Immunopharmacology Lecture Notes PDF

PM 716 Pharmacology I Immunopharmacology Chapter 55 RMRocco, PhD 1  Lecture Outline Immune System Innate (primitive) Acquired (adaptive) (1) cell mediated (2) humoral Antibodies as drugs Monoclonals...

PM 716 Pharmacology I Immunopharmacology Chapter 55 RMRocco, PhD 1  Lecture Outline Immune System Innate (primitive) Acquired (adaptive) (1) cell mediated (2) humoral Antibodies as drugs Monoclonals Polyclonals (specific and nonspecific) Abnormal Immune Response to Drugs Hypersensitivity Reactions (Type I-IV) 2  Lecture Outline Autoimmunity Immunodeficiency Replacement Immunosuppression Agents Cytotoxic Agents Immunomodulating Agents 3  Immune System Components INNATE (PRIMITIVE) Immune System First line of defense against bacterial, viral infections. Most infections eliminated. Immunity is nonspecific and has no memory. Infectious agent activates NK cells and macrophages which start a cascade of events. 4  Innate Immunity Complement proteins bind to bacterial cell surface, create pores which starts apoptotic cell death (called opsonization) Binding of proteins to bacterial cell surface creates recognition sites which allows phagocytes to kill bacteria through free radical destruction and lysozyme digestion. 5  Innate Immunity Summary Major actions: phagocytosis of bacteria Primitive immune system, has no memory Cells: granulocytes, monocytes, NK cells Mediators: complement and cytokines Specificity: None Enhanced by repeated exposure: No Major Function: phagocytosis 6 Macrophages Precursors to monocytes released from bone marrow into peripheral blood as mature monocyte. ~ 400/uL blood (1-10% of total WBC in blood. Bacteria (left) being digested by a macrophage. 7  Measurement of Innate Immunity Phagocytosis Assays Coat mouse macrophages to titer well plate. Use macrophages from mice exposed to toxic agent at test doses. Add to plate radioactive (Cr51) chicken red cells. Macrophages engulf red cells (if macrophages have not been damaged by toxic agent). Wash plate, count the radioactivity. Same assay can be done with fluorescent beads instead of red cells. 8  Acquired (Adaptive) Immunity The Antibody Production System (1) Cell mediated Immunity (CMI) min role of antibodies viable T-cell lymphocytes are required 9  (1) Cell Mediated Immunity Bacteria or soluble antigen binds to Antigen presenting cell (lymphocyte) (APC Cells) T-Cell (CD4) recognizes APC-antigen complex, produces cytokines which activate cytolytic T Lymphocytes (CD8) cells lyse bacteria. 10  Acquired Immunity (2) Humoral Immunity Antibody production Antibody binds to antigen/bacteria in solution. CD4 activated cytokines release B cells to produce antibody. Bacteria with antibody attached activates the complement cascade. 11 Lymphocytes Classified by the CD surface receptors Total ~ 2200/uL T-Cells ~ 1500/uL CD4 Helper ~ 900/uL CD8 Suppr ~ 600/uL B Cells(antb) ~300/uL NK Cells ~ 400/uL 12  Acquired Immunity Learned immunity Antibody production Cells: lymphocytes, macrophages, NK cells Mediators: antibodies and cytokines Specificity: Yes, very high (antibody) Enhanced by repeated exposure: Yes (vaccines ) Prior exposure is required: (no immune response on first exposure). 13  Measurement of Acquired Immunity Plaque Antibody Forming Cell (PFC) Assay (1) Expose animal to toxin (mouse) (2) Inject sheep RBC into exposed mouse. (3) Sheep RBC will mobilize Immune system in mouse. (4) Remove mouse spleen B cells (antb making cells) (5) Mouse spleen B cells will be making anti-sheep IgM antibody (6) Add mouse B cells to agar plate in which agar contains fresh sheep cells. (7) Mouse anti-sheep antb will lyse sheep cells in agar and creat clear zones (holes). (8) Count the number and size of the clear zones. 