Objectives

1. Objectives Summarize the principles for use of immunosuppression in allogeneic stem cell transplant (SCT) Compare and contrast commonly used medications used for immunosuppression Describe monitoring parameters and common adverse effects associated with immunosuppression

2. Immunosuppression in Bone Marrow Transplant Ashley Newland, PharmD Hematology/Oncology Pharmacist Specialist VCU Medical Center November 8, 2011

3. Cells of the Immune System

4. When a pathogen enters the body, mammals initially attempt to eliminate it by innate (natural) immunity. If the pathogen has previously infected the animal, adaptive (acquired) immunity then operates to specifically exterminate the returning invaderWhen a pathogen enters the body, mammals initially attempt to eliminate it by innate (natural) immunity. If the pathogen has previously infected the animal, adaptive (acquired) immunity then operates to specifically exterminate the returning invader

5. Use of Immunosuppression Allogeneic stem cell transplant Prevention of rejection Component of conditioning regimen Eradicates host T-cells to allow acceptance of donor cells Prevention of graft versus host disease (GVHD) Pre- & post-transplant medications Suppresses donor T-cells to minimize recognition of host cells as foreign Treatment of GVHD Suppresses the host immune system and creates space in the marrow to facilitate engraftment. Prep regimen eradicates immunologically active host tissues such as lymphocytes and macrophages to prevent the development of host versus graft rejection Cyclophosphamide is immunosuppressive After infusion of stem cells immunosuppressant are given to prevent or minimize GVHD. Post-tx meds typically slowly tapered off by 6 months or until GVHD resolvedSuppresses the host immune system and creates space in the marrow to facilitate engraftment. Prep regimen eradicates immunologically active host tissues such as lymphocytes and macrophages to prevent the development of host versus graft rejection Cyclophosphamide is immunosuppressive After infusion of stem cells immunosuppressant are given to prevent or minimize GVHD. Post-tx meds typically slowly tapered off by 6 months or until GVHD resolved

6. Pathophysiology of GVHD The disease is indicative of exaggerated but typical inflammatory mechanisms mediated by DONOR lymphocytes infused into recipient. They function appropriately in view of the foreign environment they encounter. The recipient�s tissues, which have been damaged by disease, infection and chemotherapy release proinflammatory cytokines and chemokinds � tnf alpha, IL1, 6 which increase receptors on antigen presenting cells Activation of APCs Donor t cell activation, proliferation, differentiation, migration Target tissue destruction The disease is indicative of exaggerated but typical inflammatory mechanisms mediated by DONOR lymphocytes infused into recipient. They function appropriately in view of the foreign environment they encounter. The recipient�s tissues, which have been damaged by disease, infection and chemotherapy release proinflammatory cytokines and chemokinds � tnf alpha, IL1, 6 which increase receptors on antigen presenting cells Activation of APCs Donor t cell activation, proliferation, differentiation, migration Target tissue destruction

7. Medications used for immunosuppression

8. Alemtuzumab Anti CD52 monoclonal antibody CD52 expressed on: B and T lymphocytes Monocytes Macrophages NK cells Dendritic cells Functions of mAbs controlled by the Fc region include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (not shown). Certain mAbs can lyse cells (for example, T cells or B cells) through complement activation, whereas other mAbs can bind to Fc receptors and mediate cell lysisFunctions of mAbs controlled by the Fc region include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (not shown). Certain mAbs can lyse cells (for example, T cells or B cells) through complement activation, whereas other mAbs can bind to Fc receptors and mediate cell lysis

9. Alemtuzumab Adverse Effects Infusion related reactions Chills, dyspnea, fevers, hypotension, rigors May be fatal Premedicate with acetaminophen, diphenhydramine, � corticosteroid Hypersensitivity reactions Cytokine release syndrome Opportunistic infections Requires anti-infective prophylaxis CRS results in multiple organ damage CD4 is generally >200 after 2-5 months, but cd4 and cd8 lymphocyte counts may not be back to baseline for more than 1 yr Infections with bacterial, viral, fungal and protozoan infections. PCP px and herpesCRS results in multiple organ damage CD4 is generally >200 after 2-5 months, but cd4 and cd8 lymphocyte counts may not be back to baseline for more than 1 yr Infections with bacterial, viral, fungal and protozoan infections. PCP px and herpes

