IMMUNOPHARMACOLOGY

William K. Nichols, Ph.D.

Types of Drugs
_

_
_

Immunosuppressants Immunostimulants Immunomodulators

_ _ _ _ _

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

Major Classes of Immunosuppressant Drugs

_ _ _

Glucocorticoids Calcineurin inhibitors Antiproliferative/Antimetabolic Agents Biologics (Antibodies)

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)

Major Steps in Immune Responses

1-

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

MAJOR STEPS IN IMMUNE RESPONSES

Antigen

IL-2

CD8 T cell

1 2 IL-1
antigen presenting cell (macrophage, dendritic cell)

4 3 IL-2
CD4 T helper cell
primed CD4 T helper cell

cytotoxic T cells

plasma cells

IL-2

B cell

SITES OF ACTION OF IMMUNOSUPPRESSIVE DRUGS

Antigen

IL-2
C

CD8 T cell 4

1 X A
B IL-1

X
primed CD4 T helper cell

cytotoxic T cells

X
D

3
D

IL-2

antigen presenting cell

CD4 T helper cell

X
cytokines

plasma cells

Sites of Action of Immunosuppressants Inhibiting T Cell Activation

_
_

Drug
Glucortiocoids

_ _

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)

Cyclosporine and Tacrolimus Sirolimus

Mycophenolate Mofetil

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.)

Drugs Prolonging Organ Transplantation

Cyclosporine

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

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

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
hepatotoxicity, hirsutism,

nephrotoxicity,

neurotoxicity Drug interactions due to induction and inhibition of hepatic cytochrome P450

Cyclosporine (Neoral)
Pharmacokinetics
microemulsion
(capsules

of cyclosporine

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

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)

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)

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

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 G1S phase transition

Consequences of TOR Action

Lymphocyte
T

cell proliferation & differentiation

cells B cells

Antibody

production Mesenchymal cell proliferation

Vascular

smooth muscle cells Endothelial cells Fibroblasts

Properties of TOR Inhibitors such 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

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)

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

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

Mycophenolate Mofetil
Adverse

Effects

diarrhea,

leukopenia and CMV infections increased incidence of lymphomas and other malignancies

New Immunosuppressants
_ _

Mizoribine (investigational)
_

Inhibitor of purine nucleotide synthesis Inhibitor of de nove pyrimidine synthesis Antimonocytic that decreases MHC antigen expression Calcineurin inhibitor like cyclosporine Approved for topical treatment of eczema

Brequinar (investigational)
_

15-Deoxyspergualin (investigational)
_

Pimecrolimus (Elidel)
_ _

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

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

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
polyclonal

Globulin

antibody similiar to OKT3

Antithymocyte
used

Globulin-Rabbit

to treat acute renal transplant rejection

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

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

Other Antibody Preparations

Rh(D)
for

Immune Globulin

Rh (neg.) mother after delivery of Rh(pos.) baby

Abciximab
for

surface receptor on activated platelets to prevent restenosis after coronary angioplasty CD20 on pre-B and mature B cells to treat nonhodgkins lymphoma

Rituximab
for

Antibodies as Immunosuppressive Agents

IL-2 Receptor Antibodies

Basiliximab
Chimeric

(Simulect)

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

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

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

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

Corticosteroids
Prednisone

used most often orally Methylprednisolone used parenterally Numerous available preparations

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)

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)

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

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)

Antimetabolites
Immunosuppresion

by inhibition of lymphocyte proliferation and cause bone marrow suppression

Azathioprine

(Imuran) Cyclophosphamide (Cytoxan)

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
alpha

(uses)

(anticancer uses) beta (relapsing type multiple sclerosis) gamma (chronic granulomatous disease)

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

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 Recombinant long-acting erythropoetin (3X epoetin)

Darbopoetin alpha (Aranesp)

_

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

Other Immunostimulants
_

Thymic Hormones
_

Improve primary immune deficiency in children

Levamisole stimulates phagocytosis and T cell production of cytokines BCG is viable strain of Mycobacterium bovis that enhances macrophage activity BCG used for bladder cancer and melanomas

Synthetic Stimulants
_

Adjuvants of bacterial origin

_

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

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)

Normal T Cell Response

T Cell Anery (No Response)

Inhibitory and Stimulatory APC Molecules

Drug Highlights
Calcineurin
Cyclosporine
multiple

Inhibitors
(Sandimmune, Neoral)

toxicities (nephrotoxicity, hepatotoxicity, neurotoxicity, hyperlipidemia, hypertension, hyperglycemia, hirsutism, gingival hyperplasia

Tacrolimus

(Prograf)

nephrotoxicity,

hepatotoxicity, neurotoxicity, hyperlipidemia, hypertension, hyperglycemia, alopecia

Drug Highlights
Sirolimus
Unique

(Rapamune)

mechanism involves inhibition of T cell activation by antigen and IL-2 hyperlipidemia; reduced platelets, rbcs, lymphocytes
Mycophenolate
Selective

mofetil (CellCept)

suppression of T and B cell proliferation leukopenia, diarrhea, CM V infection, lymphomas

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)

Drug Highlights
OKT3

(Muromonab-CD3)

monoclonal

antibody selective for T cells inhibits cytotoxic T cell function

Antilymphocyte
polyclonal

Globulin (ALG)

so less selective than OKT3

Antithymocyte
composed

Globulin (Thymoglobulin)

of multiple antibodies so more diverse targets that affect T cell activation, homing and cytotoxicity

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

Drug Highlights of Cytokines

G-CSF
treat

(Filgrastim)(Neupogen)
(Sargramostim)(Leukine)
recovery after bone marrow transplant

neutropenia

GM-CSF
myeloid

Interferon

Alpha (Roferon, Intron)

anticancer

uses (malignant melanoma, Kaposis sarcoma, renal cell carcinoma, hairy cell leukemia)

Interferon
relapsing

Beta (Avonex, Rebif)

type MS

Interferon
chronic

Gamma (Actimmunex)

granulomatous disease

Renal Transplant Protocols

_

Individualized for patient age, condition, other drugs type of organ transplant
_ _ _

Living identical matched donor Living closely matched donor Cadaver donor

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

Use Thymoglobulin or OKT3 for induction

If Cadaver Donor
_

See www.med.utah.edu/transplantprotocol

Problems Noted for Drugs (U/U)

_

Mycophenolate (Cellcept)
_

Dose carefully or severe leukopenia Hemolytic uremic syndrome, anemia Poor oral absorption, Nephrotoxicity Drug interactions involving CYP3A4 metabolism Nephrotoxicity Drug interactions involving CYP3A4 metabolism

Sirolimus (Rapamune)
_

Cyclosporine (Sandimmune, Neoral, Gengraf)

_ _

Tacrolimus (Prograf)
_ _

Renal Transplant Protocols

_ _ _ _ _

Univ of Utah Using steroid-free protocols (children) Pre-Transplant Immunosuppression

_

Tacrolimus (Prograf) and Mycophenolate (Cellcept) ATG-rabbit (Thymoglobulin)

Induction of Immunosuppression
_

Maintance Immunosuppression
_ _

Tacrolimus and Mycophenolate plus methylprednisolone for 6 doses, then taper over 7 days

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