14  ELISA ASSAY Coat plate with RBC (or fragments) Fluorescent tag or Enzyme Attached to anti-mouse antib Mouse anti-sheep antb if present Sheep red cells. 15 FDA Guidance for immunotoxicity testing of all in vivo implants. 16 Plasma Cells and Antibodies Plasma Cells 17  Abnormal Immune Response Hypersensitivity Reactions are of two types Antibody mediated, Types I, II, and III Cell mediated, Type IV 18  Type I Antb Mediated immediate, minutes after exposure IgE antibody mediated IgE binds to basophils (plasma) and mast cells and when antigen binds to IgE, cells degranulate and release histamine. anaphylaxis, asthma, hay fever, urticaria (hives), drugs, insect bites, some foods 19  Type II Antb Mediated Foreign antigen forms complex with IgM or IgG antibody. This complex binds to red cells, activates the complement system and red cell destruction (lysis) occurs. From incompatable blood transfusion, penicillin allergy (neoantigen), Rh incompatability. 20  Type III Antb Mediated Antigen-Antb complex precipitates onto basement membranes and destroys tissues (kidney etc) due to the release of complement. 21  Type IV Delayed Cell mediated, not antibody, response occurs 2-3 days after exposure to the antigen. Cells (macrophages) mobilize at site of exposure, release cytokines that set up the reaction. Contact hypersensitivities, poison ivy reactions. 22  Autoimmunity Antb response against “self” Rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type 1 diabetes. Antibodies in these various diseases are directed against different self proteins, such as basement membranes, DNA, CNS myelin, etc. 23  Immunodeficiency Diseases Decrease in immune function often leads to death from infection. Many are genetic in origin. HIV is acquired because the virus targets and destroys CD4 cells required for proper antibody production. 24  Immunosuppressive Drugs Drugs used to suppress the immune system: Cortisone: the gold standard for immunosuppression. Covered extensively in Chapter 39. 25  Immunosuppresive Drugs Calcineurin Inhibitors Cyclosporine: peptide antibiotic, made organ transplant possible, blocks activation of T-cells, drug inhibits calcineurin (a phosphatase enzyme) which prevents activation of T-cell specific transcription factors. Given iv or po. Too numerous ADRs to mention. 26  Immunosuppressive Drugs Calcineurin Inhibitors: Tacrolimus: macrolide antibiotic, MOA similar to cyclosporine, used iv, po and topical cream for psoriasis. 27  Immunosuppressive Drugs Proliferation Signal Inhibitors Sirolimus (sir OH li mus)and Everolimus: drugs block molecular target of rapamycin (mTOR) signaling mechanism, this leads to inhibition of T-cell proliferation. Profound myelosuppression can occur. 28  Immunosuppressive Drugs Mycophenolate Mofetil: inhibits T and B cell response through inhibition of purine synthesis. 29  Immunosuppressive Drugs Thalidomide: used in over 40 different diseases, drug inhibits angiogenesis, is anti- inflammatory, inhibits tumor necrosis factor alpha (TNF alpha), reduces neutrophil phagocytosis. Highly regulated due to possibility of birth defects. 30  Immunosuppressive Drugs Cytotoxic Agents Azathioprine: prodrug of mercaptopurine, drug blocks purine nucleic acid synthesis which blocks cell proliferation. Cyclophosphamide: alkylating agent, destroys proliferating lymphoid cells,. 31  Immunosuppressive Drugs Pyrimidine Synthesis Inhibitors Leflunomide and Teriflunomide: both inhibit pyrimidine synthesis which blocks lymphocyte activation. Used mostly for RA at present. 