10. Antithymocyte Globulin (ATG) T cell depleting antibody When used as a prep regimen, levels can still be detected at up to 1 month. T cell depletion is long lived Proposed mechanisms through which ATG can interfere with the immune response. (1) T-cell depletion in blood and peripheral lymphoid tissues through complement-dependent lysis and T-cell activation and apoptosis; (2) induction of B-cell apoptosis; (3) modulation of key cell-surface molecules (adhesion and chemokine receptors) that mediate leukocyte/endothelium interactions; (4) Interference with DC functional properties (maturation and migration); and (5) Induction of Treg and NK-T cells. An ever-growing body of evidence supports important immunoregulatory functions of Treg in maintaining both self-tolerance and tolerance toward autoantigens52 and alloantigens.53 Thus, efforts are currently ongoing to optimize in vitro methods for Treg generationT cell depleting antibody When used as a prep regimen, levels can still be detected at up to 1 month. T cell depletion is long lived Proposed mechanisms through which ATG can interfere with the immune response. (1) T-cell depletion in blood and peripheral lymphoid tissues through complement-dependent lysis and T-cell activation and apoptosis; (2) induction of B-cell apoptosis; (3) modulation of key cell-surface molecules (adhesion and chemokine receptors) that mediate leukocyte/endothelium interactions; (4) Interference with DC functional properties (maturation and migration); and (5) Induction of Treg and NK-T cells. An ever-growing body of evidence supports important immunoregulatory functions of Treg in maintaining both self-tolerance and tolerance toward autoantigens52 and alloantigens.53 Thus, efforts are currently ongoing to optimize in vitro methods for Treg generation

11. Antithymocyte Globulin Polyclonal antibodies active against T cells Administration Infuse over at least 6 hours Premedicate with acetaminophen, corticosteroids, and an antihistamine Rabbit ATG (Thymoglobulin�) and equine ATG (Atgam �) are NOT interchangeable Inc risk of infection d/t delayed immune reconstitutionInc risk of infection d/t delayed immune reconstitution

12. Antithymocyte Globulin Adverse effects Infusion-related reactions Fever, chills, headache Hypersensitivity reactions Cytokine release syndrome Increased risk of infections Serum sickness Serum sickness is an immune system reaction Symptoms Fever General ill feeling Hives Itching Joint pain Rash Swollen lymph nodes Note: Symptoms usually do not develop until 7 - 21 days after the first dose of antiserum or exposure to the medication. However, some people may develop symptoms in 1 - 3 days if they have previously been exposed to the substance. Signs and tests The lymph nodes may be enlarged and tender to the touch. The urine may contain blood or protein. Blood tests may show immune complexes or signs of blood vessel inflammation. Treatment Corticosteroid creams or ointments or other soothing skin medications may relieve discomfort from itching and rash. Antihistamines may shorten the length of illness and help ease rash and itching. Nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen or naproxen, may relieve joint pain. Corticosteroids taken by mouth (such as prednisone) may be prescribed for severe cases. Medications causing the problem should be stopped, and future use of the medication or antiserum should be avoided. Serum sickness is an immune system reaction Symptoms Fever General ill feeling Hives Itching Joint pain Rash Swollen lymph nodes Note: Symptoms usually do not develop until 7 - 21 days after the first dose of antiserum or exposure to the medication. However, some people may develop symptoms in 1 - 3 days if they have previously been exposed to the substance. Signs and tests The lymph nodes may be enlarged and tender to the touch. The urine may contain blood or protein. Blood tests may show immune complexes or signs of blood vessel inflammation. Treatment Corticosteroid creams or ointments or other soothing skin medications may relieve discomfort from itching and rash. Antihistamines may shorten the length of illness and help ease rash and itching. Nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen or naproxen, may relieve joint pain. Corticosteroids taken by mouth (such as prednisone) may be prescribed for severe cases. Medications causing the problem should be stopped, and future use of the medication or antiserum should be avoided.

