Chapter 35: Immunosuppressants, Tolerogens, and Immunostimulants

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 The fifth principle, which is common to all drugs, is that a drug should be reduced or withdrawn if its toxicity exceeds its benefit.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 t_1/2. 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 t_1/2 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 t_1/2 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.

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.

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 t_1/2 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.

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.

Cyclosporine

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 t_1/2 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.

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).

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 t_1/2 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.

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.

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 t_1/2 of azathioprine is ∼10 minutes, while that of its metabolite, 6-mercaptopurine, is ∼1 hour. Other metabolites have a t_1/2 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.

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.

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 t_1/2 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.

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.

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.

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.

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.

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.

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.

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).

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 t_1/2 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 t_1/2 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).

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.

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.

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.

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.

In preclinical studies, inhibition of the co-stimulatory signal has been shown to induce tolerance (Weaver et al., 2008). Experimental approaches to inhibit co-stimulation include a recombinant fusion protein molecule, CTLA4-Ig, and anti-CD80 and/or anti-CD86 mAbs. The antibodies h1F1 and h3D1 are humanized anti-CD80 and anti-CD86 mAbs, respectively. In vitro, h1F1 and h3D1 block CD28-dependent T-cell proliferation and decrease mixed lymphocyte reactions. These mAbs must be used in tandem, because either CD80 or CD86 is sufficient to stimulate T cells via CD28. In nonhuman primates, anti-CD80 and anti-CD86 mAbs were proven effective in renal transplantation, either as monotherapy or in combination with steroids or cyclosporine (Weaver et al., 2008), but did not induce durable tolerance. A phase I study of h1F1 and h3D1 in renal transplant recipients was performed in patients receiving maintenance therapy consisting of cyclosporine, MMF, and steroids (Vincenti, 2002). Although the results of this study showed that h1F1 and h3D1 are relatively safe and possibly effective, clinical development was not further pursued.

CTLA4-Ig (abatacept) contains the binding region of CTLA4, which is a CD28 homolog, and the constant region of the human IgG₁. CTLA4-Ig competitively inhibits CD28. Numerous animal studies have confirmed the efficacy of CTLA4-Ig in inhibiting alloimmune responses, resulting in successful organ transplantation. More recently, CTLA4-Ig was shown to be effective in the treatment of rheumatoid arthritis. However, CTLA4-Ig was less effective when utilized in nonhuman primate models of renal transplantation. Belatacept (LEA29Y) (Figure 35–5) is a second-generation CTLA4-Ig with two amino acid substitutions. Belatacept has higher affinity for CD80 (2-fold) and CD86 (4-fold), yielding a 10-fold increase in potency in vitro as compared to CTLA4-Ig. Preclinical renal transplant studies in nonhuman primates showed that belatacept did not induce tolerance but did prolong graft survival. In a large phase II clinical trial, belatacept was administered intravenously initially every 2 weeks then every 4 or 8 weeks without calcineurin inhibitors and compared to a cyclosporine-based regimen (Vincenti et al., 2005). Belatacept showed comparable efficacy to cyclosporine but was associated with better renal function. Recent reports from phase III trials show similar results to phase II except that the more intense regimen was no more efficacious than the lower intensity regimen but was associated with more infections and posttransplant lymphoproliferative disease (PTLD) (Emamaullee et al., 2009). Because of the risk of PTLD, EBV negative patients should not be treated with belatacept. Belatacept may be approved for maintenance biologic therapy in renal transplantation soon.

Figure 35–5.

Structure of belatacept, a CLTA4Ig congener. For details, see the text and Figure 35–4.

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A second co-stimulatory pathway involves the interaction of CD40 on activated T cells with CD40 ligand (CD154) on B cells, endothelium, and/or antigen-presenting cells (Figure 35–4). Among the purported activities of anti-CD154 antibody treatment is the blockade of B7 expression induced by immune activation. Two humanized anti-CD154 monoclonal antibodies have been used in clinical trials in renal transplantation and auto-immune diseases. The development of these antibodies, however, is on hold because of associated thromboembolic events. An alternative approach to block the CD154-CD40 pathway is to target CD40 with monoclonal antibodies. These antibodies are undergoing trials in non-Hodgkin's lymphoma but are also likely to be developed for auto-immunity and transplantation.

Donor Cell Chimerism

Another promising approach is induction of chimerism (co-existence of cells from two genetic lineages in a single individual) by any of a variety of protocols that first dampen or eliminate immune function in the recipient with ionizing radiation, drugs such as cyclophosphamide, and/or antibody treatment and then provide a new source of immune function by adoptive transfer (transfusion) of bone marrow or hematopoietic stem cells (Starzl et al., 1997). Upon reconstitution of immune function, the recipient no longer recognizes new antigens provided during a critical period as "nonself." Such tolerance is long lived and is less likely to be complicated by the use of calcineurin inhibitors. Although the most promising approaches in this arena have been therapies that promote the development of mixed or macrochimerism, in which substantial numbers of donor cells are present in the circulation, some microchimerization approaches also have shown promise in the development of long-term unresponsiveness.

