Immunosuppressants

1. Immunosuppressants

2. Very, very powerful Immunosuppressants available • Non-Pharmacological: • Irradiation • Plasmapheresis  removal of • autoantibodies in diseases like MG • Pharmacological: • deplete T cells or B cells completely with anti-CD3 • or anti-CD20 antibodies • chemotherapy • calcineurin inhibitors, steroids Problem: Immunosuppression (infections)

3. Side-Effects of Immunosuppressants • General Side Effect: Immunosuppression • Reactivation of latent viral infections • (e.g. herpes, varicella, CMV, PML) • Bacterial and fungal infections • Bone marrow suppression • Increased risk of cancer • Specific Side Effects: • Nephrotoxicity of the calcineurin inhibitors • Metabolic changes with steroids • “cytokine release syndrome” with anti-CD3 Ab

4. Corticosteroids • broad anti-inflammatory effects • High dose pulses of i.v. methylprednisolone (SOLU-MERDOL, A-METHAPRED) used to reverse acute transplant rejection and acute exacerbations of autoimmune diseases like MS or SLE • also used to suppress allergic reactions to biologics (e.g. first-dose cytokine storm in transplantation with muromonad-CD3 or thymoglobulin) Invaluable for short-term use!!!

5. Cytotoxic Agents Are typically covered in Oncology. For this lecture it is sufficient you to know that all cytotoxic drugs that are used for cancer therapy also hit all fast dividing cells in the body: • immune system • gut mucosa • hair follicles Cyclophosphamide and methotrexate often used in RA and MS

6. Calcineurin Inhibitors: Cyclosporine Tacrolimus Bind to immunophilins (cyclophilin or FK506-binding protein [FKBP]) forming a complex that inhibits calcineurin Calcineurin does not dephosphorylate NFAT NFAT does not translocate to the nucleus Inhibition of T cell proliferation and IL-2 production

7. Cyclosporine (Cyclosporin A) • cyclic 11 amino acid polypeptide (very lipophilic, orally absorbed) • very effective against T cell dependent responses, less effective against B cells Therapeutic uses: • kidney, liver, heart and other organ transplantation • severe cases of RA • severe disabling psoriasis where other therapies have failed Pharmacokinetics: • Administered i.v. (SANDIMMUNE Injection) or orally • oral availability 20-50% [Different formulations like SANDIMMUNE gelatine capsules and NEORAL microemulsion are NOT bioequivalent; should not be freely substituted!!!] • plasma peak between 1.5 to 2 h; t1/2 5-18 hours • extensively metabolized through CYP3A4; excreted through bile •  Plasma drug levels must be monitored, drug interactions avoided and doses adjusted for hepatic insufficiency

8. Drug Interactions with Cyclosporine • any drug that is metabolized through CYP3A4 affects CsA metabolism • CYP3A4 blockers (ketoconazole, fluconazole, verapamil, idinavir, grape fruit juice) increase CsA plasma levels • CYP3A4 inducers (phenobarbital, phenytoin) reduce CsA plasma levels Cyclosporine Side Effects • Nephrotoxicity: occurs in nearly all patients  cessation or modification of therapy • Hypertension: occurs in ~50% of kidney and 100% of heart transplant patients • Hyperlipidemia • Tremor • Hirsutism and hypertrichosis • Diabetogenic in combination with glucocorticoids

9. Tacrolimus (FK506) • macrolide antibiotic produced by Streptomyces tsukunaensis • available for oral and i.v. administration (PROGRAF) • t1/2 ~12 hours, PK variable. Metabolized through CYP3A4 (same interactions like CsA) Therapeutic Uses: • Similar to CsA but much less commonly used • “Rescue Therapy” in patients who show rejection • despite therapeutic CsA plasma levels Side Effects: • Same as CsA, nephrotoxicity is limiting

