Goodman & Gilman's The Pharmacological Basis of Therapeutics, 12e > Chapter 35. Immunosuppressants, Tolerogens, and Immunostimulants >

The Immune Response

The immune system evolved to discriminate self from nonself. Multicellular organisms were faced with the problem of destroying infectious invaders (microbes) or dysregulated self (tumors) while leaving normal cells intact. These organisms responded by developing a robust array of receptor-mediated sensing and effector mechanisms broadly described as innate and adaptive. Innate, or natural, immunity is primitive, does not require priming, and is of relatively low affinity, but is broadly reactive. Adaptive, or learned, immunity is antigen specific, depends on antigen exposure or priming, and can be of very high affinity. The two arms of immunity work closely together, with the innate immune system being most active early in an immune response and adaptive immunity becoming progressively dominant over time. The major effectors of innate immunity are complement, granulocytes, monocytes/macrophages, natural killer cells, mast cells, and basophils. The major effectors of adaptive immunity are B and T lymphocytes. B lymphocytes make antibodies; T lymphocytes function as helper, cytolytic, and regulatory (suppressor) cells. These cells are important in the normal immune response to infection and tumors but also mediate transplant rejection and auto-immunity. Immunoglobulins (antibodies) on the B-lymphocyte surface are receptors for a large variety of specific structural conformations. In contrast, T lymphocytes recognize antigens as peptide fragments in the context of self major histocompatibility complex (MHC) antigens (called human leukocyte antigens [HLAs] in humans) on the surface of antigen-presenting cells, such as dendritic cells, macrophages, and other cell types expressing MHC class I (HLA-A, -B, and -C) and class II antigens (HLA-DR, -DP, and -DQ) in humans. Once activated by specific antigen recognition via their respective clonally restricted cell-surface receptors, both B and T lymphocytes are triggered to differentiate and divide, leading to release of soluble mediators (cytokines, lymphokines) that perform as effectors and regulators of the immune response.

The impact of the immune system in human disease is enormous. Developing vaccines against emerging infectious agents such as human immunodeficiency virus (HIV) and Ebola virus is among the most critical challenges facing the research community. Immune system–mediated diseases are significant medical problems. Immunological diseases are growing at epidemic proportions that require aggressive and innovative approaches to develop new treatments. These diseases include a broad spectrum of auto-immune diseases, such as rheumatoid arthritis, type I diabetes mellitus, systemic lupus erythematosus, and multiple sclerosis (MS); solid tumors and hematological malignancies; infectious diseases; asthma; and various allergic conditions. Furthermore, one of the great therapeutic opportunities for the treatment of many disorders is organ transplantation. However, immune system–mediated graft rejection remains the single greatest barrier to widespread use of this technology. An improved understanding of the immune system has led to the development of new therapies to treat immune system–mediated diseases.

This chapter briefly reviews drugs used to modulate the immune response in three ways: immunosuppression, tolerance, and immunostimulation. Four major classes of immunosuppressive drugs are discussed: glucocorticoids (Chapter 42), calcineurin inhibitors, anti-proliferative and antimetabolic agents (Chapter 61), and antibodies. The

"holy grail" of immunomodulation is the induction and maintenance of immune tolerance, the active state of antigen-specific nonresponsiveness. Approaches expected to overcome the risks of infections and tumors with immunosuppression are reviewed. These include co-stimulatory blockade, donor-cell chimerism, soluble HLAs, and antigen-based therapies. A general discussion of the limited number of immunostimulant agents is presented, followed by an overview of active and passive immunization, and concluding with a brief case study of immunotherapy for MS.Immunosuppression

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:

glucocorticoids calcineurin inhibitors anti-proliferative/antimetabolic agents biologicals (antibodies)

These drugs have met with a high degree of clinical success in treating conditions such as acute immune rejection of organ transplants and severe 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.

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)

IL, interleukin; mTOR, mammalian target of rapamycin. 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 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.

Adrenocortical Steroids

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- B, 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.

Calcineurin Inhibitors

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.

Figure 35–1.

Chemical structures of immunosuppressive drugs.