32  Antibodies as Drugs Monoclonals: inject mouse with antigen isolate antibody producing B lymphocytes from mouse spleen fuse B cell with human B lymphocyte from bone marrow cancer (myeloma) [hybridoma cells] grow hybridoma cells, harvest antibody 33  Antibodies as Drugs Mouse: full constant region (Fc) and full antigen binding region (Fab) Chimeric: ~66% human (Fc region replaced with human sequence) Humanized: 90+% human (Fc and much of the Fab replaced with human sequence. Mouse contributes the 7-10 amino acids in binding recognition sites on Fab) 34  Antibodies as Drugs An alternative to mouse monoclonal: Fuse human genes for antibody production into a mouse. Inject mouse with antigen. Collect antibody from cultured cells (bioreactor). 35  Antibody Drug Examples abciximab (ReoPro) (chimeric) binds to GP IIb/IIIa receptor on platelets and prevents clotting during surgery etc. rituximab (Rituxan) (chimeric) binds CD20 receptor on B lymphs to block cells in Non- Hodgkin’s lymphoma. Trastuzumab (tras TU zoo mab) (Herceptin) (humanized) binds to HER-2/neu receptors on breast cancer cells, blocks replication. 36  Antibody Drug Examples infliximab (Remicade) (chimeric) binds to TNF alpha to block TNF mediated (cytokine) inflammation used in RA, Crohn’s disease etanercept (Enbrel) (fusion protein, human constant IgG linked to TNF alpha receptor). Binds TNF as above, used in RA. Adalimumab (a da LIM yo mab) (Humira) (Humanized) binds TNF alpha as above, used in RA. 37  Monoclonal Antibodies Trastuzumab: recombinant DNA-derived humanized monoclonal antb that binds to epidermal growth factor receptor HER-2/neu. Drug blocks overepressed receptor in some breast cancers which inhibits protein synthesis required for tumor growth. 38  Monoclonal Antibodies Infliximab monoclonal binds to TNF-alpha. TNF alpha is a cytokine which initiates inflammtory response. In RA 52% of patients showed improvement of arthritic symptoms. 39  Infliximab Lipsky, P. E. et al Infliximab and methotrexate in the treatment of rheumatoid arthritis New Engl J Med 343:1594-1602 (2000) Randomized, two year, double blind, placebo controlled clinical trial. 40  Infliximab Measures of improvement included: number of tender and swollen joints in 68 joints examined patient’s global assessment of disease status health assessment of disability ESR blood and CRP plasma levels 41  Infliximab Treatments N = 81-88 per group placebo + methotrexate 3 mg Inf/kg every 8 wks + methotrexate 3 mg Inf/kg every 4 wks + methotrexate 10 mg Inf/kg every 8 wks + methotrexate 10 mg Inf/kg every 4 wks + methotrexate 42  Infliximab Results at 54 weeks, the number of patients showing 50-70 % improvement Placebo + Mtx 10/88 (11%) 3 mg Inf 8 wk + Mtx 31/86 (36%) 3 mg Inf 4 wk + Mtx 51/86 (59%) 10 mg Inf 8 wk + Mtx64/87 (74%) 10 mg Inf 4 wk + Mtx57/81 (70%) 43  Immune Globulins as Drugs Passive immunization obtained with the injection of specific pooled immune globulins. Used when disease is present, in patient with decreased immune response, or in patients at high risk for contracting the disease and there is no time for vaccines to work. Protection lasts 1-3 months and drugs are mostly purified IgG fractions from pooled human donors. 44  Immune Globulins Specific immune globulin preparations are available for a wide variety of conditions: CMV Hepatitis B Rabies Tetanus Respiratory syncytial virus 45  Immune Globulins Immune globulin (IgG) iv (Gamimune) a polyclonal human immunoglobulin obtained from pools of thousands of healthy donors no specific antigen in patient is target of the drug drug at 2g/kg modulates the patient immune response, decreases immune response, lowers T cell numbers. used in numerous immune disorders. 46  Case Study A 30-year old female with one living child, age 6. Her child and husband are Rh positive and she is Rho (D) and Du negative. She is now in her ninth month of pregnancy and is in delivery. Her earlier Rh antibody test was negative. What immunotherapy is appropriate? 47  Antibodies as Drugs Rho (D) Immune Globulin Prevents Rh hemolytic anemia of the new born. One of the first major advances in immunopharmacology. 