10. Sirolimus (Rapamycin) RAPAMUNE • Macrocyclic lactone produced by Streptomyces hygroscopicus (soil of Easter Islands) • Originally developed by Wyeth as an antifungal when it was found to be an immunosuppressant • Mechanism of Action: • Inhibits T cell activation downstream of the IL2 receptor by binding to the immunophilin FKBP-12 • sirolismus-FKBP-12 complex inhibits the protein kinase mTOR (mammalian target of rapamycin), which is a key enzyme in cell-cycle progression • cell cycle arrested in G1-S phase transition • PK: • oral availability ~15%, plasma peak after 1-2 hours, t1/2 = 62 h, metabolized by CYP3A4

11. Sirolimus continued • Therapeutic Uses (introduced in 1999): • used in combination with calcineurin inhibitors and glucocorticoids for organ transplantation (Synergy!!) • in patients experiencing or at high risk for calcineurin-inhibitor associated nephrotoxicity used with glucocorticoids and mycophenolate mofetil to avoid permanent renal damage • also coated onto stents to prevent restenosis following angioplasty • Side Effects: • increase in cholesterol and triglycerides • delayed wound healing ( often started delayed after surgery) • much less nephrotoxic than calcineurin inhibitors but carries warning about associated nephrotoxicity since Oct. 2008

12. Cyclosporine and Sirolismus (rapamycin) are synergistic  Often combined to reduce CsA toxicity

13. Azathioprine (IMURAN) Mechanism of Action (Antimetabolite): Cleaved by nucleophiles like glutathione to 6-mercaptopurine  Inhibits purine synthesis and thus cell proliferation • Therapeutic Uses: • in combination for organ transplantation • severe RA • Side Effects: • bone marrow suppression (leukopenia, sometimes thrombocytopenia) • hepatotoxicity • increased risk of infections and cancer

14. Mycophenolate Mofetil (CELLCEPT) • Prodrug (2-morpholinoethyl ester) that is rapidly hydrolyzed to mycophenolic acid (MPA), which is a reversible inhibitor of ionosine monophosphate dehydrogenase (IMPDH), an enzyme involved in guanine nucleotide synthesis • T and B cells highly dependent on guanine nucleotide synthesis ( other cells can use other pathways)  MPA inhibits T cell and B cell proliferation as well as antibody production • Therapeutic Uses: • Prevention of transplant rejection (typically in combination with glucocorticoids and calcineurin inhibitors) • approved for renal transplant in 1995, other transplants later; indications might widen in the future • Side Effects: • Leukopenia • Increased incidence of infections (in June 2006 Roche and FDA issued • warning about PML; 17 cases, 7 fatal)

15. Progressive Multifocal Leukoencephalopathy (PML) • PML is a rare demyelinating disease of the CNS that usually is fatal or leads to severe disability. PML is caused by reactivation of the JC virus, a polyomavirus that resides latently in 70-90% of adults worldwide. • PML presents with ataxia, hemiparesis, confusion, apathy, cognitive deficiencies and radiographic evidence of white matter disease. • PML should be considered in any transplant or autoimmune disease patient who is on immunosuppression and who presents with neurological symptoms. • Other than reducing the dose of immunosuppressant there are no interventions that can stop or treat PML. [see also Natalizumab for MS treatment]

16. FTY720 sequesters T cells in lymph nodes and prevents them from recirculating Will probably become a new drug for combination therapy FTY720 • Fingolimod (FTY720) is sphingosine-1-phosphate receptor 1 agonist that prevents egress of lymphocytes from lymph nodes • was not superior to calcineurin inhibitors in Phase-3 clinical trial for kidney transplantation as a monotherapy • Very encouraging effects in Phase-2 clinical trial for MS (relapse rate reduced by 80%); various other trials going on