Figure 35–2.

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 1-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 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  (TGF- ), 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 Andantimetabolic 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

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

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.

Figure 35–3.

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.

Monoclonal Antibodies

Anti-CD3 Monoclonal Antibodies

Antibodies directed at the chain of CD3, a trimeric molecule adjacent to the T-cell receptor on the surface of human T lymphocytes, have been used with considerable efficacy since the early 1980s in human transplantation. The original mouse IgG2a antihuman CD3 monoclonal antibody, muromonab-CD3 (OKT3, ORTHOCLONE OKT3), still is used to reverse glucocorticoid-resistant rejection episodes (Cosimi et al., 1981).

Mechanism of Action

Muromonab-CD3 binds to the chain of CD3, a monomorphic component of the T-cell receptor complex involved in antigen recognition, cell signaling, and proliferation. 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- [TNF- ], IL-2, IL-6, and interferon- [IFN- ]), which are released by

activated T cells and/or monocytes. In several studies, the production of TNF- has been shown to be the major cause of the toxicity (Herbelin et al., 1995). The symptoms usually are worst with the first dose; frequency and severity decrease with subsequent doses. Common clinical manifestations include high fever, chills/rigor, headache, tremor, nausea, vomiting, diarrhea, abdominal pain, malaise, myalgias, arthralgias, and generalized weakness. Less common complaints include skin reactions and cardiorespiratory and 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 -chain epitope than that recognized and bound by daclizumab (Vincenti et al., 1998). Similar results were obtained in studies with basiliximab (Amlot et al., 1995). These findings indicate that therapy with the anti IL-2R mAbs results in a relative decrease of the expression of the chain, either from depletion of coated lymphocytes or modulation of the chain secondary to decreased expression or increased shedding. There also is recent evidence that the 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-2R 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 g/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 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- in their joints, while patients with Crohn's disease have elevated levels of TNF- 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-  monoclonal antibody containing a human constant region and a murine variable region. It binds with high affinity to TNF- and prevents the cytokine from binding to its receptors.

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- . Etanercept contains the ligand-binding portion of a human TNF- receptor fused to the Fc portion of human IgG1, and binds to TNF- and prevents it from interacting with its receptors. It is approved in the 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.

Il-1 Inhibition

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-1  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.

Alefacept

(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.

Targeting B Cells

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.Tolerance

Immunosuppression has concomitant risks of opportunistic infections and secondary tumors. Therefore, the ultimate goal of research on organ transplantation and auto-immune diseases is to induce and maintain immunological tolerance, the active state of antigen-specific nonresponsiveness (Krensky and Clayberger, 1994). Tolerance, if attainable, would represent a true cure for conditions discussed earlier in this section without the side effects of the various immunosuppressive therapies. The calcineurin inhibitors prevent tolerance induction in some, but not all, preclinical models (Van Parijs and Abbas, 1998). In these same model systems, sirolimus does not prevent tolerance and may even promote tolerance induction (Li et al., 1998). Several other promising approaches are being evaluated in clinical trials. Because they remain experimental, they are discussed only briefly here.

Co-Stimulatory Blockade

Induction of specific immune responses by T lymphocytes requires two signals: an antigen-specific signal via the T-cell receptor and a co-stimulatory signal provided by the interaction of molecules such as CD28 on the T lymphocyte and CD80 and CD86 on the antigen-presenting cell (Figure 35–4; Khoury et al., 1999).

Figure 35–4.

Co-stimulation. A. Two signals are required for T-cell activation. Signal 1 is via the T-cell receptor (TCR), and signal 2 is via a co-stimulatory receptor–ligand pair. Both signals are required for T-cell activation. Signal 1 in the absence of signal 2 results in an inactivated T cell. B. One important co-stimulatory pathway involves CD28 on the T cell and B7-1 (CD80) and B7-2 (CD86) on the antigen-presenting cell (APC). After a T cell is activated, it expresses additional co-stimulatory molecules. CD152 is CD40 ligand, which interacts with CD40 as a co-stimulatory pair. CD154 (CTLA4) interacts with CD80 and CD86 to dampen or downregulate an immune response. Antibodies against CD80, CD86, and CD152 are being evaluated as potential therapeutic agents. CTLA4-Ig, a chimeric protein consisting of part of an immunoglobulin molecule and part of CD154, also has been tested as a therapeutic agent. (Adapted with permission from Clayberger, C., and Krensky, A.M. Mechanisms of allograft rejection. In, Immunologic Renal Diseases. (Nielson, E.G., and Couser, W.G., eds) Lippincott-Raven, Philadelphia, 2001. (http://lww.com).)

