CME Clinical Immunology

Oral immunosuppressive drugs The adaptive immune system of both T and B cells relies on the non-specific uptake of antigen by antigen presenting Sujoy Khan MRCP, Immunology Specialist pathological process is the key to under- cells (APCs) (eg monocytes and den- Registrar standing effective immunosuppressive dritic cells). Specific inhibitors of this WA Carrock Sewell PhD FRCP MRCPath, strategies. For example, understanding process are not commercially available. Consultant Immunologist and Professor of the role of the activated CD4+ T cell in Drugs such as minocycline4 and chloro- Immunology cellular rejection of grafts has revolu- quine4,5 can inhibit the processing of Path Links Immunology, Scunthorpe General tionised transplant therapies. antigens by APCs, which is the reason for Hospital; University of Lincoln, Lincoln Many immunosuppressive drugs are their anti-inflammatory effect. used for unlicensed indications. It is diffi- Clin Med 2006;6:352–5 cult to achieve a balance between ade- Non-specific immunosuppressive quate control of the disease process and drugs overimmunosuppression leading to Understanding the immune infections and malignancy. This review Corticosteroids influence numerous gene network examines the commoner agents in use, transcription and post-transcriptional highlighting which part of the immune events and inhibit cytokine production The last few decades have witnessed system they modify, their most important and leukocyte migration, and thus exert remarkable achievements in the develop- side effects and monitoring strategies anti-inflammatory and systemic effects. ment of immunosuppressive drugs, (Tables 1–3). They are used extensively in many fields made possible by significant advances in molecular immunology and improved understanding of the mechanisms by Table 1. Side effect profile of common immunosuppressive agents. which cells communicate. The identifica- tion of the relevant cells involved in a Drug Side effects/ Special precautions

Ciclosporin A Very common (≥10%): nephrotoxicity, tremor, headache, ↑BP, ↑lipids Key Points Common (≥1% to <10%): fatigue, anorexia, nausea, vomiting, abdominal pain, diarrhoea, gingival hyperplasia Rare: confusion, haematuria, palpitations, paraesthesiae, myotoxicity Many drugs are immunosuppressive; Creatinine >130 µmol/l: adjust dose; >180 µmol/l: withhold knowledge of how they work, Tacrolimus Frequent: anaemia, nephrotoxicity, neurotoxicity, hyperkalaemia, tremor, combined with understanding the nausea, fever, diarrhoea, hypertension, hyperglycaemia immunopathology of the disease, Less frequent: vomiting, ascites, pancreatitis, alopecia, cardiomyopathy allows better selection of an ECG (or echo) for arrhythmias: dose reduction or discontinue effective agent Sirolimus Common: oedema, hyperlipidaemia (worse in diabetics), hypertension Some immunosuppressive agents Less common/rare: rash, venous thromboembolism, skin lymphoma require blood level monitoring to Monitoring of blood levels during hepatic impairment recommended reduce toxicity, but the drug level Everolimus Common: leukopenia, thrombocytopenia, hyperlipidaemia is not proportional to the degree Significantly increased risk of rejection at trough levels <3ng/ml of immunosuppression Halve dosage in hepatic impairment

Combinations of agents are usually Mycophenolate Common: haematological (anaemia, leukopenia, thrombocytopenia), GI more effective than single agents; (nausea, vomiting, diarrhoea, haemorrhagic gastritis, erosive enterocolitis) they reduce the incidence of Fewer GI side effects with enteric-coated mycophenolate sodium toxicity by allowing the use of Reduce dose in patients with decreased GFR lower doses Dose-related bone marrow suppression, hepatic impairment, nausea, changes in hair colour/texture, pancreatitis, hepatic veno-occlusive Excess immunosuppression may disease (rare) manifest as infections, but an Prior testing with TPMT assay (phenotype or genotype) recommended increased incidence of malignancy Common: GI, haematological, mucocutaneous toxicity pneumonitis (5%): should also be borne in mind stop methotrexate Obtain baseline chest radiograph Several new immunosuppressive agents are in advanced stages of Common: nausea, vomiting, alopecia, bone marrow suppression, development and may have novel bladder toxicity, infertility, carcinogenicity (most are dose-dependent) and unexpected applications Adequate hydration (for oral and iv), mesna administration during iv bolus therapy reduces bladder toxicity