17. Antithymocyte Globin (THYMOGLUBIN) • Purified gamma globulin fraction from the serum of rabbits immunized with human thymocytes • contains cytotoxic Abs that bind to CD2, CD3, CD4, CD8, CD11a, CD18, CD25, CD45 and MHC class I and II (and probably a lot more!!) • Abs deplete circulating lymphocytes by direct cytotoxicity (complement and cell mediated) and block cell surface receptors • Therapeutic Uses: • Approved for acute renal transplant rejection • Often used immediately after kidney transplant if graft function is delayed to avoid early use of nephrotoxic calcineurin inhibitors • Toxicity: • Polyclonal rabbit Ab  xenogenic protein elicits acute allergic reactions (fever, chill, hypotension)  premedication with glucocorticoids, acetaminophen and antihistamines • Anti-ATG Abs develop but do not limit repeated use

18. Anti-CD3 Monoclonal Antibodies • Antibodies directed against the e chain of the T cells receptor CD3  treatment induces rapid internalization of the TCR and is followed by depletion and extravasation of T cells from the blood and lymphoid organs (redistribution to the lungs) • Muromonab-CD3 (OKT3) original mouse IgG2a introduced in 1986 • Fully humanized Ab: hOKTg1; less cytokine release • Therapeutic Uses: • Mouse Ab still used to reverse glucocorticoid resistant rejection episodes (repeated use results in neutralizing Abs) • Humanized Ab for treatment and prevention of acute rejection • Phase-2/3 clinical trials for prevention of T1DM • Side Effects: • “cytokine release syndrome” typically 30 min after infusion (TNFa, IL2, IL6, INFg from activated T cells and macrophages)  high fever, chills, tremor (worst on first dose) • Glucocorticoid administration on first dose is now standard

19. Rituximab (RITUXAN) • monoclonal anti-CD20 Ab approved in 1997 for the treatment of B-cell non-Hodgkin lymphomas • binds to CD20 on the surface of B cells and seems to induce apoptosis of CD20+ cells and/or induce complement and cell mediated cytotoxicity (ADCC), drastically reduces number of circulating B cells • Therapeutic Uses: • Leukemia, lymphoma [see Hem/Onc] • In combination with methotrexate for severe RA • Clinical trials ongoing for various autoimmune diseases including idiopathic autoimmune hemolytic anemia, MS, T1DM, Sjogren's syndrome, SLE • Side Effects: • Infusion reactions similar to anti-CD3 Abs • Immunosupppression [carries Box Warning about PML]

20. Anti-CD25 (Anti-IL-2 Receptor) • Daclizumab (ZENAPAX): humanized chimeric monoclonal Ab • Basiliximab (SIMULET): murine-human chimeric monoclonal Ab • both Abs seem to inhibit activated T cells without significant depletion • Therapeutic Uses: • Prophylaxis of acute transplant rejection (pre-operatively and afterwards in biweekly intervals) • Combination for maintenance therapy with other immuno-suppressants (azathioprine, glucocorticoids, cyclosporine) • Side Effects: • no cytokine release syndrome • anaphylaxis can occur

21. Abatacept (ORENCIA) • fusion protein composed of human immunoglobulin fused to the extra-cellular domain of CTLA-4 • Costimulation of CD28 through B7-1/2 (CD80/CD86) is necessary for full activation of naïve T cells; Engagement of the related receptor CTLA-4 inhibits/reduces T cell activation  Abatacept prevents co-stimulation and can potentially induce tolerance

22. Abatacept continued • Therapeutic Uses: • Currently approved for RA patients who have failed methotrexate and anti-TNF reagents • Currently in clinical trials for T1DM prevention, colitis, psoriasis and transplant rejection • Side Effects: • usual risk of increased infections and malignancies • should not be used in combination with TNF-antagonist (no additional benefit and greatly increased risk of infections)