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.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.Immunostimulation

General Principles

In contrast to immunosuppressive agents that inhibit the immune response in transplant

rejection and auto-immunity, a few immunostimulatory drugs have been developed with applicability to infection, immunodeficiency, and cancer. Problems with such drugs include systemic (generalized) effects at one extreme or limited efficacy at the other.

Immunostimulants

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.

Thalidomide

Thalidomide (THALOMID) is best known for the severe, life-threatening birth defects it caused when administered to pregnant women. For this reason, it is available only under a restricted distribution program and can be prescribed only by specially registered physicians who understand the risk of teratogenicity if thalidomide is used during pregnancy. Thalidomide should never be taken by women who are pregnant or who could become pregnant while taking the drug. Nevertheless, it is indicated for the treatment of patients with erythema nodosum leprosum (Chapter 56) and multiple myeloma. In addition, it has orphan drug status for mycobacterial infections, Crohn's disease, HIV-associated wasting, Kaposi sarcoma, lupus, myelofibrosis, brain malignancies, leprosy, graft-versus-host disease, and aphthous ulcers.

Its mechanism of action is unclear (see Figure 62–4). Reported immunological effects vary substantially under different conditions. For example, thalidomide has been reported to decrease circulating TNF- in patients with erythema nodosum leprosum but to increase it in patients who are HIV seropositive. Alternatively, it has been suggested that the drug affects angiogenesis (Paravar and Lee, 2008). The anti–TNF- effect has led to its evaluation as a treatment for severe, refractory rheumatoid arthritis.

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.

Bacillus Calmette-GuéRin (BCG)

Live BCG (TICE BCG, THERACYS) is an attenuated, live culture of the bacillus of Calmette and Guérin strain of Mycobacterium bovis that induces a granulomatous reaction at the site of administration. By unclear mechanisms, this preparation is active against tumors and is indicated for the treatment and prophylaxis of carcinoma in situ of the urinary bladder and for prophylaxis of primary and recurrent stage Ta and/or T1 papillary tumors after transurethral resection (Patard et al., 1998). Adverse effects include hypersensitivity, shock, chills, fever, malaise, and immune complex disease.

Recombinant Cytokines

Interferons

Although interferons ( , , and ) initially were identified by their antiviral activity, these agents also have important immunomodulatory activities (Ransohoff, 1998). The interferons bind to specific cell-surface receptors that initiate a series of intracellular events: induction of certain enzymes, inhibition of cell proliferation, and enhancement of immune activities, including increased phagocytosis by macrophages and augmentation of specific cytotoxicity by T lymphocytes.

Recombinant IFN- -2b (INTRON A) is obtained from Escherichia coli by recombinant expression. It is a member of a family of naturally occurring small proteins with molecular weights of 15,000-27,600 Da, produced and secreted by cells in response to viral infections and other inducers. IFN- -2b is indicated in the treatment of a variety of tumors, including hairy cell leukemia, malignant melanoma, follicular lymphoma, and AIDS-related Kaposi sarcoma (Sinkovics and Horvath, 2000). It also is indicated for infectious diseases, chronic hepatitis B, and condylomata acuminata. In addition, it is supplied in combination with ribavirin (REBETRON) for treatment of chronic hepatitis C in patients with compensated liver function not treated previously with IFN- -2b or who have relapsed after IFN- -2b therapy (Lo Iacono et al., 2000).

Flu-like symptoms, including fever, chills, and headache, are the most common adverse effects after IFN- -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 interferons carry a boxed warning regarding development of pulmonary hypertension.