KEY WORDS: azathioprine, ciclosporin, BP = blood pressure; GFR = glomerular filtration rate; GI = gastrointestinal; iv = intravenous; TPMT = mycophenolate, oral thiopurine methyltransferase. immunosuppressive drugs

352 Clinical Medicine Vol 6 No 4 July/August 2006 CME Clinical Immunology of medicine and have been reviewed channels. Calcineurin inhibitors cause Several forms of CSA are available widely elsewhere.6 Since all nucleated apoptosis of activated T cells as well as including microemulsions and capsules. cells express glucocorticoid receptor, inhibiting the production of the key CSA has a narrow therapeutic window, adverse effects associated with long-term Tcell cytokine IL-2. with wide inter- and intrapatient phar- use are inevitable and adequate prophy- Calcineurin inhibitors such as macokinetics. It is essential always to pre- laxis to protect bone must be considered. ciclosporin (CSA) and tacrolimus revolu- scribe CSA by brand name as there are Metabolites of cyclophosphamide tionised transplantation surgery but are wide differences in bioavailability alkylate DNA bases and affect both T and associated with significant dose-limiting between products.10 B cells. Low doses of cyclophosphamide side effects. Blood calcineurin inhibitor affect cell-mediated immunity more concentrations correlate with toxicity but Mammalian target of rapamycin than bolus doses which tend to impair do not directly translate into degree of antibody responses. Cyclophosphamide immunosuppression. T cell counts and Tcell function is also controlled by bolus doses drop the B cell counts and Tcell proliferation assays do not correlate another enzyme, mammalian target of significantly more CD8+ T cells than with immunosuppression either, rapamycin (mTOR), which is essential CD4+T cells.7 Cyclophosphamide works although tests such as ImmuKnow9 are for regulating intracellular signalling synergistically with steroids and other being assessed and may be commercially through the IL-2 receptor. Inhibition of immunosuppressive agents like azathio- available in the near future. mTOR results in arrest of cell-cycle prine (AZA). Methotrexate is a folic acid antagonist, given once weekly, which inhibits the Table 2. Therapeutic drug levels and useful laboratory tests in monitoring of enzyme dihydrofolate reductase. Its main immunosuppressive agents.* effect is inhibition of DNA synthesis but Drug Therapeutic levels Useful laboratory tests** it also interferes with RNA and protein synthesis and causes clonal deletion of Ciclosporin A 100–300 ng/l (12-hour pre-dose U&Es (↑creat, ↑K+, ↓Mg2+, activated T cells.8 Methotrexate sup- trough whole blood) ↑uric acid), LFTs (cholestasis) presses inflammatory responses through Mark separate lumens for administration and level testing in release of adenosine; this induces apop- iv regimens tosis of activated lymphocytes and Tacrolimus 5–15ng/ml (whole blood trough U&Es (↑creat, ↑K+, ↓Mg2+), inhibits the synthesis of and levels for adult and paediatric liver LFTs (hepatic dysfunction), pyrimidines. Methotrexate is well and renal transplant); ↑glucose absorbed orally, but subcutaneous, intra- >30 ng/ml: toxic muscular or intravenous bioavailability Sirolimus Pre-dose (trough) blood-levels FBC (all lineages affected), is higher. with CSA: 4–12 µg/l; pre-dose lipid profile (↑cholesterol, levels of CSA: 12–20 µg/l ↑triglycerides), U&Es (↑creat) Everolimus Trough: 3–8 ng/l FBC (anaemia, ↓platelets), Specific immunosuppressive >8 ng/l: increased toxicity1 lipid profile (↑cholesterol, drugs ↑triglycerides) Mycophenolate Trough ≥2 mg/l: less rejection FBC weekly first 4 weeks, The advent of HIV and AIDS forcibly early post-transplant (cardiac then twice monthly for demonstrated the importance of the T allograft);2 levels >10 mg/l: 2 months, then monthly for helper cell in controlling the immune risk of side effects a year response. T cells recognise antigen pre- Azathioprine Drug levels not routinely available FBC weekly first 4 weeks, sented to them by APCs using the T cell TPMT metabolises azathioprine; then 3-monthly, ↑ receptor (TCR). Following TCR engage- risk of myelosuppression in low LFTs (cholestasis), U&Es activity and homozygous-deficient ment, a complex cascade of signals patients results in activation and proliferation of Methotrexate Drug levels not routinely available FBC, LFTs, creat every the T cell. This process is critically 4–8 weeks dependent upon the concentration of Rising procollagen III intracellular calcium. aminopeptide levels may predict hepatic fibrosis23 Cyclophosphamide Drug levels not routinely available FBC, U&Es, urinalysis every Calcineurin inhibitors 2–4 weeks