Soluble HLA

In the pre-cyclosporine era, blood transfusions were shown to be associated with improved outcomes in renal transplant patients (Opelz and Terasaki, 1978). These findings gave rise to donor-specific transfusion protocols that improved outcomes (Opelz et al., 1997). After the introduction of cyclosporine, however, these effects of blood transfusions disappeared, presumably due to the efficacy of this drug in blocking T-cell activation. Nevertheless, the existence of tolerance-promoting effects of transfusions is irrefutable. It is possible that this effect is due to HLA molecules on the surface of cells or in soluble forms. Recently, soluble HLA and peptides corresponding to linear sequences of HLA molecules have been shown to induce immunological tolerance in animal models via a variety of mechanisms (Murphy and Krensky, 1999).

antigens

Specific antigens provided in a variety of forms (generally as peptides) induce immunological tolerance in preclinical models of diabetes mellitus, arthritis, and MS. Clinical trials of such approaches are under way. The past decade has witnessed a revolution in our understanding of the basis of immune tolerance. It is now well established that antigen/MHC complex binding to the T cell–receptor/CD3 complex coupled with soluble and membrane-bound co-stimulatory signals initiates a cascade of signaling events that lead to productive immunity. In addition, the immune response is regulated by a number of negative signaling events that control cell survival and expansion. In vitro and preclinical in vivo studies have demonstrated that one can selectively inhibit immune responses to specific antigens without the associated toxicity of established immunosuppressive therapies (Van Parijs and Abbas, 1998). With these insights comes the promise of specific immune therapies to treat the vast array of immune disorders from auto-immunity to transplant rejection. These new therapies will take advantage of a combination of drugs that target the primary T-cell receptor–mediated signal, either by blocking cell-surface receptor interactions or inhibiting early signal transduction events. The drugs will be combined with therapies that effectively block co-stimulation to prevent cell expansion and differentiation of those cells that have engaged antigen while maintaining a non-inflammatory milieu.

Levamisole. Levamisole (ergamisol) was synthesized originally as an anthelmintic but appears to "restore" depressed immune function of B lymphocytes, T lymphocytes, monocytes, and macrophages. Its only clinical indication was as adjuvant therapy with 5-fluorouracil after surgical resection in patients with Dukes' stage C colon cancer (Moertel et al., 1990). Because of its risk for fatal agranulocytosis, levamisole was withdrawn from the U.S. market in 2005.

Lenalidomide. Lenalidomide (revlimid), 3-(4-amino-1-oxo 1,3-dihydro-2H-isoindol-2-yl) piperidine-2,6-dione, is a thalidomide analog with immunomodulatory and anti-angiogenic properties. Lenalidomide is FDA approved for the treatment of patients with transfusion-dependent anemia due to low- or intermediate risk myelodysplastic syndromes associated with a deletion 5q cytogenetic abnormality with or without additional cytogenetic abnormalities.

The usual starting dose is 10 mg/day. Because lenalidomide causes significant neutropenia and thrombocytopenia in almost all patients, patients have to be closely monitored with weekly blood counts and lenalidomide dose adjusted according to the labeling information. Lenalidomide also is associated with a significant risk for deep vein thrombosis. Lenalidomide carries the same risk of teratogenicity as thalidomide, and pregnancy has to be avoided. Lenalidomide's availability is limited to a special distribution program administered by the manufacturer.

Flu-like symptoms, including fever, chills, and headache, are the most common adverse effects after IFN-α-2b administration. Adverse experiences involving the cardiovascular system (e.g., hypotension, arrhythmias, and rarely cardiomyopathy and myocardial infarction) and CNS (e.g., depression, confusion) are less frequent side effects. All α interferons carry a boxed warning regarding development of pulmonary hypertension.

Administration of aldesleukin has been associated with serious cardiovascular toxicity resulting from capillary leak syndrome, which involves loss of vascular tone and leak of plasma proteins and fluid into the extravascular space. Hypotension, reduced organ perfusion, and death may occur. An increased risk of disseminated infection due to impaired neutrophil function also has been associated with aldesleukin treatment.

