IMMUNOPHARMACOLOGY

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IMMUNOPHARMACOLOGY

• William K. Nichols, Ph.D.

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Types of Drugs

• Immunosuppressants
• Immunostimulants
• Immunomodulators
• Induction of tolerance (tolerogens)
• Cytokines
• Hematopoetic Growth Factors
• Antibodies targeting key cell receptors/ligands
• Antibody-mediated drug delivery

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Major Classes of Immunosuppressant Drugs

• Glucocorticoids
• Calcineurin inhibitors
• Antiproliferative/Antimetabolic Agents
• Biologics (Antibodies)

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Different Principles for drugs used for cancer vs
immunosuppression

• 1- cancer cell proliferation is unstimulated and
  unsynchronized
• 2- immune respsonse involves cell proliferation
  that partially synchronized
• 3- cytotoxic drugs given in low daily doses that
  continuous for immunosuppression
• 4- cytotoxic drugs given in high pulse course
  every 3-6 weeks (allows recovery)

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Major Steps in Immune Responses

• 1- Antigen recognition
• 2- IL-1 production
• 3- IL-2 and other cytokine expression
• 4- lymphocyte proliferation differentiation

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MAJOR STEPS IN IMMUNE RESPONSES
CD8 T cell
Antigen
IL-2
cytotoxic T cells
1
4
3
2
primed CD4 T helper cell
IL-2
IL-1
antigen presenting cell (macrophage, dendritic
cell)
CD4 T helper cell
plasma cells
4
IL-2
B cell
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SITES OF ACTION OF IMMUNOSUPPRESSIVE DRUGS
CD8 T cell
4
Antigen
IL-2
X
cytotoxic T cells
1
E
X
C
A
X
3
2
primed CD4 T helper cell
X
X
D
D
IL-2
IL-1
B
antigen presenting cell
CD4 T helper cell
4
plasma cells
X
cytokines
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Sites of Action of Immunosuppressants Inhibiting
T Cell Activation

• Target
• GRE of DNA (regulate gene transcription, inhibit
  transcription)
• Calcineurin (inhibit the phosphatase required for
  IL-2 transcription)
• Protein kinase involved in cell-cycle progression
  (inhibits mTOR and inhbits IL-2 signaling)
• Inosine monophosphate dehydrogenase (inhibits de
  novo guanine nucleotide synthesis)

• Drug
• Glucortiocoids
• Cyclosporine and Tacrolimus
• Sirolimus
• Mycophenolate Mofetil

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Inhibitors of Immune Response (site of action)

• A- Immune Globulin (antigen recognition)
• B- Corticosteroids (IL-1 production, cell
  proliferation)
• C- OKT3 ,ATG (T cell receptors/surface prot.)
• D- Cyclosporine, Tacrolimus, (1L-2 gene expr.),
  Sirolimus (IL-2 signal transduction)
• E- Rapamycin, Mycophenolate (T cell prolif.),
  Azathioprine,Cyclophosphamide (all cell prolif.)

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Drugs ProlongingOrgan Transplantation

• Cyclosporine (Neoral)
• Tacrolimus (FK506, Prograf)
• Sirolimus (Rapamune)
• Mycophenolate mofetil (Cellcept)
• Prednisone, Methylprednisolone

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Cyclosporine (Neoral, Gengraf)

• Structure
• lipophilic cyclic peptide
• Mechanism
• inhibits transcription of IL-2 gene plus other
  cytokine expression (IL-3, gamma interferon)
• site of action is a binding protein that inhibits
  calcineurin (a phosphatase) involved in signal
  transduction upon antigen stimulation of T cell
  receptor

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Cyclosporine (Neoral)

• Pharmacokinetics
• variable, incomplete oral absorption
• extensive hepatic metabolism, excreted in bile
• used alone or in combination with prednisone and
  azathioprine (or other antineoplastic drugs)
• Adverse Effects
• nephrotoxicity, hepatotoxicity, hirsutism,
  neurotoxicity
• Drug interactions due to induction and inhibition
  of hepatic cytochrome P450

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Cyclosporine (Neoral)

• Pharmacokinetics
• microemulsion of cyclosporine
• (capsules and oral solution)
• improved oral absorption initial preps avail.
• 60 oral availability Neoral vs 30 for
  Sandimmune
• terminal half-life approx. 8.4 hours vs 19 hours
  for Sandimmune