The calcium-dependent phosphatase cal- *Drug levels may be monitored using a variety of approaches; local laboratories should be consulted cineurin is critical for diverse T cell sig- regarding the timing of samples in relation to the dose and acceptable blood levels. nalling activities such as interleukin **Brackets denote common abnormalities. creat = creatinine; CSA = ciclosporin; FBC = full blood count; iv = intravenous; LFT = liver function test; (IL)-2 production, apoptosis, cyto- TPMT = thiopurine methyltransferase; U&E = urea and electrolyte. skeletal deployment and control of ion

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Table 3. Effect of immunosuppressive drugs on T cells and immunoglobulin (Ig) block DHOD activity.17 is levels. used as an immunomodulatory agent and Drug T cells Ig is licensed as a disease-modifying anti- rheumatic drug in .18 Ciclosporin A ↓CD4 T cells Total Igs not significantly altered, but specific The malononitrilamide FK778 is a new antibody levels may fall class of low molecular weight immuno- ↓ Tacrolimus CD4 T cells Total Igs not significantly altered, but specific suppressants that block both T cell and antibody levels may fall, usually less than with 19 ciclosporin humoral immune responses. Sirolimus ↓CD4 T cells Not established hypogammaglobulinaemia seen when used with mycophenolate Newer immunomodulatory Everolimus ↓ Not established agents Mycophenolate ↓ May cause hypogammaglobulinaemia Thalidomide and its analogues, lenalido- Azathioprine ↓ May cause severe hypogammaglobulinaemia mide and CC-4047 (Actimid) provide Methotrexate ↓(variable counts) May cause hypogammaglobulinaemia (rare) immunomodulation through inhibition ↓ Cyclophosphamide (CD8 more May cause hypogammaglobulinaemia of tumour necrosis factor-α production than CD4) ↓ ↓ in monocytes and costimulatory effects Glucocorticoids (CD4 more IgG and IgA levels with long-term use + 20 than CD8) on human CD8 Tcells. Mizoribine, like mycophenolate, inhibits inosine monophosphate dehy- drogenase; long-term use seems to pre- progression and inhibition of prolifera- thrombocytopenia. These side effects are vent renal relapses and reduce tion of T cells, B cells and vascular generally dose-dependent and resolve in prednisolone requirement among severe smooth muscle in response to cytokine 7–10 days with dose reduction. proliferative lupus nephritis patients. A signals. Two mTOR inhibitors, sirolimus Individuals with a polymorphism of few anecdotal reports show mizoribine is (previously known as rapamycin) and the thiopurine methyl transferase effective in steroid-resistant nephrotic everolimus, are used in various centres enzyme (TPMT) cannot metabolise AZA syndrome.21 for transplant immunosuppression. effectively, leading to severe side effects. 