, neurotoxicity (e.g., tremor, headache, motor disturbances, seizures), GI complaints, hypertension, hyperkalemia, hyperglycemia, and diabetes all are associated with tacrolimus use. As with cyclosporine, nephrotoxicity is limiting. Tacrolimus has a negative effect on pancreatic islet β 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. Diarrhea and alopecia are commonly noted in patients on concomitant mycophenolate therapy.
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. Co-administration 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. Because tacrolimus is metabolized mainly by CYP3A, the potential interactions described in the following section for cyclosporine also apply for tacrolimus.
Chemistry. Cyclosporine (cyclosporin A), a cyclic polypeptide of 11 amino acids, is produced by the fungus Beauveria nivea. 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 auto-immunity. It preferentially inhibits antigen-triggered signal transduction in T lymphocytes, blunting expression of many lymphokines, including IL-2, and the expression of anti-apoptotic proteins. Cyclosporine forms a complex with cyclophilin, a cytoplasmic-receptor protein present in target cells (Figure 35-2). 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 β (TGF-β), a potent inhibitor of IL-2–stimulated T-cell proliferation and generation of cytotoxic T lymphocytes (CTLs) (Khanna et al., 1994).
Disposition and Pharmacokinetics. Cyclosporine can be administered intravenously or orally. The intravenous preparation (sandimmune, others) 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 is absorbed slowly, with 20-50% bioavailability. A modified microemulsion formulation (neoral) has become the most widely used preparation. It has more uniform and slightly increased bioavailability compared to the original formulation. It is provided as 25-mg and 100-mg soft gelatin capsules and a 100-mg/mL oral solution. Because the original and microemulsion formulations are not bioequivalent, they cannot be used interchangeably without supervision by a physician and monitoring of drug concentrations in plasma. Generic preparations of both NEORAL and SANDIMMUNE, now widely available, are bioequivalent by FDA criteria. When switching between 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. Because SANDIMMUNE and NEORAL differ in terms of their pharmacokinetics and definitely are 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.
Blood is most conveniently sampled before the next dose (a C0 or trough level). Although convenient to obtain, C0 concentrations do not reflect the area under the drug concentration curve (AUC) as a measure of 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) (Cole et al., 2003). 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 if 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 generally is biphasic, with a terminal t1/2 of 5-18 hours (Noble and Markham, 1995). After intravenous infusion, clearance is ∼5-7 mL/min/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 1.5-2 hours (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 ∼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 reportedly is as high as 3-5 L/kg in solid-organ transplant recipients.
Only 0.1% of cyclosporine is excreted unchanged in urine. Cyclosporine is extensively metabolized in the liver by CYP3A and to a lesser degree by the GI tract and kidneys. At least 25 metabolites have been identified in human bile, feces, blood, and urine. 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 ∼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. Its use in dermatology is discussed in Chapter 65. 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 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 or tacrolimus 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. Dosing 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 can 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. In psoriasis, cyclosporine is indicated for treatment of adult immunocompetent patients with severe and disabling disease for whom other systemic therapies have failed. 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. Cyclosporine reportedly is effective in Behçet'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 and hypertension; tremor, hirsutism, hyperlipidemia, and gum hyperplasia also are frequently encountered. Hypertension occurs in ∼50% of renal transplant and almost all cardiac transplant patients. Hyperuricemia may lead to worsening of gout, increased P-glycoprotein activity, and hypercholesterolemia. Nephrotoxicity occurs in the majority of patients and is the major reason for cessation or modification of therapy (Nankivell et al., 2003). Recent reviews of calcineurin inhibitor nephrotoxicity are available (Burdmann et al., 2003). Combined use of calcineurin inhibitors and glucocorticoids is particularly diabetogenic, although this apparently is more problematic in patients treated with tacrolimus (see "Tacrolimus" section earlier). Especially at risk are obese patients, African-American or Hispanic transplant recipients, or those with a 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).
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 CYP3A, may impact cyclosporine blood concentrations.