Although most vaccines have targeted infectious diseases, a new generation of vaccines may provide complete or limited protection from specific cancers or auto-immune diseases. Because T cells optimally are activated by peptides and co-stimulatory ligands that are present on antigen-presenting cells (APCs), one approach for vaccination has consisted of immunizing patients with APCs expressing a tumor antigen. The first generation of anticancer vaccines used whole cancer cells or tumor-cell lysates as a source of antigen in combination with various adjuvants, relying on host APCs to process and present tumor-specific antigens (Sinkovics and Horvath, 2000). These anticancer vaccines resulted in occasional clinical responses and are being tested in prospective clinical trials. Second-generation anticancer vaccines utilized specific APCs incubated ex vivo with antigen or transduced to express antigen and subsequently reinfused into patients. In laboratory animals, immunization with dendritic cells previously pulsed with MHC class I–restricted peptides derived from tumor-specific antigens led to pronounced antitumor cytotoxic T-lymphocyte responses and protective tumor immunity (Tarte and Klein, 1999). Finally, multiple studies have demonstrated the efficacy of DNA vaccines in small- and large-animal models of infectious diseases and cancer (Lewis and Babiuk, 1999). The advantage of DNA vaccination over peptide immunization is that it permits generation of entire proteins, enabling determinant selection to occur in the host without having to restrict immunization to patients bearing specific HLA alleles. However, a safety concern about this technique is the potential for integration of the plasmid DNA into the host genome, possibly disrupting important genes and thereby leading to phenotypic mutations or carcinogenicity. A final approach to generate or enhance immune responses against specific antigens consists of infecting cells with recombinant viruses that encode the protein antigen of interest. Different types of viral vectors that can infect mammalian cells, such as vaccinia, avipox, lentivirus, adenovirus, or adenovirus-associated virus, have been used.

Specific immune globulin preparations are available for hepatitis B, rabies, tetanus, varicella-zoster, cytomegalovirus, botulism, and respiratory syncytial virus. Rho(D) immune globulin is a specific hyperimmune globulin for prophylaxis against hemolytic disease of the newborn due to Rh incompatibility between mother and fetus. All such plasma-derived products carry the theoretical risk of transmission of infectious disease.

Rho(D) Immune Globulin. The commercial forms of Rho(D) immune globulin (Table 35–2) consist of IgG containing a high titer of antibodies against the Rh(D) antigen on the surface of red blood cells. All donors are carefully screened to reduce the risk of transmitting infectious diseases. Fractionation of the plasma is performed by precipitation with cold alcohol followed by passage through a viral clearance system (Bowman, 1998).

Mechanism of Action. Rho(D) immune globulin binds Rho antigens, thereby preventing sensitization (Peterec, 1995). Rh-negative women may be sensitized to the "foreign" Rh antigen on red blood cells via the fetus at the time of birth, miscarriage, ectopic pregnancy, or any transplacental hemorrhage. If the women go on to have a primary immune response, they will make antibodies to Rh antigen that can cross the placenta and damage subsequent fetuses by lysing red blood cells. This syndrome, called hemolytic disease of the newborn, is life-threatening. The form due to Rh incompatibility is largely preventable by Rho(D) immune globulin.

Therapeutic Use. Rho(D) immune globulin is indicated whenever fetal red blood cells are known or suspected to have entered the circulation of an Rh-negative mother unless the fetus is known to also be Rh negative. The drug is given intramuscularly. The t_1/2 of circulating immunoglobulin is ∼21-29 days.

Toxicity. Injection-site discomfort and low-grade fever have been reported. Systemic reactions are extremely rare, but myalgia, lethargy, and anaphylactic shock have been reported. As with all plasma-derived products, there is a theoretical risk of transmission of infectious diseases.

Although the mechanism of action of IVIG in immune modulation remains largely unknown, proposed mechanisms include modulation of expression and function of Fc receptors on leukocytes and endothelial cells, interference with complement activation and cytokine production, provision of anti-idiotypic antibodies (Jerne's network theory), and effects on the activation and effector function of T and B lymphocytes. Although IVIG is effective in many auto-immune diseases, its spectrum of efficacy and appropriate dosing (especially duration of therapy) are unknown. Additional controlled studies of IVIG are needed to identify proper dosing, cost-benefit, and quality-of-life parameters.

A number of other new immunomodulatory therapies have either recently been approved by the FDA or are completing phase III trials. The monoclonal antibody, natalizumab (tysabri), directed against the adhesion molecule α₄ integrin, antagonizes interactions with integrin heterodimers containing α₄ integrin, such as α₄β₁ integrin that is expressed on the surface of activated lymphocytes and monocytes. Preclinical data suggest that an interaction of α₄β₁ integrin with vascular-cellular adhesion molecule (VCAM)-1 is critical for T-cell trafficking from the periphery into the CNS (Steinman, 2004); thus, blocking this interaction would hypothetically inhibit disease exacerbations. Phase III clinical trials demonstrated a significant decrease in the number of new lesions as determined by magnetic resonance imaging and clinical attacks in MS patients receiving natalizumab (Polman et al., 2006). Postmarketing use of natalizumab has been associated with the development of progressive multifocal leukoencephalopathy, and availability has been limited to a special distribution program (TOUCH) administered by the manufacturer. Monoclonal antibodies directed against the IL-2 receptor and against CD52 (alemtuzumab; CAMPATH) are also in phase III clinical trials. The pharmacotherapy of MS has been reviewed by De Jager and Hafler (2007); the utility of immunotherapy for auto-immune diseases has been reviewed by Steinman (2004).