15-Deoxyspergualin has properties Their side effect profile is different to Screening for this is now possible in similar to CSA and tacrolimus. It inhibits that of calcineurin inhibitors, causing advance of AZA therapy by measuring the activation of APCs and monocytes less nephro- and neurotoxoicity but a TPMT activity (phenotype) or genotype. without affecting IL-2 synthesis.22 greater incidence of hyperlipidaemia and Patients with intermediate TPMT FTY 720 is a synthetic molecule struc- bone marrow suppressive effects. Both activity or who are heterozygotes need a turally related to myriocin, a fungal calcineurin and mTOR inhibitors can be 50% reduction in the standard dose; metabolite. FTY 720 reduces the number combined, thereby reducing the dose- those with homozygous TPMT defi- of circulating lymphocytes, especially limiting calcineurin inhibitor side ciency should not receive AZA or only Thelper cells, by downregulation of effects.11 extremely low doses.13,14 sphingosine receptors and redirecting A subset of T cells, CD4+CD25+ regula- Mycophenolic acid inhibits inositol them to the lymph nodes. FTY 720 has tory T cells (TREG), play an important role monophosphate dehydrogenase, pre- shown considerable promise in pro- in allograft tolerance; their functional venting de novo biosynthesis in a longing renal allograft transplant sur- activity depends on signalling via the IL-2 similar way to AZA. Its function is unique vival23 and is undergoing trials in receptor. Rapamycin promotes apoptosis in that it blocks IL-2-mediated prolifera- pancreatic islet cell transplantation for of alloreactive T cells and strongly pre- tion but does not inhibit cell survival and type 1 diabetes. serves TREG, in contrast to CSA, which Fas-mediated apoptosis of activated 15 may favour the use of rapamycin in the Tlymphocytes. Mycophenolic acid- Practicalities of 12 induction of tolerance. based drugs include mycophenolate immunosuppression mofetil (MMF) and a newer agent, Treatment with immunosuppressive De novo nucleotide synthesis mycophenolate sodium, an enteric- therapies requires an understanding of inhibitors coated prodrug of mycophenolic acid associated with fewer gastrointestinal side the speed of onset of the drugs and effec- AZA is enzymatically converted and effects than MMF.16 tive monitoring systems (Table 2). incorporated into DNA, inhibiting Dihydroorotate dehydrogenase (DHOD) Corticosteroids, acting within hours, purine biosynthesis and proliferation of is a key enzyme in de novo pyrimidine syn- enable rapid symptom control. Bolus T and B lymphocytes. The major side thesis. Leflunomide, FK778 (a lefluno- dose cyclophosphamide begins to work effect of AZA is therefore myelosuppres- mide derivative), brequinar sodium and within days. Other drugs need to be sion, including leukopenia, anaemia and atovaquone are chemically distinct but all started early because their onset of action