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 juice inhibits CYP3A and the P-glycoprotein multidrug efflux pump and should be minimized by patients taking cyclosporine because 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 others (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 (also see "Drug Interactions" in the sirolimus section) 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 co-administered with nonsteroidal anti-inflammatory drugs (NSAIDs) and other drugs that cause renal dysfunction; elevation of methotrexate levels when the two drugs are co-administered; and reduced clearance of other drugs, including prednisolone, digoxin, and statins.
ISATX247. This is a new oral semisynthetic structural analog of cyclosporine. The cyclosporine molecule is modified at the first amino acid residue. It is more potent on a weight basis than cyclosporine in vitro for calcineurin inhibition. Some preclinical studies show reduced nephrotoxicity, and thus the drug is in clinical development as a primary immunosuppressive drug. Phase 2 clinical trials are in process and thus far show similar or less nephrotoxicity with less frequent glucose intolerance compared to tacrolimus-treated patients (Vincenti and Kirk, 2008).
Janus Kinase Inhibitors/CP-690550. Cytokine receptors are enticing targets for modulation by new small immunosuppressive molecules. Janus kinase (JAK) inhibitors are a class of drugs that inhibit important cytoplasmic tyrosine kinases that are involved in cell signaling. The molecule CP-690550 currently is in clinical trials. As an immunosuppressive drug, this compound inhibits JAK3, which is found primarily on hematopoietic cells. In preclinical studies, this JAK3 inhibitor has been tolerated without nephrotoxicity, malignancy, or other important side effects. To date, all studies have shown non-inferiority with other standard immunosuppressive regimens (Vincenti and Kirk, 2008).
Protein Kinase C Inhibitors/AEB071. Various isoforms of PKC are important mediators in signaling pathways distal to the T-cell receptor and co-stimulators. AEB071 is a low-molecular-weight compound that blocks T-cell activation by inhibition of PKC, thus producing immunosuppression by a different mechanism than calcineurin inhibitors. Clinical studies are ongoing. Early trials using PKC inhibitors in combination with calcineurin inhibitors (CNIs) followed by discontinuation of the CNI had to be stopped because acute rejections occurred when the CNIs were discontinued (Vincenti and Kirk, 2008).
Anti-Proliferative and Antimetabolic Drugs
Sirolimus. Sirolimus (rapamycin; RAPAMUNE) is a macrocyclic lactone (Figure 35–1) produced by Streptomyces hygroscopicus.
Mechanism of Action. Sirolimus inhibits T-lymphocyte activation and proliferation downstream of the IL-2 and other T-cell growth factor receptors (Figure 35–2). 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 mTOR, which is a key enzyme in cell-cycle progression. 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 auto-immune diseases, but its effect also lasts several months after discontinuing therapy, suggesting a tolerizing effect (see "Tolerance") (Groth et al., 1999). A newer indication for sirolimus is the avoidance of calcineurin inhibitors, even when patients are stable, to protect kidney function (Flechner et al., 2008).
Disposition and Pharmacokinetics. After oral administration, sirolimus is absorbed rapidly and reaches a peak blood concentration within ∼1 hour after a single dose in healthy subjects and within ∼2 hours after multiple oral doses in renal transplant patients. Systemic availability is ∼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. 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 >90% of the immunosuppressive effect. The blood t1/2 after multiple doses in stable renal transplant patients is 62 hours (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 usually in combination with a reduced dose of calcineurin inhibitor and glucocorticoids. In patients experiencing or at high risk for calcineurin inhibitor–associated nephrotoxicity, sirolimus has been used with glucocorticoids and mycophenolate to avoid permanent renal damage. Sirolimus dosing regimens are relatively complex with blood levels generally targeted between 5-15 ng/mL. It is recommended that the daily 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. Although 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 anti-proliferative action (Smith 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, mouth ulcer, hypokalemia, proteinuria, and GI 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. Sirolimus is not recommended in liver and lung transplants due to the risk of hepatic artery thrombosis and bronchial anastomotic dehiscence, respectively.