13. Calcineurin inhibitors CNIs (cyclosporin and tacrolimus) bind to their respective immunophilins (cyclophilin or FKBP), and inhibit calcineurin. Calcineurin is then unable to dephosphorylate NFAT, which will prevent translocation of NFAT to the nucleus and thereby production of IL-2. Sirolimus is an mTOR inhibitor. It binds to FKBP and inhibits mTOR, which in turn inhibits transition of the cell cycle from G1 to S phase. MPA and LFL are also cell-cycle inhibitors, and act via inhibition of nucleotide synthesis. CNIs (cyclosporin and tacrolimus) bind to their respective immunophilins (cyclophilin or FKBP), and inhibit calcineurin. Calcineurin is then unable to dephosphorylate NFAT, which will prevent translocation of NFAT to the nucleus and thereby production of IL-2. Sirolimus is an mTOR inhibitor. It binds to FKBP and inhibits mTOR, which in turn inhibits transition of the cell cycle from G1 to S phase. MPA and LFL are also cell-cycle inhibitors, and act via inhibition of nucleotide synthesis.

14. Calcineurin Inhibitors Inhibit T cell activation by suppressing production of IL-2 IV Administration Non-PVC tubing Continuous infusion over 24 hours IV:PO conversion = ~1:3 Therapeutic Drug Monitoring (TDM) PO: trough levels (30 min prior to dose) IV: be sure to waste sufficient amount to avoid falsely elevated levels Binds to FKPB-12 and complexes with calcineurin dependent proteins to inhibit calcineurin phosphatase activity Binds to FKPB-12 and complexes with calcineurin dependent proteins to inhibit calcineurin phosphatase activity

15. Calcineurin Inhibitors: Adverse Effects Nephrotoxicity Hypertension Hyperglycemia Hypercholesterolemia Hypomagnesemia Hyperkalemia HUS/TTP CNS toxicity Tremor Posterior reversible encephalopathy syndrome (PRES) HUS/TTP-hemolytic uremic syndrome/TTP- increasing LDH, decreasing platelets, increasing Tbili, decreasing Hgb) TMA: severe thrombocytopenia, hemolysis, renal dysfxn, neurotoxicity (increased risk if sirolimus used in combo with tacrolimus) PRES � seizures, changes on MRI, mental status changesHUS/TTP-hemolytic uremic syndrome/TTP- increasing LDH, decreasing platelets, increasing Tbili, decreasing Hgb) TMA: severe thrombocytopenia, hemolysis, renal dysfxn, neurotoxicity (increased risk if sirolimus used in combo with tacrolimus) PRES � seizures, changes on MRI, mental status changes

16. Calcineurin Inhibitors: Drug Interactions Many others CYP3A4 inducers and inhibitors Tac=CYP 3A4 substrateTac=CYP 3A4 substrate

17. Calcineurin Inhibitors: Cyclosporine Dosing 3 mg/kg CIVI over 24 hours (initial) 5-6 mg/kg PO every 12 hours (initial) Modified ? non-modified May mix oral solution with orange juice TDM 150-350 ng/ml Adverse effects Hirsutism/hypertrichosis Gingival hyperplasia Usually begin on D-2 & continue for 6 months or more depending on GVHD Modified product (Neoral, Gengraf) has increased bioavailability vs non-modified (SandImmune) & are not interchangeable Hypertrichosis (werewolf syndrome�excessive hair growth) These ADRs do not occur with tacrolimus Cost ~$500/monthUsually begin on D-2 & continue for 6 months or more depending on GVHD Modified product (Neoral, Gengraf) has increased bioavailability vs non-modified (SandImmune) & are not interchangeable Hypertrichosis (werewolf syndrome�excessive hair growth) These ADRs do not occur with tacrolimus Cost ~$500/month

18. Calcineurin Inhibitors: Tacrolimus Dosing 0.03 mcg/kg CIVI over 24 hours (initial) 90 mcg/kg PO every 12 hours (initial) TDM 5-15 ng/ml Usually begin on D-2 & continue for 6 months or more depending on GVHD Cost ~$700/monthUsually begin on D-2 & continue for 6 months or more depending on GVHD Cost ~$700/month

19. Methotrexate Mechanism of action Induces apoptosis of activated lymphocytes Blocks dihydrofolate reductase to inhibit purine synthesis Dosing 5-15 mg/m2 IVP on D+1, 3, 6, 11 +/- leucovorin rescue Adverse effects Mucositis Myelosuppression Hepatotoxicity Leucovorin Leucovorin