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Tacrolimus (Prograf)

• Structure
• macrolide (structure like erythromycin)
• Mechanism
• similiar to cyclosporine except binds to
  different protein that inhibits calcineurin (a
  phosphatase enzyme involved in gene transcription
  of IL-2, gamma interferon and other cytokines)

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Tacrolimus (Prograf)

• Bioavailability
• given by IV infusion or orally
• used concomitantly with corticosteroids
• Adverse Effects
• nephrotoxicity, increased risk of lymphomas,
  hypersensitivity, hyperglycemia, GI complaints,
  hypertension, neurotoxicity (tremor, headache,
  motor disturbances, seizures)

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Sirolimus (Rapamune)

• Structure
• macrolide similiar to tacrolimus
• Mechanism
• binds to immunophilin protein that binds to a key
  regulatory kinase required for T cell activation
• (new unique mechanism to inhibit T lymphocyte
  activation by IL-2)
• different site of action than cyclosporine and
  tacrolimus

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Sirolimus (Rapamune)

• Inhibits mammalian target of rapamycin (mTOR)
• mTOR is a protein kinase that plays pivotal role
  in IL-2 receptor responses
• IL-2 binds to its receptor on T cells and leads
  to mTOR activation
• mTOR initiates cascade of events (including
  cyclin dependent kinases) that promote T
  lymphocyte proliferation and differentiation
• Inhibition of mTOR blocks IL-2 dependent
  cell-cycle progression at G1?S phase transition

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Consequences of TOR Action

• Lymphocyte cell proliferation differentiation
• T cells
• B cells
• Antibody production
• Mesenchymal cell proliferation
• Vascular smooth muscle cells
• Endothelial cells
• Fibroblasts

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Properties of TOR Inhibitorssuch as Sirolimus
(Rapamune)

• Selective blockade of cytokine signal
  transduction
• Inhibition of T cell division and proliferation
• Potent and effective immunosuppression
• Potential for synergy with other
  immunosuppressants

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Sirolimus (Rapamune)

• other theoretical actions include
• blockade of B cell Ig synthesis
• inhibition of antibody-dependent cellular
  toxicity
• inhibition of lymphocyte activated killer cells
• inhibition of natural killer cells
• inhibition of immune and nonimmune cell
  proliferation (via inhibition of growth factor
  signaling) (may explain antitumor actions)

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Sirolimus (Rapamune)

• Bioavailability
• low oral absorption
• hepatic metabolism by CYP4503A4 (drug
  interactions may occur)
• long half-life (60 hours)
• Adverse Effects
• thrombocytopenia, hyperlipidemia, rash
• lacks direct end organ toxicity but increased
  incidence impaired renal function when combined
  with cyclosporine

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Mycophenolate Mofetil (CellCept)

• Structure
• derivative of mycophenolic acid
• Mechanism
• inhibits inosine monophosphate dehydrogenase
  involved in de novo synthesis of purines
• selectively suppressess T- and B-cell
  proliferation
• Also suppresses some macrophage functions (may
  explain anti-inflammatory actions)
• Pharmacokinetics
• oral absorption and hepatic metabolism

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Mycophenolate Mofetil

• Adverse Effects
• diarrhea, leukopenia and CMV infections
• increased incidence of lymphomas and other
  malignancies

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New Immunosuppressants

• Mizoribine (investigational)
• Inhibitor of purine nucleotide synthesis
• Brequinar (investigational)
• Inhibitor of de nove pyrimidine synthesis
• 15-Deoxyspergualin (investigational)
• Antimonocytic that decreases MHC antigen
  expression
• Pimecrolimus (Elidel)
• Calcineurin inhibitor like cyclosporine
• Approved for topical treatment of eczema

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New Class of Immunosuppressant

• FTY720 (prodrug requires phosphorylation)
• Sphingosine 1-phosphate receptor (S1P-R) agonist
  
• Reduces recirculation of lymphocytes from
  lymphatic system to the blood
• Lymphocyte homing action which reversibly
  sequesters host lymphocytes into lymph nodes
• Useful in combination therapy but not alone
• Toxicity lymphopenia, decreased heart rate

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Alemtuzumab (Campath)