Drug Interactions. Because 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. As noted above, cyclosporine and sirolimus interact, and their administration should be separated by time. Dose adjustment may be required when sirolimus is co-administered with diltiazem or rifampin. Dose adjustment apparently is not required when sirolimus is co-administered 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 [40-O-(2-hydroxyethyl)-rapamycin] is closely related chemically and clinically to sirolimus but has distinct pharmacokinetics. The main difference is a shorter t1/2 and thus a shorter time to achieve steady-state concentrations of the drug. Dosage on a milligram per kilogram basis is similar to that of sirolimus. Aside from the shorter t1/2, 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 that enhances toxicity and reduces rejection. The toxicity of everolimus and the drug interactions reported to date seem to be the same as with sirolimus.
Azathioprine. Azathioprine (imuran, others) is a purine antimetabolite. It is an imidazolyl derivative of 6-mercaptopurine (see Figure 61–11).
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 (Chapter 61). A fraudulent nucleotide, 6-thio-IMP, is converted to 6-thio-GMP and finally to 6-thio-GTP, which is incorporated into DNA. Cell proliferation thereby is 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 1-2 hours after administration. The t1/2 of azathioprine is ∼10 minutes, while that of its metabolite, 6-mercaptopurine, is ∼1 hour. Other metabolites have a t1/2 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.
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. Although the dose of azathioprine required to prevent organ rejection and minimize toxicity varies, 3-5 mg/kg/day is the usual starting dose. Lower initial doses (1 mg/kg/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 25-33% of the usual dose; it is best not to use these two drugs together. Adverse effects resulting from co-administration of azathioprine with other myelosuppressive agents or angiotensin-converting enzyme inhibitors include leukopenia, thrombocytopenia, and anemia as a result of myelosuppression.
Mycophenolate mofetil (MMF; cell-cept) is the 2-morpholinoethyl ester of mycophenolic acid (MPA).
Mechanism of Action. MMF is a prodrug that is rapidly hydrolyzed to the active drug, MPA, a selective, noncompetitive, reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH), 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. MMF 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 t1/2 of MPA is ∼16 hours. Negligible (<1%) amounts of MPA are excreted in the urine. 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 after transplant), plasma concentrations of MPA after a single dose of MMF are about half of those found in healthy volunteers or stable renal transplant patients.
Therapeutic Uses. MMF is indicated for prophylaxis of transplant rejection, and it typically is used in combination with glucocorticoids and a calcineurin inhibitor but not with azathioprine. 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/day). A higher dose, 1.5 g twice daily (3 g/day), may be recommended for African-American renal transplant patients and all liver and cardiac transplant patients. MMF is increasingly used off label in systemic lupus.
Toxicity. The principal toxicities of MMF are gastrointestinal and hematologic. These include leukopenia, pure red cell aplasia, diarrhea, and vomiting. There also is an increased incidence of some infections, especially sepsis associated with cytomegalovirus; progressive multifocal leukoencephalopathy also has been reported in conjunction with the administration of MMF. Tacrolimus in combination with MMF has been associated with activation of polyoma viruses such as BK virus, which can cause interstitial nephritis difficult to distinguish from acute rejection (Hirsch et al., 2002). Excessive immunosuppression is suspected to be responsible for this adverse effect, not necessarily this widely used drug combination. The use of mycophenolate in pregnancy is associated with congenital anomalies and increased risk of pregnancy loss. Women of childbearing potential taking mycophenolate must use effective contraception.
Drug Interactions. Potential drug interactions between MMF and several other drugs commonly used by transplant patients have been studied. There appear to be no untoward effects produced by combination therapy with cyclosporine, trimethoprim–sulfamethoxazole, or oral contraceptives. Unlike cyclosporine, tacrolimus delays elimination of MMF by impairing the conversion of MPA to MPAG. This may enhance GI toxicity. Co-administration with antacids containing aluminum or magnesium hydroxide leads to decreased absorption of MMF; thus, these drugs should not be administered simultaneously. MMF 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.
A delayed-release tablet form of MPA (myfortic) also is available. It does not release MPA under acidic conditions (pH <5) such as in the stomach but is highly soluble in neutral pH present in the intestine. The enteric coating results in a delay in the time to reach maximum MPA concentrations and may improve GI tolerability, although data are sparse and not convincing (Darji et al., 2008).