23. Anti-TNF Reagents • Infliximab (REMICADE): chimeric anti-TNF-a monoclonal Ab containing a human constant and a murine variable region; binds with high affinity to TNF-a and prevents it from binding to its receptor • Etanercept (ENBREL): Fusion protein of Fc portion of human IgG1 and ligand binding portion of TNF-a receptor • Adalimimab (HUMIRA): fully humanized recombinant monoclonal anti-TNF-a antibody for i.v. use • Therapeutic Uses: • Infliximab and Adalimimab: approved for psoriasis, Crohn's disease, ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis and ulcerative colitis • Etanercept (various formulations including autoinjector pen): approved for RA; $4.3 billion sales in 2006!! (7th top selling drug) • Side Effects: • Increase incidence of lymphomas and infections [Black box warning] • Rare cases of demylelination (contra-indicated in MS)

24. Other Biologics [Recently introduced or in Development] • Efalizumab (RAPTIVA): targets LFA-1 and blocks T cell adhesion and activation • Anakinra (KINERET): IL-1 receptor antagonist approved for RA • Alefacept (AMEVIVE): Fusion protein that targets CD2, approved for moderate to severe psoriasis • Campath-1H (ALEMTUZUMAB): humanized anti-CD52 Ab  depletes T cells, B cells, macrophages and NK cells; approved for renal transplantation • Anti-IL-12/IL23 antibody: clinical trials for MS and RA There will be many more new immunosuppressants in future as we understand the immune system better.

25. Therapy of Organ Transplantation • For solid organs like heart and kidney it is rarely possible to achieve perfect HLA matching because of limited supply of organs (exception: live kidney donation from identical twin)  Strong immunosuppression necessary • At most transplant centers intensive biologic induction therapy followed by maintenance therapy Induction Therapy (delays use of nephrotoxic agents): • Anti-CD3 Abs ( anti-CD25) • Antithymocyte globin • plasmapheresis if high titers of anti-HLA antibodies Maintenance Therapy (typically triple therapy at low doses for synergy): • calcineurin inhibitor + glucocorticoid + mycophenolate mofetil • sirolimus + glucocorticoid + mycophenolate mofetil • calcineurin inhibitor + sirolimus + third agent

26. Therapy of Organ Transplantation • Therapy of Established or Acute Rejection: • High dose methylprednisolone • Anti-CD3 Abs ( anti-CD25) • Antithymocyte globin • Calcineurin Inhibitors and kidney transplants: • Ultrasound guided biopsy best way to differentiate nephro-toxicity (too much CsA) from rejection (too little CsA) Future?? Currently a lot of research going on into the possibility of achieving tolerance with sirolimus (spares Tregs in contrast to CsA) and/or abatacept

27. Therapy of Autoimmune Diseases • Typically “milder” immunosuppression used than for prevention of transplant rejection • Choice of therapy should be guided by: • Knowledge about specific pathology of autoimmune disease • Careful weighing of benefits of therapy versus side effects since treatment is typically for the rest of the patient’s life Example of a “bad” strategy: In the late 1980s and early 1990s cyclosporine was used to prevent T1DM during the “honeymoon” period  very effective at preventing T1DM but abandoned because of nephrotoxicity

28. Example: Rheumatoid Arthritis American College of Rheumatology recommends that treatment should be chosen based on: • how long a patient has had rheumatoid arthritis • the severity of rheumatoid arthritis symptoms • potential for side effects • tuberculosis screening (required before starting biologic DMARDs) • cost of treatment (real issue with biologics!!) • patient preference regarding treatment options DMARD = disease modifying anti-rheumatic drugs (as opposed to NSADs like aspirin that purely treat the symptoms)

29. The 2008 ACR Recommendations for Rheumatoid Arthritis Treatments: • Initiating treatment with methotrexate or leflunomide for most RA patients • Methotrexate plus Plaquenil (hydroxychloroquine) for RA patients with moderate to high disease activity • Treatment with TNF-a antagonist (etanercept, infliximab, adalimumab) plus methotrexate for patients with early rheumatoid arthritis (symptoms for less than 3 months) and high disease activity • For RA with moderate to long disease duration, TNF-a antagonists for those who failed to get a satisfactory response from methotrexate therapy • Abatacept and rituximab should be reserved for patients with at least moderate disease activity and inadequate treatment response to other agents • Treatment with methotrexate or the biologics should not be resumed or started during an active bacterial or viral infection • Severe flair-ups: CsA or methylprednisolone