48  Case Study Give 2 mL im 300 mcg anti-Rho (D) immune globulin within 24-72 h post partum. This clears any fetal Rh positive red cells from her circulation. This prevents mother from developing antibodies that might harm future pregnancy. 49  Rho (D) Immune Globulin Pathogenesis: Hemolytic Disease of the Newborn Rh Neg mother carries an Rh Pos baby to term (first pregnancy). Babies Rh Pos RBCs enter’s mother’s blood (especially during delivery) Mother makes antibodies against Rh Pos Red Cells. A second pregnancy with Rh Pos baby, mother’s antibodies destroys baby’s blood. 50 Hemolytic Disease of the Newborn RhoGAM® administered within 24-48 h after delivery. Drug blocks mother’s immune response to Rh Pos red cells by binding to Rh Pos cells in her blood (the antigens are blocked). Drug is concentrate of human IgG antibody to Rho (D) antigen found on Rh Pos red cells (baby’s rbcs). 51  Rho(D) Immune Globulin Hemolytic disease of the new born also called erythroblastosis fetalis. Drug blocks the mother’s immune response to baby’s Rh Pos cells. Dose is usually 2 mL im (~ 300 ug anti-Rh o (D) IgG). 52 Rho (D) Immune Globulin Vincent Freda (1928 - 2003) Columbia First in US to perform aminocentesis First to perform fetal surgery. Developed RhoGAM. Won Lasker Award 1980. 53  Abnormal Immune Responses Hypersensitivity Immediate or Delayed Immediate Hypersensitivity (min to hrs) Type I Hypersensitivity IgE attached to mast cells binds antigen (pollen etc etc) and degranulation leads to histamine release. Found with asthma, hay fever, insect venoms, foods and drugs (anaphylaxis). 54 Immunologic Reactions to Drugs (Drug Allergy) Type I: IgE-mediated acute reaction to drugs (and pollens, stings). IgE attached to mast cells and release histamine. Type II: Drug forms neoantigen with patient proteins. Type III: Drug causes serum sickness (IgG and complement activation) Type IV: Cell-mediated allergy to topical drugs (allergic contact dermatitis). 55  Drug Allergy Type I (Immediate Allergy) Patient develops IgE antibodies to Drug- Protein complex (Neoantigen). Some patients develop IgE, sone IgG response, reasons unknown. IgE bound to mast cells and eosinophils release histamine when drug binds to IgE antibody. 56  Drug Allergy Type I (Immediate Allergy) Test for allergy to drug with scratch test. Dilute solution of drug on tip of needle used to scratch the skin. Look for immediate wheal (edema) and flare (increased blood flow) at site of the scratch. Prednisone will block IgE synthesis. Epinephrine relaxes bronchiolar smooth muscle and contracts vascular smooth muscle. 57  Drug Allergy Type I (Immediate Allergy) Desensitization to the Drug(s) (penicllin, insulin) Start drug at very low doses for days or weeks then increase to therapeutic doses. Anaphylaxis can occur before therapeutic dose achieved. Concept based on the saturation of all available IgE on mast cells which then allows full dose of drug to be given. 58  Drug Allergy Type II (Autoimmune) Drug induces an autoimmune syndrome. Example, systemic lupus erythematosus with procainamide. Methyldopa induces autoimmune hemolytic anemia Antibodies (IgG) to the drug are demonstrated in plasma. 59  Drug Allergy Type III Reactions (Serum Sickness) Characteristics of serum sickness include skin eruptions, fever, arthritis, lymph node enlargement Symptoms subside when drug removed. Plasmapheresis removes drug from circulation, glucocorticoids reduce antibody response. IgG and IgM antibodies involved. 60 Mast Cells Mast Cells in the lung Mast Cell, allergen mediated IgE binds to cell surface. Antigen binding promotes release of agents that contract airway smooth muscle 61  Immuno Model of Asthma Airway mucosa Antigen Mast Cell IgE Antb Histamine Tryptase Leukotrienes C4 and D4 Prostaglandins 62 Eosinophils Cytokines also released by antigen binding to mast cell-IgE cause activation of eosinophils. Eosinophils release additional factors which stimulate edema, mucus hypersecretion, smooth muscle contraction. 