• New Immunosuppressant
• Recombinant DNA-derived humanized monoclonal
  antibody
• Binds to CD52. a nonmodulating antigen present on
  surface of all T and B cells
• Some bone marrow cells express CD52 including
  some CD34 cells
• Produces profound T cell depletion
• Used for for selected leukemias and lymphomas
• also for stem cell transplant procedures

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Antibodies Used for Acute Rejection of Organ
Transplants

• OKT3 (Muromonab-CD3)
• monoclonal antibody to CD3 on T cell
• inhibits cytotoxic T killer cell function
• opsonizes circulating T lymphocytes and enhances
  their removal
• used to prevent or reverse acute graft rejection
• Antilymphocyte Globulin
• polyclonal antibody similiar to OKT3
• Antithymocyte Globulin-Rabbit
• used to treat acute renal transplant rejection

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Antithymocyte Globulin-Rabbit (Thymoglobulin)

• Rabbit gamma immune globulin preparation
• Composed of antibodies to variety of T cell
  markers
• Mechanisms
• removal of T cells from circulation
• modulation of T cell activation, homing and
  cytotoxicity
• decreases cytokine induced reactions

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Adverse Effects of Antibody Preps

• Hypersensitivity reactions may occur with
  nonhuman antibodies resulting in chills, fever,
  thrombocytopenia, erythema, pruritis
• Problem with murine monoclonal antibody called
  OKT3 is formation of anti-OKT3 antibodies limit
  its action so only given by IV infusion for 7-14
  days

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Other Antibody Preparations

• Rh(D) Immune Globulin
• for Rh (neg.) mother after delivery of Rh(pos.)
  baby
• Abciximab
• for surface receptor on activated platelets to
  prevent restenosis after coronary angioplasty
• Rituximab
• for CD20 on pre-B and mature B cells to treat
  non-hodgkins lymphoma

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Antibodies as Immunosuppressive Agents
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IL-2 Receptor Antibodies

• Basiliximab (Simulect)
• Chimeric murine monoclonal antibody against human
  IL-2 receptor alpha subunit of activated T to
  block T cell
• Blocks activation and inhibits clonal expansion
  of T cells
• Used to induce immunosuppression and to prolong
  organ transplants in combination with
  immunosuppressants

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Other IL-2 Receptor Antibodies

• Daclizumab (Zenapax)
• a humanized immunoglobulin similar to Basiliximab
  which blocks IL-2 receptor
• Formed by splicing complementary portions of
  light and heavy chain variable regions of murine
  antibody into human-derived Fab framework and
  fusing the Fab to the Fc portion of human IgG

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Advantages of Chimeric and Humanized Antibodies

• Reduce immunogenicity without sacrificing
  affinity
• Allow complement fixation to occur by using the
  human Fc region instead of murine Fc
• Resulting in ADCC and activation of phagocytic
  cells
• Humanization of Fab fragment may decrease binding
  affinity compared to initial murine antibody
• Baciliximab has higher affinity for IL-2 receptor
  than Daclizumab

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Other Antibodies (cont.)

• Rho(D) Immune Globulin
• used to suppress immune response of Rh(neg.)
  mother after delivery of Rh (pos.) baby
• Given within 72 hours after birth of Rh(pos.)
  baby to prevent hemolytic anemia of newborn that
  may occur in subsequent pregnancies

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Corticosteroids

• Prednisone used most often orally
• Methylprednisolone used parenterally
• Numerous available preparations

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Corticosteroid Actions

• Inhibition of IL-1 and TNF gene expression and
  synthesis
• Decreased activation of T lymphocytes by
  decreasing IL-1 release
• Decreased neutrophil functions esp chemotaxis
• Decreased antibody production (high doses)
• Decreased release of kinins and proinflammatory
  eicosanoids (prostaglandins and leukotrienes)

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Corticosteroid Immunosuppression

• Decreased cell-mediated immune reactions that
  mediate rejection of organ transplants
• Mechanisms
• decreased activation of T lymphocytes by
  inhibition of IL-1 synthesis by macrophages
• decreased lymphocyte mobilization out of
  lymphoid organs (lymphopenia)

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Corticosteroid Adverse Reactions

• All commonly occur because high doses used for
  immunosuppression
• Suppression of hypothalmic-pituitary adrenal axis
  (HPA) function
• Osteoporosis
• Hypertension
• Weight gain
• Hyperglycemia
• Euphoric personality changes
• Cataracts

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Clinical Concerns with Corticosteroids