IFN- -1b (ACTIMMUNE) is a recombinant polypeptide that activates phagocytes and induces their generation of oxygen metabolites that are toxic to a number of microorganisms. It is indicated to reduce the frequency and severity of serious infections associated with chronic granulomatous disease and to delay the time to progression in severe malignant osteopetrosis. IFN- -1b is not effective and may increase mortality in patients with idiopathic pulmonary fibrosis. Adverse reactions include fever, headache, rash, fatigue, GI distress, anorexia, weight loss, myalgia, and

depression.

IFN- -1a (AVONEX, REBIF), a 166–amino acid recombinant glycoprotein, and IFN- -1b (BETASERON), a 165–amino acid recombinant protein, have antiviral and immunomodulatory properties. They are FDA- approved for the treatment of relapsing MS to reduce the frequency of clinical exacerbations (see "Multiple Sclerosis"). The mechanism of their action in MS is unclear. Flu-like symptoms (e.g., fever, chills, myalgia) and injection-site reactions have been common adverse effects.

Further discussion of the use of these and other interferons in the treatment of viral diseases can be found in Chapter 58.

Interleukin-2

Human recombinant IL-2 (aldesleukin, PROLEUKIN; des-alanyl-1, serine-125 human IL-2) is produced by recombinant DNA technology in E. coli (Taniguchi and Minami, 1993). This recombinant form differs from native IL-2 in that it is not glycosylated, has no amino-terminal alanine, and has a serine substituted for the cysteine at amino acid 125 (Doyle et al., 1985). The potency of the preparation is represented in International Units in a lymphocyte proliferation assay such that 1.1 mg of recombinant IL-2 protein equals 18 million IU. Aldesleukin has the following in vitro biological activities of native IL-2: enhancement of lymphocyte proliferation and growth of IL-2-dependent cell lines, enhancement of lymphocyte-mediated cytotoxicity and killer cell activity, and induction of IFN- activity (Whittington and Faulds, 1993). In vivo administration of aldesleukin in animals produces multiple immunological effects in a dose-dependent manner. Cellular immunity is profoundly activated with lymphocytosis, eosinophilia, thrombocytopenia, and release of multiple cytokines (e.g., TNF, IL-1, IFN- ). Aldesleukin is indicated for the treatment of adults with metastatic renal cell carcinoma and melanoma.

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.

Immunization

Immunization may be active or passive. Active immunization involves stimulation with an antigen to develop immunological defenses against a future exposure. Passive immunization involves administration of pre-formed antibodies to an individual who is already exposed or is about to be exposed to an antigen.

Vaccines

Active immunization, vaccination, involves administration of an antigen as a whole, killed (inactivated) organism; attenuated (live) organism; or a specific protein or peptide constituent of an organism. Booster doses often are required, especially when killed organisms are used as the immunogen. In the U.S., vaccination has sharply curtailed or

practically eliminated a variety of major infections, including diphtheria, measles, mumps, pertussis, rubella, tetanus, Haemophilus influenzae type b, and pneumococcus.

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.

Immune Globulin

Passive immunization is indicated when an individual is deficient in antibodies because of a congenital or acquired immunodeficiency, when an individual with a high degree of risk is exposed to an agent and there is inadequate time for active immunization (e.g., measles, rabies, hepatitis B), or when a disease is already present but can be ameliorated by passive antibodies (e.g., botulism, diphtheria, tetanus). Passive immunization may be provided by several different products (Table 35–2). Nonspecific immunoglobulins or highly specific immunoglobulins may be provided based on the indication. The protection provided usually lasts 1-3 months. Immune globulin is derived from pooled plasma of adults by an alcohol-fractionation procedure. It contains largely IgG (95%) and is indicated for antibody-deficiency disorders, exposure to infections such as hepatitis A and measles, and specific immunological diseases such as immune thrombocytopenic purpura and Guillain-Barré syndrome. In contrast, specific immune globulins ("hyperimmune") differ from other immune globulin preparations in that donors are selected for high titers of the desired antibodies.