Other Anti-Proliferative and Cytotoxic Agents. Many of the cytotoxic and antimetabolic agents used in cancer chemotherapy (Chapter 61) are immunosuppressive due to their action on lymphocytes and other cells of the immune system. Other cytotoxic drugs that have been used off label as immunosuppressive agents include methotrexate, cyclophosphamide, thalidomide (thalomid), and chlorambucil (leukeran). Methotrexate is used for treatment of graft-versus-host disease, rheumatoid arthritis, psoriasis, and some cancers. Cyclophosphamide and chlorambucil are used in leukemia and lymphomas and a variety of other malignancies. Cyclophosphamide also is FDA approved for childhood nephrotic syndrome and is used widely for treatment of severe systemic lupus erythematosus and other vasculitides such as Wegener's granulomatosis. Leflunomide (arava, others) is a pyrimidine-synthesis inhibitor indicated for the treatment of adults with rheumatoid arthritis (Prakash and Jarvis, 1999). This drug has found increasing empirical use in the treatment of polyomavirus nephropathy seen in immunosuppressed renal transplant recipients. There are no controlled studies showing efficacy compared with control patients treated with only 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.
Fingolimod (FTY720). This is the first agent in a new class of small molecules, sphingosine-1-phosphate receptor (S1P-R) agonists (Figure 35–1). This S1P receptor prodrug reduces recirculation of lymphocytes from the lymphatic system to the blood and peripheral tissues, including inflammatory lesions and organ grafts.
Therapeutic Uses. The drug has not been as effective as standard regimens in phase III trials, and further drug development has been limited (Vincenti and Kirk, 2008).
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. Although FTY720 sequesters lymphocytes, it 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 pharmacological 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 in up to 30% of patients. In most patients, the heart rate returns to baseline within 48 hours, with the remainder returning to baseline thereafter.
Biological Immunosuppression Antibodies and Fusion Receptor Protein
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 (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 now is possible to make essentially unlimited amounts of a single antibody of a defined specificity (Figure 35–3). 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.
Generation of monoclonal antibodies. Mice are immunized with the selected antigen, and spleen or lymph node is harvested and B cells separated. These B cells are fused to a suitable B-cell myeloma that has been selected for its inability to grow in medium supplemented with hypoxanthine, aminopterin, and thymidine (HAT). Only myelomas that fuse with B cells can survive in HAT-supplemented medium. The hybridomas expand in culture. Those of interest based on a specific screening technique are then selected and cloned by limiting dilution. Monoclonal antibodies can be used directly as supernatants or ascites fluid experimentally but are purified for clinical use. HPRT, hypoxanthine–guanine phosphoribosyl transferase. (Reproduced with permission from Krensky A.M. and Clayberger C. Transplantation immunobiology. In, Pediatric Nephrology, 5th ed. (Avner E.D., Harmon W.E., Niauder P., eds) Lippincott Williams & Wilkins, Philadelphia, 2004. (http://lww.com).)
The first-generation murine monoclonal antibodies have been replaced by newer humanized or fully human monoclonal antibodies that lack antigenicity, have a prolonged t1/2, and can be mutagenized to alter their affinity to Fc receptors. Another class of biological agents being developed for both auto-immunity and transplantation are fusion receptor proteins. These agents usually consist of the ligand-binding domains of receptors bound to the Fc region of an immunoglobulin (usually IgG1) to provide a longer t1/2. Examples of such agents include abatacept (CTLA4-Ig) and belatacept (a second-generation CTLA4-Ig), discussed later in "Co-stimulatory Blockade." Thus, polyclonal and monoclonal antibodies as well as fusion receptor proteins have a place in immunosuppressive therapy.
Antithymocyte Globulin. ATG 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
ATG 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. ATG 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/day (over 4-6 hours) for 7-14 days. Mean T-cell counts fall by day 2 of therapy. ATG 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 4-6 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.
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. 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 complement 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).
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 (<1 minute) for 10-14 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 ∼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. 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 ATG.