• Growth inhibition in pediatric transplants
• Cataracts (10 incidence)
• Bone disease (inhibition of osteoblastic
  activity, decreased calcium absorption, increased
  urinary calcium excretion)
• Diabetes (insulin-resistance, gluconeogenesis)
• Hyperlipidemia (40-60 posttransplant accelerated
  atherogenesis, increased incidence if combined
  with calcineurin inhibitors and sirolimus)
• Hypertension (60-80 in transplant patients)
• Increased cardiovascular risk factors
• Predisposition to infection (decr. PMN, T cell
  activity)

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Antimetabolites

• Immunosuppresion by inhibition of lymphocyte
  proliferation and cause bone marrow suppression
• Azathioprine (Imuran)
• Cyclophosphamide (Cytoxan)

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Immunostimulatory Cytokines

• Interleukins
• IL-2 (enhance antitumor actions of cytotoxic T
  cells and NK cells)
• Colony Stimulating Factors
• G-CSF (neutropenia) and GM-CSF (bone marrow
  transplant patients)
• Interferons (uses)
• alpha (anticancer uses)
• beta (relapsing type multiple sclerosis)
• gamma (chronic granulomatous disease)

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Interferon Uses

• Interferon Alpha (prod. by leukocytes)
• (antiviral, antiproliferative)
• malignant melanoma, renal cell carcinoma, hairy
  cell leukemia, Kaposis sarcoma
• Interferon Beta (prod. by fibroblasts)
• (antiviral, antiproliferative)
• relapsing type MS
• Interferon Gamma (prod. by lymphocytes)
• (stimulates NK cells and macrophages)
• chronic granulomatous disease

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Other Hematopoetic Growth Factors

• Erythropoietin alpha (Epoetin alpha) (Procrit)
• Produced by recombinant DNA technology
• Stimulates division and differention of erythroid
  progenitor cells
• Used for anemia due to renal failure or cancer
  chemotherapy
• Adverse effects include hypertension, headache,
  hypersensitivity reactions are rare
• Darbopoetin alpha (Aranesp)
• Recombinant long-acting erythropoetin (3X epoetin)

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Cytokine Inhibitors

• TNF inhibitors (disease modifiers to treat
  rheumatoid arthritis)
• Etanercept (Enbrel)
• Recombinant version of TNF receptor
• Infliximab (Remicade)
• Chimeric human/murine anti-TNF monoclonal
  antibody
• Anakinra (Kineret)
• Human IL-1 receptor antagonist
• Disease modifier agent for Rheumatoid arthritis

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Other Immunostimulants

• Thymic Hormones
• Improve primary immune deficiency in children
• Synthetic Stimulants
• Levamisole stimulates phagocytosis and T cell
  production of cytokines
• Adjuvants of bacterial origin
• BCG is viable strain of Mycobacterium bovis that
  enhances macrophage activity
• BCG used for bladder cancer and melanomas

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Targeted Immunotherapy

• Antibody-mediated delivery systems
• Radiolabeled antibodies
• Types of antibodies in trials
• Anti-CD20 for B cell lymphomas
• Anti-vascular endothelial cell growth factor
• Anti-fibroblast growth factor
• Anti-body to F19 on surface of activated
  fibroblasts

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New Approaches for Tolerance

• Interference with costimulatory signals required
  for T cell activation
• Two signals required for T cell activation
• Signal 1 via T cell receptor
• Signal 2 via costimulatory receptor-ligand pair
• Antibodies to costimulator receptors (on T cell)
  or ligands (on antigen presenting cell)
• Anti-CTLA4 (blocks B7 binding to T cell CD28)
• Anti-CD40 (inhibits macrophage and endothelial
  activation by blocking T cell CD40 ligand binding
  to macrophage CD40)

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Normal T Cell Response
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T Cell Anery (No Response)
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Inhibitory and Stimulatory APC Molecules
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Drug Highlights

• Calcineurin Inhibitors
• Cyclosporine (Sandimmune, Neoral)
• multiple toxicities (nephrotoxicity,
  hepatotoxicity, neurotoxicity, hyperlipidemia,
  hypertension, hyperglycemia, hirsutism, gingival
  hyperplasia
• Tacrolimus (Prograf)
• nephrotoxicity, hepatotoxicity, neurotoxicity,
  hyperlipidemia, hypertension, hyperglycemia,
  alopecia