20. MTX is thought to inhibit DHRFR by an accumulation of oxidized folates at the expense of reduced folates owing to the continued synthetic function of TS dTMP (thymidylate) ? dTDP ? dTTP ? DNA MTX depletes dTTP and purine nucleotides and interferes with cellular capacity to repair DNA ? strand breaks. Intracellular accumulation of dUMP which can be converted to dUTP which is then incorporated into DNA, resulting in inhibition of chain longation and DNA synthesis. There can then be excision repair of the DNA containing misincorporated dUTP moieties by uracil DNA glycosylase, which leads to further DNA fragmentation. Dose dependent reductions in methionine synthase enzyme activity (catalyzes folate dependent reaction where 5methyltetrahydrofolate serves as critical one carbon carrier methyl donor and mediates conversion of homocysteine to methionine.) Inhibition of methionine synthase leads to inhibition of downstream pathways including transmethylation reactions, polyamine biosynthesis, protein synthesis, or all three. This simplified figure illustrates the interconnectedness of folate metabolism and proteins for which functional polymorphisms have been identified. Polymorphisms have been found that are associated with pharmacogenetic outcomes in three key proteins in these pathways: the drug transporter protein reduced folate carrier (RFC); the regulatory enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR); and the drug target thymidylate synthase. Key enzymes are denoted as ovals, substrates as rectangles. Red ovals denote enzymes with genetic polymorphisms that have been investigated in pharmacogenetic studies. Orange ovals denote enzymes for which functional genetic polymorphisms have been described. 5-FU, 5-fluorouracil; AICAR, 5-aminoimidazole-4-carboxamine ribonucleotide; AICARFT, AICAR formyltransferase; CBS, cystathionine-�-synthase; DHF, dihydrofolate; DHFR, DHF reductase; dTMP, deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate; GAR, glycinamide ribonucleotide; GART, phosphoribosylglycinamide formyltransferase; hFR, human folate receptor; MTX, methotrexate; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SHMT, serine hydroxymethyltransferase; THF, tetrahydrofolate; X, various substrates for methylation. MTX is thought to inhibit DHRFR by an accumulation of oxidized folates at the expense of reduced folates owing to the continued synthetic function of TS dTMP (thymidylate) ? dTDP ? dTTP ? DNA MTX depletes dTTP and purine nucleotides and interferes with cellular capacity to repair DNA ? strand breaks. Intracellular accumulation of dUMP which can be converted to dUTP which is then incorporated into DNA, resulting in inhibition of chain longation and DNA synthesis. There can then be excision repair of the DNA containing misincorporated dUTP moieties by uracil DNA glycosylase, which leads to further DNA fragmentation. Dose dependent reductions in methionine synthase enzyme activity (catalyzes folate dependent reaction where 5methyltetrahydrofolate serves as critical one carbon carrier methyl donor and mediates conversion of homocysteine to methionine.) Inhibition of methionine synthase leads to inhibition of downstream pathways including transmethylation reactions, polyamine biosynthesis, protein synthesis, or all three. This simplified figure illustrates the interconnectedness of folate metabolism and proteins for which functional polymorphisms have been identified. Polymorphisms have been found that are associated with pharmacogenetic outcomes in three key proteins in these pathways: the drug transporter protein reduced folate carrier (RFC); the regulatory enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR); and the drug target thymidylate synthase. Key enzymes are denoted as ovals, substrates as rectangles. Red ovals denote enzymes with genetic polymorphisms that have been investigated in pharmacogenetic studies. Orange ovals denote enzymes for which functional genetic polymorphisms have been described. 5-FU, 5-fluorouracil; AICAR, 5-aminoimidazole-4-carboxamine ribonucleotide; AICARFT, AICAR formyltransferase; CBS, cystathionine-�-synthase; DHF, dihydrofolate; DHFR, DHF reductase; dTMP, deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate; GAR, glycinamide ribonucleotide; GART, phosphoribosylglycinamide formyltransferase; hFR, human folate receptor; MTX, methotrexate; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SHMT, serine hydroxymethyltransferase; THF, tetrahydrofolate; X, various substrates for methylation.