Table 35–2 Selected Immune Globulin Preparations

GENERIC NAME COMMON SYNONYMS

ORIGIN BRAND NAME

Antithymocyte globulin

ATG Rabbit THYMOGLOBULIN 

Botulismimmune globulin intravenous

BIG-IV Human BABYBIG 

Cytomegalovirus immune globulin intravenous

CMV-IGIV Human CYTOGAM 

Hepatitis B immune globulin

HBIG Human HEPAGAM B, HYPERHEP B S/D, NABI-HB 

Immune globulin intramuscular

Gamma globulin, IgG, IGIM

Human GAMASTAN S/D 

Immune globulin intravenous

IVIG Human CARIMUNE NF, FLEBOGAMMA 5%, GAMMAGARD LIQUID, GAMUNEX, IVEEGAM EN, OCTAGAM, PRIVIGEN 

Immune globulin subcutaneous

IGSC Human VIVAGLOBIN 

Lymphocyte immune globulin

ALG, antithymocyte globulin (equine), ATG (equine)

Equine ATGAM 

Rabies immune globulin

RIG Human HYPERRAB S/D, IMOGAM RABIES–HT 

Rho(D) immune globulin intramuscular

Rho[D] IGIM Human HYPERRHO S/D, RHOGAM 

Rho(D) immune globulin intravenous

Rho[D] IGIV Human RHOPHYLAC, WINRHO SDF 

Rho(D) immune globulin microdose

Rho[D] IG microdose

Human HYPERRHO S/D MICRODOSE, MICRHOGAM 

Tetanus immune globulin

TIG Human BAYTET 

Vaccinia immune globulin intravenous

VIGIV Human Generic

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 t1/2 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.

Intravenous Immunoglobulin (Ivig)

In recent years, indications for the use of IVIG have expanded beyond replacement therapy for agammaglobulinemia and other immunodeficiencies to include a variety of bacterial and viral infections, and an array of auto-immune and inflammatory diseases as diverse as thrombocytopenic purpura, Kawasaki disease, and auto-immune skin, neuromuscular, and neurological 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 Case Study: Immunotherapy for Multiple Sclerosis

Clinical Features and Pathology

MS is a demyelinating inflammatory disease of the CNS white matter that displays a triad of pathogenic symptoms: mononuclear cell infiltration, demyelination, and scarring (gliosis). The peripheral nervous system is uninvolved. The disease, which may be episodic or progressive, occurs in early to middle adulthood with prevalence increasing from late adolescence to 35 years of age and then declining. MS is roughly 3-fold more common in females than in males and occurs mainly in higher latitudes of the temperate climates. Epidemiologic studies suggest a role for environmental factors in the pathogenesis of MS; despite many suggestions, associations with infectious agents have proven inconclusive, even though several viruses can cause similar demyelinating diseases in laboratory animals and humans. A stronger linkage is the genetic one: people of northern European ancestry have a higher susceptibility to MS, and studies in twins and siblings suggest a strong genetic component of susceptibility to MS.

Specifically, MS is a complex genetic disease in which multiple allelic variants lead to disease susceptibility. Although there is long-range linkage disequilibrium in the MHC region, HLA-DR2 clearly is associated with risk of developing MS (p = 10–228), as is HLA-B*4402. Genome-wide association studies have identified predominantly immune-related variants associated with disease risk, including the IL-2RA chain (p = 10–27), IL-7R chain (p = 10–20), CLEC16A (p = 10–15), CD58 (LFA-3, p = 10–10), and CD226 (p = 10–8) (IMSGC, 2007; IMSGC, 2008; Hafler, 2008). It is estimated that >200 common allelic variants will be uncovered as genome-wide association studies become properly powered. Interestingly, these variants are strikingly common among the different auto-immune diseases.