354 Clinical Medicine Vol 6 No 4 July/August 2006 CME Clinical Immunology is considerably slower. Effective blood collagen III aminopeptide for monitoring urine methyltransferase status in rheuma- levels of calcineurin inhibitors are patients with psoriasis receiving long-term tology patients receiving azathioprine. reached in days, but sustained effects methotrexate: a multicentre audit and Rheumatology (Oxford) 2004;43:13–8. health economic analysis. Br J Dermatol 14 Cara CJ, Pena AS, Sans M et al. Reviewing may be seen only after several weeks, 2005;152:444–50. the mechanism of action of thiopurine whereas AZA may take two or three 4Kalish RS, Koujak S. Minocycline inhibits drugs: towards a new paradigm in clinical months to peak effect. As non-steroid antigen processing for presentation to practice. Med Sci Monit 2004;10: RA247–54. agents begin to take effect, the steroid human T cells: additive inhibition with 15 Nakamura M, Ogawa N, Shalabi A et al. dose can gradually be tapered. at therapeutic concentrations. Positive effect on T-cell regulatory apoptosis Clin Immunol. 2004;113:270-7. by mycophenolate mofetil. Clin Transplant A good general principle is that pow- 5Accapezzato D, Visco V, Francavilla V et al. 2001;15(Suppl 6):36–40. erful combinations of immunosuppres- Chloroquine enhances human CD8+ Tcell 16 Behrend M, Braun F. Enteric-coated sants (eg methyl prednisolone with responses against soluble antigens in vivo. mycophenolate sodium: tolerability profile cyclophosphamide) are used initially, JExp Med2005;202:817–28. compared with mycophenolate mofetil. with milder agents (eg AZA or 6Boumpas DT, Chrousos GP, Wilder RL, Drugs 2005;65:1037–50. Cupps TR, Balow JE. Glucocorticoid 17 Hansen M, Le Nours J, Johansson E et al. methotrexate) replacing them once dis- therapy for immune-mediated diseases: Inhibitor binding in a class 2 dihydroorotate ease control is established. However, this basic and clinical correlates. Review. Ann dehydrogenase causes variations in the approach may not be required in all clin- Intern Med 1993;119:1198–208. membrane-associated N-terminal domain. ical settings. Disease-specific, evidence- 7Lacki JK, Mackiewicz SH, Leszczynski P, Review. Protein Sci 2004;13:1031–42. based guidelines should be consulted Muller W. The effect of intravenous 18 Li EK, Tam LS, Tomlinson B. Leflunomide cyclophosphamide pulse on peripheral in the treatment of rheumatoid arthritis. frequently as new studies emerge. blood lymphocytes in lupus erythematosus Review. Clin Ther 2004;26:447–59. patients. Rheumatol Int 1997;17:55–60. 19 Fitzsimmons WE, First MR. FK778, a syn- 8Genestier L, Paillot R, Fournel S et al. thetic malononitrilamide. Yonsei Med J The future Immunosuppressive properties of metho- 2004;45:1132–5. Oral immunosuppressive drugs are trexate: apoptosis and clonal deletion of 20 Crane E, List A. Immunomodulatory drugs. activated peripheral T cells. J Clin Invest Review. Cancer Invest 2005;23:625–34. widely used for both licensed and unli- 1998;102:322–8. 21 Yokota S. Mizoribine: mode of action and censed indications. All physicians need 9Hooper E, Hawkins DM, Kowalski RJ et al. effects in clinical use. Review. Pediatr Int to be aware of the possible uses, interac- Establishing pediatric immune response 2002;44:196–8. tions and side effects of these drugs as zones using the Cylex ImmuKnow assay. 22 Holcombe H, Mellman I, Janeway CA Jr, patients may present acutely with infec- Clin Transplant 2005;19:834–9. Bottomly K, Dittel BN. The immunosup- 10 Johnston A, Belitsky P, Frei U et al. Potential pressive agent 15-deoxyspergualin functions tious, malignant or other complications clinical implications of substitution of by inhibiting progression and related to their immunosuppression. generic cyclosporine formulations for cytokine production following naive T cell cyclosporine microemulsion (Neoral) in activation. J Immunol 2002;169:4982–9. transplant recipients. Review. Eur J Clin 23 Budde K, Schmouder RL, Brunkhorst R et References Pharmacol 2004;60:389–95. al. First human trial of FTY720, a novel 11 Nashan B. Maximizing the clinical outcome immunomodulator, in stable renal trans- 1Mabasa VH, Ensom MH. The role of thera- with mTOR inhibitors in the renal trans- plant patients. J Am Soc Nephrol 2002;13: peutic monitoring of everolimus in solid plant recipient: defining the role of cal- 1073–83. organ transplantation. Ther Drug Monit cineurin inhibitors. Review. Transpl Int 2005;27:666–76. 2004;17:279–85. 2 Yamani MH, Starling RC, Goormastic M et 12 Coenen JJ, Koenen HJ, van Rijssen E, al. The impact of routine mycophenolate Hilbrands LB, Joosten I. Rapamycin, and mofetil drug monitoring on the treatment of not cyclosporin A, preserves the highly sup- cardiac allograft rejection. Transplantation pressive CD27+ subset of human 2000;69:2326–30. CD4+CD25+ regulatory T cells. Blood 3Chalmers RJ, Kirby B, Smith A et al. 2006;107:1018–23. Replacement of routine liver biopsy by pro- 13 Clunie GP, Lennard L. Relevance of thiop-

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