21. Mycophenolate mofetil Mechanism of action Inhibits lymphocyte proliferationby blocking purine synthesis Dosing 1000 mg PO/IV every 12 hours Drug interactions Calcium & magnesium Adverse effects Nausea, vomiting, diarrhea Myelosuppression Most side effects are dose-dependent DI: separate calcium & magnesium supplements by ~2hrs Cash price ~$700/monthMost side effects are dose-dependent DI: separate calcium & magnesium supplements by ~2hrs Cash price ~$700/month

22. Cytostatic effect on T and B lymphocytes. Is an inhibitor of inosine monophosphate dehydrogenase (IMPDH) which inhibits de novo guanosine nucleotide synthesis. T and B lymphocytes are dependent on this pathway for proliferation Cytostatic effect on T and B lymphocytes. Is an inhibitor of inosine monophosphate dehydrogenase (IMPDH) which inhibits de novo guanosine nucleotide synthesis. T and B lymphocytes are dependent on this pathway for proliferation

23. Corticosteroids Mechanism of action Affect number & function of B-cells & T-cells Dosing Systemic Methylprednisolone or prednisone 0.5-2 mg/kg IV/PO daily Taper when applicable Topical Budesonide-SR 3 mg PO every 8-12 hours (gut GVHD) Triamcinolone cream 0.1% to body +/- hydrocortisone 1% to face (skin GVHD) MOA: affects lymphocytes by inhibiting transcription factors (activator protein-1 & nuclear factor) Increased infection risk includes PCP, fungal infectionsMOA: affects lymphocytes by inhibiting transcription factors (activator protein-1 & nuclear factor) Increased infection risk includes PCP, fungal infections

24. Corticosteroid Adverse Effects Short term Hyperglycemia Mood disturbance, psychosis Insomnia Hypertension Fluid retention Skin atrophy Gastric ulcers Long term Adrenal suppression Moon facies Weight gain Osteoporosis Buffalo hump Cataracts Myopathy Infections

25. Sirolimus Mechanism of action Inhibits proliferation of lymphocytes by blocking m-TOR Dosing 12 mg PO x 1 then 4 mg PO once daily Therapeutic Drug Monitoring (TDM) 3-12 ng/ml Trough levels (30 min prior to dose) m-TOR (mammalian target of rapamycin) is a kinase that is responsible for growth & proliferation of lymphocytes; blunting of immune system by sirolimus is much less than with tacrolimus/cyclosporine so cannot be used as monotherapy No IV product; cost ~$1000/monthm-TOR (mammalian target of rapamycin) is a kinase that is responsible for growth & proliferation of lymphocytes; blunting of immune system by sirolimus is much less than with tacrolimus/cyclosporine so cannot be used as monotherapy No IV product; cost ~$1000/month

26. Inhibits cellular response to IL-2 thereby decreasing protein synthesis & ultimately lymphocyte proliferation (keeps lymphocytes from moving from G1 [synthesis of components for DNA synthesis) to S [DNA synthesis] phase) **Big difference b/w CIs & sirolimus: --CIs inhibit production of cytokines (ie. IL-2) --sirolimus blocks effects of cytokines on cell proliferationInhibits cellular response to IL-2 thereby decreasing protein synthesis & ultimately lymphocyte proliferation (keeps lymphocytes from moving from G1 [synthesis of components for DNA synthesis) to S [DNA synthesis] phase) **Big difference b/w CIs & sirolimus: --CIs inhibit production of cytokines (ie. IL-2) --sirolimus blocks effects of cytokines on cell proliferation

27. Sirolimus Drug interactions Similar to calcineurin inhibitors(CYP 3A4) Adverse effects Hyperlipidemia Myelosuppression Pneumonitis Thrombotic microangiopathy m-TOR (mammalian target of rapamycin) is a kinase that is responsible for growth & proliferation of lymphocytes; blunting of immune system by sirolimus is much less than with tacrolimus/cyclosporine so cannot be used as monotherapym-TOR (mammalian target of rapamycin) is a kinase that is responsible for growth & proliferation of lymphocytes; blunting of immune system by sirolimus is much less than with tacrolimus/cyclosporine so cannot be used as monotherapy

28. Additional Immunosuppressants: Treatment for GVHD TNFa blockers Etanercept, infliximab Pentostatin Alefacept Many drugs under investigation for treatment of acute and chronic GVHD

29. Infection Prevention Use appropriate anti-infective prophylaxis throughout immunosuppressive therapy Pneumocystis carinii pneumonia Fungal infections Viral infections

30. Summary Immunosuppression is utilized in allogeneic SCT to prevent rejection and GVHD, and for the treatment of GVHD Calcineurin inhibitors and sirolimus require TDM and close monitoring for side effects and drug interactions Infectious complications are common, making appropriate anti-infective prophylaxis important

31. Thank You! Ashley Newland, PharmD anewland@mcvh-vcu.edu