There also is substantial evidence of an auto-immune component to MS: in MS patients, there are activated T cells that are reactive to different myelin antigens, including myelin basic protein (MBP). In addition, there is evidence for the presence of auto-antibodies to myelin oligodendrocyte glycoprotein (MOG) and to MBP that can be eluted from the CNS plaque tissue, although it appears unlikely that high-affinity auto-antibodies are present in the circulation. These antibodies may act with pathogenic T cells to produce some of the cellular pathology of MS. The neurophysiological result is altered conduction (both positive and negative) in myelinated fibers within the CNS (cerebral white matter, brain stem, cerebellar tracts, optic nerves, spinal cord); some alterations appear to result from exposure of voltage-dependent K+channels that normally are covered by myelin.

Attacks are classified by type and severity and likely correspond to specific degrees of CNS damage and pathological processes. Thus, physicians refer to relapsing-remitting MS (the form in 85% of younger patients), secondary progressive MS (progressive neurological deterioration following a long period of relapsing-remitting disease), and primary progressive MS ( 15% of patients, wherein deterioration with relatively little inflammation is apparent at onset). De Jager and Hafler (2007) have reviewed current concepts of the etiology, natural history, and current therapy of MS.

Pharmacotherapy for MS

Specific therapies are aimed at resolving acute attacks, reducing recurrences and exacerbations, and slowing the progression of disability (Table 35–3). Nonspecific therapies focus on maintaining function and quality of life. For acute attacks, pulse glucocorticoids often are employed (typically, 1 g/day of methylprednisolone administered intravenously for 3-5 days). There is no evidence that tapered doses of oral prednisone are useful or even desirable.

Table 35–3 Pharmacotherapy of Multiple Sclerosis

THERAPEUTIC AGENT

BRAND NAME (DOSE, REGIMEN)

INDICATIONS RESULTS MECHANISM OF ACTION

IFN- -1a AVONEX (30 g, IM, weekly)

REBIFF (22 or 44 g, SC, 3 times weekly)

Treatment of RRMS

Reduction of relapses by one-third

Reduction of new MRI T2 lesions and the volume of enlarging T2 lesions

Reduction in the number and volume of Gd-enhancing lesions

Slowing of brain atrophy

Acts on blood-brain barrier by interfering with T-cell adhesion to the endothelium by binding VLA-4 on T cells or by inhibiting the T-cell expression of MMP

Reduction in T cell activation by interfering with HLA class II and co-stimulatory molecules B7/CD28 and CD40:CD40L

Immune deviation of Th2 over Th1 cytokine profile

IFN- -1b BETASERON (0.25 mg, SC, every other day after 6-week titration) 

Treatment of RRMS

Same as IFN- -1a, above

Same as IFN- -1a, above

Glatiramer acetate COPAXONE (20 mg, SC, daily) 

Treatment of RRMS

Reduction of relapses by one-third

Reduction in the number

Induces T-helper type 2 cells that enter the CNS; mediates bystander suppression at sites of inflammation

and volume of Gd-enhancing lesions

Mitoxantrone NOVANTRONE, generic (12 mg/m2, as short [5–15 minute] IV infusion every 3 months) 

Worsening forms of RRMS

SPMS

Reduction in relapses by 67%

Slowed progression on EDSS, ambulation index, and MRI disease activity

Intercalates DNA (see Chapter 61)

Suppresses cellular and humoral immune response

EDSS, Expanded Disability Status Scale, a neurologic assessment scale for MS pathology. Gd, gadolinium, used in Gd-enhanced MRI to assess the number and size of inflammatory brain lesions; IFN, interferon; IM, intramuscularly; IV, intravenously; MMP, matrix metalloprotease; MS, multiple sclerosis; RRMS, relapsing-remitting MS; SC, subcutaneously; SPMS, secondary progressive MS; MRI, magnetic resonance imaging. For reducing the recurrence of relapsing-remitting attacks, immunomodulatory therapies are approved: -1 interferons [IFN- -1a, IFN- -1b], and glatiramer acetate (GA; COPAXONE). The interferons suppress the proliferation of T lymphocytes, inhibit their movement into the CNS from the periphery, and shift the cytokine profile from pro- to anti-inflammatory types.