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Drug Highlights

• Sirolimus (Rapamune)
• Unique mechanism involves inhibition of T cell
  activation by antigen and IL-2
• hyperlipidemia reduced platelets, rbcs,
  lymphocytes
• Mycophenolate mofetil (CellCept)
• Selective suppression of T and B cell
  proliferation
• leukopenia, diarrhea, CM V infection, lymphomas

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Clinical Concerns with Corticosteroids

• Growth inhibition in pediatric transplants
• Cataracts (10 incidence)
• Bone disease (inhibition of osteoblastic
  activity, decreased calcium absorption, increased
  urinary calcium excretion)
• Diabetes (insulin-resistance, gluconeogenesis)
• Hyperlipidemia (40-60 posttransplant accelerated
  atherogenesis, increased incidence if combined
  with calcineurin inhibitors and sirolimus)
• Hypertension (60-80 in transplant patients)
• Increased cardiovascular risk factors
• Predisposition to infection (decr. PMN, T cell
  activity)

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Drug Highlights

• OKT3 (Muromonab-CD3)
• monoclonal antibody selective for T cells
• inhibits cytotoxic T cell function
• Antilymphocyte Globulin (ALG)
• polyclonal so less selective than OKT3
• Antithymocyte Globulin (Thymoglobulin)
• composed of multiple antibodies so more diverse
  targets that affect T cell activation, homing and
  cytotoxicity

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Drug Highlights

• Genetically Engineered Anti-IL-2 Receptor
  Antibodies
• Inhibit clonal expansion of T cells
• Advantages include
• reduced immunogenicity
• ability to promote ADCC
• Basiliximab (Simulect) chimeric antibody
• Daclizumab (Zenapax) humanized chimeric antibody

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Drug Highlights of Cytokines

• G-CSF (Filgrastim)(Neupogen)
• treat neutropenia
• GM-CSF (Sargramostim)(Leukine)
• myeloid recovery after bone marrow transplant
• Interferon Alpha (Roferon, Intron)
• anticancer uses (malignant melanoma, Kaposis
  sarcoma, renal cell carcinoma, hairy cell
  leukemia)
• Interferon Beta (Avonex, Rebif)
• relapsing type MS
• Interferon Gamma (Actimmunex)
• chronic granulomatous disease

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View Notes
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Renal Transplant Protocols

• Individualized for patient age, condition, other
  drugs
• type of organ transplant
• Living identical matched donor
• Living closely matched donor
• Cadaver donor

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Renal Transplant Protocols (U/U)

• Three Drug Combinations (Living Donor)
• Cyclosporine(Gengraf) or Tacrolimus (Prograf)
• Mycophenolate (Cellcept)
• Corticosteroids (IV initially, reduce to oral as
  taper)
• If Identical Match
• Use Basilixamab (Simulect) for induction
• If Cadaver Donor
• Use Thymoglobulin or OKT3 for induction
• See www.med.utah.edu/transplantprotocol

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Problems Noted for Drugs (U/U)

• Mycophenolate (Cellcept)
• Dose carefully or severe leukopenia
• Sirolimus (Rapamune)
• Hemolytic uremic syndrome, anemia
• Cyclosporine (Sandimmune, Neoral, Gengraf)
• Poor oral absorption, Nephrotoxicity
• Drug interactions involving CYP3A4 metabolism
• Tacrolimus (Prograf)
• Nephrotoxicity
• Drug interactions involving CYP3A4 metabolism

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Renal Transplant Protocols

• Univ of Utah
• Using steroid-free protocols (children)
• Pre-Transplant Immunosuppression
• Tacrolimus (Prograf) and Mycophenolate (Cellcept)
• Induction of Immunosuppression
• ATG-rabbit (Thymoglobulin)
• Maintance Immunosuppression
• Tacrolimus and Mycophenolate
• plus methylprednisolone for 6 doses, then taper
  over 7 days

63
New Immunosuppressant

• Alemtuzumab (Campath)
• Recombinant DNA-derived humanized monoclonal
  antibody
• Binds to CD52. a nonmodulating antigen present on
  surface of all T and B lymphocytes
• Some bone marrow cells express CD52 including
  some CD34 cells
• Produces profound T cell depletion
• Used for selected leukemias (CLL), and lymphomas,
  plus stem cell transplant procedures