30. Example: Multiple Sclerosis Chronic, immune-mediated demyelinating disease of the CNS that is characterized by inflammation, gliosis and axonal damage Most common neurological disease in young adults (age of onset 15-50 years); affects 400 000 people in the US and about 2.5 million worldwide

31. CD3 CD4 MS is a T cell mediated disease • myelin-antigen specific T cells with a Th1 profile can be isolated both from the blood and the CSF of MS patients • Although myelin-antigen specific T cells are also present in the blood of healthy controls and occur with the same frequency, their activation state in MS patients is different. Myelin-reactive MS patients T cells secrete larger amounts of IL-2, IFN-g and TNF-a than T cells from controls, and are predominantly of a Th1/Th0 memory phenotype Extravasating T cells in a postmortem brain section from a MS patient

32. FDA APPROVED TREATMENT OPTIONS FOR MS Immunomodulatory drugs: • IFN-b • Copaxone (Glatiramer acetate) • Natalizumab Immunosuppressive drugs: • Acute severe attack: methylprednisolone • Cyclophosphamide and mitoxantrone for secondary progressive and primary progressive MS [Diagnosis and symptoms of MS will be covered in Neurology]

33. Interferon-b • naturally occurring cytokine with still unknown mechanism of action: - suppresses T cell proliferation - reduces T cell migration into the CNS - changes cytokine profile from Th1 to Th2 (?) • like most proteins interferons are potentially immunogenic (especially when produced recombinantly  altered glycosylation) and neutralizing Abs appear in many patients  still hotly debated whether these antibodies reduce efficacy of INF-b

34. Glatiramer Acetate (Copaxone) • random polymer of glutamic acid, lysine, alanine and tyrosine (which compose MBP) with unclear mechanism: • induction of tolerance to MBP (?) • induces a population of GA-reactive Th2-type “regulatory” T cells that secrete Th2 cytokines in the brain and create an “anti-inflammatory milieu” either spontaneously or after cross-stimulation with myelin antigens (?) • long-term treatment also seems to induce serum Abs

35. 35 32% 32% 31% 29% 29% 30 P<0.0001 P=0.002* P=0.0004 P=0.007 P<0.0001 25 20 % Reduction in relapse rates 18% P=0.04 15 10 5 0 Avonex 30 mcg Betaseron 875-1000 mcg Copaxone 140 mg Rebif 66 mcg Rebif 132 mcg Phase III Trials: Reduction in Annual Relapse Rate INF-b INF-b *For patients who had been in the study for 2 years. Jacobs et al. Ann Neurol. 1996;39:285; IFNB MS Study Group. Neurology. 1993;43:655; IFNB MS Study Group and University of British Columbia MS/MRI Analysis Group. Neurology. 1995;45:1277; Johnson et al. Neurology. 1995;45:1268; PRISMS Study Group. Lancet. 1998;352:1498;

36. Natalizumab (Tysabri)Anti-VLA-4 Monoclonal Antibody • 66% reduction in relapses • 92% reduction in active MRI lesions • Monthly IV administration • 2/1200 patients treated developed PML and died  Tysabri removed from the market in early 2005 March 2006: an FDA advisory committee recommended that it should return to the market (massive pressure from MS patients and NMSS)

37. Symptom Management of MS • Primary • Caused by actual demyelination within the CNS • Fatigue, tremor, incontinence, spasticity, depression  antidepressants, anti-spastics • Secondary • Caused by failure to manage the primary • Contractures, decubiti, fractures, muscle atrophy  physiotherapy • Tertiary • Psychological, social, marital, vocational, personal  antidepressants, Viagra, Life-style changes