Random polymers that contain amino acids commonly used as MHC anchors and T cell–receptor contact residues have been proposed as possible "universal APLs (altered peptide ligands)." GA is a random-sequence polypeptide consisting of four amino acids [alanine (A), lysine (K), glutamate (E), and tyrosine (Y) at a molar ratio of A:K:E:Y of 4.5:3.6:1.5:1] with an average length of 40-100 amino acids. Directly labeled GA binds efficiently to different murine H2 I-A molecules, as well as to their human counterparts, the MHC class II DR molecules, but does not bind MHC class II DQ or MHC class I molecules in vitro. In phase III clinical trials, GA, administered subcutaneously to patients with relapsing-remitting MS, decreased the rate of exacerbations by 30% (De Jager and Hafler, 2007). In vivo administration of GA induces highly cross-reactive CD4+ T cells that are immune deviated to secrete Th2 cytokines and prevents the appearance of new lesions detectable by magnetic resonance imaging. This represents one of the first successful uses of an agent that ameliorates auto-immune disease by altering signals through the T cell–receptor complex.

For relapsing-remitting attacks and for secondary progressive MS, the alkylating agent cyclophosphamide (De Jager and Hafler, 2007) and the anthracenedione-derivative mitoxantrone (NOVANTRONE, others) currently are used in patients refractory to other immunomodulators. These agents, primarily used for cancer chemotherapy, have significant toxicities (see Chapter 61 for structures and pharmacology). Although

cyclophosphamide in patients with MS may not be limited by an accumulated dose exposure, mitoxantrone generally can be tolerated only up to an accumulated dose of 100-140 mg/m2 (Crossley, 1984). However, because decreases in left-ventricular ejection fraction (LVEF) and frank congestive heart failure have occurred in patients who have received <100 mg/m2, the FDA now recommends that LVEF be evaluated before initiating therapy, prior to each dose, and annually after patients have finished treatment to detect late-occurring cardiac toxicity. The utility of interferon therapy in patients with secondary progressive MS is unclear. In primary progressive MS, with no discrete attacks and less observed inflammation, suppression of inflammation seems to be less helpful. A minority of patients at this stage will respond to high doses of glucocorticoids. Table 35–3 summarizes current immunomodulatory therapies for MS.

Each of the agents mentioned in this section has side effects and contraindications that may be limiting: infections (for glucocorticoids), hypersensitivity and pregnancy (for immunomodulators), and prior anthracycline/anthracenedione use, mediastinal irradiation, or cardiac disease (mitoxantrone). With all of these agents, it is clear that the earlier they are used, the more effective they are in preventing disease relapses. What is not clear is whether any of these agents will prevent or diminish the later onset of secondary progressive disease, which causes the more severe form of disability. Given the fluctuating nature of this disease, only long-term studies lasting decades will answer this question.

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 4 integrin, antagonizes interactions with integrin heterodimers containing 4 integrin, such as 4 1 integrin that is expressed on the surface of activated lymphocytes and monocytes. Preclinical data suggest that an interaction of 4 1 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).Clinical Summary

Most transplant centers employ some combination of immunosuppressive drugs with antilymphocyte induction therapy with either a monoclonal or polyclonal antibody agent. Maintenance immunosuppression consists of a calcineurin inhibitor (cyclosporine or tacrolimus), glucocorticoids, and an antimetabolite (azathioprine or mycophenolate). Mycophenolate has largely replaced azathioprine as part of the standard immunosuppressive regimen after transplantation. At present, a number of centers are conducting trials with new drug combinations including either cyclosporine or

tacrolimus in combination with glucocorticoids and mycophenolate, with or without antibody-induction therapy or fingolimod with cyclosporine. Sirolimus is being used to limit exposure to the nephrotoxic calcineurin inhibitors, while steroid avoidance or minimization strategies are used increasingly. Newer immunosuppressive agents are providing more effective control of rejection and permitting transplantation to become an accepted procedure with a number of different organs, including kidney, liver, pancreas, and heart. The apparent effectiveness of new drug combinations has resulted in a resurgence of interest in reducing or avoiding glucocorticoids and calcineum inhibitors.Bibliography

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