Toxicity. The major side effect of anti-CD3 therapy is the "cytokine release syndrome" (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–mediated cross-linking 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-α [TNF-α], IL-2, IL-6, and interferon-γ [IFN-γ]), which are released by activated T cells and/or monocytes. In several studies, the production of TNF-α 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 central nervous system (CNS) disorders, including aseptic meningitis. Potentially fatal 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, reduces first-dose reactions considerably, and now is 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. Anti-CD3 therapies may be limited by anti-idiotypic or antimurine antibodies in the recipient.
Currently, muromomab-CD3 rarely is used in transplantation. It has been replaced by ATG and alemtuzumab.
New-Generation Anti-CD3 Antibodies. Recently, genetically altered anti-CD3 monoclonal antibodies have been developed that are "humanized" to minimize the occurrence of anti-antibody responses and mutated to prevent binding to Fc receptors (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 Fc receptors reversed acute renal allograft rejection without causing the first-dose cytokine-release syndrome. Clinical efficacy of these agents in auto-immune diseases is being evaluated (Herold et al., 2002).
It is not clear whether any of the new generation of anti-CD3s will be developed for use in transplantation.
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 but a longer t1/2 (20 days) than basiliximab. 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 α-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 α chain, either from depletion of coated lymphocytes or modulation of the α chain secondary to decreased expression or increased shedding. There also is recent evidence that the β chain may be downregulated by the anti-CD25 antibody. Recent evidence suggests that T-regulatory cells are transiently depleted during anti-CD25 therapy (Bluestone et al., 2008).
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).
In phase III trials, daclizumab was administered in five doses (1 mg/kg given intravenously over 15 minutes in 50-100 mL of normal saline) starting immediately preoperatively, and subsequently at biweekly intervals. The t1/2 of daclizumab was 20 days, resulting in saturation of the IL-2Rα on circulating lymphocytes for up to 120 days after transplantation. In these trials, daclizumab was used with maintenance immunosuppressive regimens (cyclosporine, azathioprine, and steroids; cyclosporine and steroids). Subsequently, daclizumab was successfully used with a maintenance triple-therapy regimen—either with cyclosporine or tacrolimus, steroids, and MMF substituting for azathioprine (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 (Kahan et al., 1999). This regimen of basiliximab resulted in a concentration of ≥0.2 μg/mL, sufficient to saturate IL-2R on circulating lymphocytes for 25-35 days after transplantation.
The t1/2 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 found to be 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 an α 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 versus 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).
Alemtuzumab. Alemtuzumab (campath) 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 alemtuzumab is accepted into the clinical armamentarium for transplantation.
Anti-TNF Reagents. TNF has been implicated in the pathogenesis of several immune-mediated intestinal, skin, and joint diseases. For example, patients with rheumatoid arthritis have elevated levels of TNF-α in their joints, while patients with Crohn's disease have elevated levels of TNF-α in their stools. As a result, a number of anti-TNF agents have been developed for the treatment of these disorders.
Infliximab (remicade) is a chimeric anti–TNF-α monoclonal antibody containing a human constant region and a murine variable region. It binds with high affinity to TNF-α and prevents the cytokine from binding to its receptors.
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 U.S. for treating the symptoms of rheumatoid arthritis and is typically 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 (Chapter 47). Other FDA-approved indications include ankylosing spondylitis, plaque psoriasis, psoriatic arthritis, and ulcerative colitis. About one of six patients receiving infliximab experiences an infusion reaction characterized by fever, urticaria, hypotension, and dyspnea within 1-2 hours after antibody administration. The development of antinuclear antibodies, and rarely a lupus-like syndrome, has been reported after treatment with infliximab.
Although not a monoclonal antibody, etanercept (enbrel) is mechanistically related to infliximab because it also targets TNF-α. Etanercept contains the ligand-binding portion of a human TNF-α receptor fused to the Fc portion of human IgG1, and binds to TNF-α and prevents it from interacting with its receptors. It is approved in the U.S. for treatment of the symptoms of rheumatoid arthritis in patients who have not responded to other treatments, as well as for treatment of ankylosing spondylitis, plaque psoriasis, polyarticular juvenile idiopathic arthritis, and psoriatic arthritis. Etanercept can be used in combination with methotrexate in patients who have not responded adequately to methotrexate alone. Injection-site reactions (i.e., 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, ankylosing spondylitis, Crohn's disease, juvenile idiopathic arthritis, plaque psoriasis, and psoriatic arthritis.
Toxicity. All anti-TNF agents (i.e., infliximab, etanercept, adalimumab) increase the risk for serious infections, lymphomas, and other malignancies. For example, fatal hepatosplenic T-cell lymphomas have been reported in adolescent and young adult patients with Crohn's disease treated with infliximab in conjunction with azathioprine or 6-mercaptopurine.
Plasma IL-1 levels are increased in patients with active inflammation (Moltó and Olivé, 2009; see also Chapter 34). In addition to the naturally occurring IL-1 receptor antagonist (IL-1RA), several IL-1 receptor antagonists are in development and a few have been approved for clinical use. Anakinra is an FDA-approved recombinant, non-glycosylated form of human IL-1RA for the management of joint disease in rheumatoid arthitis. It can be used alone or in combination with anti-TNF agents such as etanercept (enbrel), infliximab (remicade), or adalimumab (humira). Canakinumab (ilaris) is an IL-1β monoclonal antibody approved by the FDA in June 2009 for Cryoprin-associated periodic syndromes (CAPS), a group of rare inherited inflammatory diseases associated with overproduction of IL-1 that includes Familial Cold Autoinflammatory and Muckle-Wells Syndromes (Lachmann et al., 2009). Canakinumab is also being evaluated for use in chronic obstructive pulmonary disease (Church et al., 2009). Rilonacept (IL-1 TRAP) is another IL-1 blocker (a fusion protein that binds IL-1) that is now being evaluated in a phase 3 study for gout (Terkeltaub et al., 2009). IL-1 is an inflammatory mediator of joint pain associated with elevated uric acid crystals.
Lymphocyte Function–Associated Antigen-1 (LFA-1) Inhibition
Efalizumab. (raptiva) is a humanized IgG1 mAb targeting the CD11a chain of lymphocyte function–associated antigen-1 (LFA-1). Efalizumab binds to LFA-1 and prevents the LFA-1–intercellular adhesion molecule (ICAM) interaction to block T-cell adhesion, trafficking, and activation.
Pretransplant therapy with anti-CD11a prolonged survival of murine skin and heart allografts and monkey heart allografts (Nakakura et al., 1996). A randomized, multicenter trial of 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; Vincenti et al., 2007). 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, three patients developed post-transplant lymphoproliferative diseases. Progressive multifocal leukoencephalopathy (PML) also has occurred during therapy with efalizumab. Although 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. Several trials are being conducted with efalizumab in renal, liver, and islet cell transplantation.
(amevive) is a human LFA-3-IgG1 fusion protein. The LFA-3 portion of alefacept binds to CD2 on T lymphocytes, blocking the interaction between LFA-3 and CD2 and interfering with T-cell activation. Alefacept is FDA approved for use in psoriasis.
Treatment with alefacept has been shown to produce a dose-dependent reduction in T-effector memory cells (CD45, RO+) but not in naïve cells (CD45, RA+). This effect has been related to its efficacy in psoriatic disease and is of significant interest in transplantation because T-effector memory cells have been associated with co-stimulation blockade resistant and depletional induction-resistant rejection. Alefacept will delay rejection in non-human primate (NHP) cardiac transplantation and has recently been shown to have synergistic potential when used with co-stimulation blockade and/or sirolimus-based regimens in NHPs (Vincenti and Kirk, 2008). A phase II randomized, open-label, parallel-group, multicenter study to assess the safety and efficacy of maintenance therapy with alefacept in kidney transplant recipients currently is under way.
Most of the advances in transplantation can be attributed to drugs designed to inhibit T-cell responses. As a result, T cell–mediated acute rejection has been become much less of a problem, while B cell–mediated responses such as antibody-mediated rejection and other effects of donor-specific antibodies have become more evident. Thus, several agents, both biologicals and small molecules with B-cell specific effects now are being considered for development in transplantation, including humanized monoclonal antibodies to CD20 and inhibitors of the two B cell–activation factors BLYS and APRIL and their respective receptors.