63  Anti-IgE Antibodies Mouse (humanized) anti-IgE monoclonal antibody blocks binding of IgE to mast cells. Omalizumab (oh mah lye ZOO mab) (anti-IgE Mab) binds to IgE, blocks binding to mast cells and prevents histamine release when cells exposed to antigens. Drug treatment for 10 weeks lowers Cp levels of IgE to below detection levels. Drug given iv or sc. 64 Omalizumab Omalizumab (oh mah lye ZOO mab) humanized monclonal antibody binds to IgE and prevents attachment to mast cells. 65  Abnormal Immune Response Type II (Immediate Hypersensitivity) Antigen binds IgM or IgG antibody (1) Incompatible blood used for transfusion Antibody binds to and hemolyzes the red cells. (2) Hemolytic Disease of the New Born (3) Penicillin induced hemolytic anemia (neoantigen formation) 66  Abnormal Immune Response Type III Immediate Hypersensitivity High levels of antigen-antb complex causes tissue damage. Complex precipitates onto basement membrane cells and activates inflammatory responses. 67  Abnormal Immune Response Delayed Hypersensitivity (2-3 days) Cell-mediated response 2 -3 days after exposure to antigen. Antigen (bacteria) in tissue induces inflammatory response, release of cytokines, infiltration of white cells, release of digestive enzymes which leads to tissue damage. 68  BCG for TB Bacille Calmette-Guerin vaccination or Bacillus Calmette-Guerin vaccine(BCG) Albert Calmette (1863-1933) and Camille Guerin (1872-1961) developed BCG in 1921. BCG made from an attenuated (weakened) strain of Mycobacterium bovis, used for TB immunization. (Close to M. tuberculosis). Drug activates macrophages to make them more effective against MTB. 69  BCG for TB Most widely used vaccine in the world. Always given in neonatal period, ineffective in adults. Children vaccinated contract TB as adults. BCG vaccination can cause false positive tuberculin skin test. In US BCG never used (low incidence of childhood TB). A positive skin test in patient who had BCG is treated as if patient has active or latent TB (major issue among recent newcomers to US). 70  Tuberculin Skin Test for TB Tuberculin = glycerin extract of tubercule bacilli. Purified Protein Derivative (PPD) is an extract of proteins from Mycobacterium tuberculosis. Mantoux (US)Test uses injection of tuberculin under the skin, examine for eruptions (tuberculin) in 24-48h. Multiple Puncture Tine Test Pos TB Test, induration (bump) (MPTT) uses pins (tines) to puncture skin (mostly Japan) Test Pos 20 y after exposure. 71  Tuberculin Skin Test A new alternative, the ELISA interferon Gamma (IFN-g) assay QuantiFERON-TB Gold® produced in Australia and FDA cleared in 2005. Patients who carry TB will have sensitized lymphocytes. 72  Tuberculin Skin Test Patient’s whole blood (lymphocytes) exposed in vitro to two proteins found only in MTB, CFP-10 and ESAT-6. Sensitized cells will release Interferon- Gamma (IFN-g). Measure IFN-g with ELISA. BCG vaccine has no CFP-10 or ESAT-6 proteins. 73  Autoimmunity T and B lymphocytes fail to distinguish between self and non-self. Autoimmune Disease include: RA, lupus erythematosus (possible drug induced), MS, Type I diabetes. 74  RA In RA, IgM (rheumatoid factors) antibodies are produced which bind to Fc portion of IgG antb. IgM-IgG Fc immune complexes activate the complement cascade causing inflammation of the joints and kidneys. 75 Complement Cascade 76  Screening Tests for RA RA Factor RI < 30 U/mL Test measures in serum the presence of the IgM antibody against the IgG Fc portion of the IgG antibody. Somewhat nonspecific and may be present in infections. Subtype IgA RA may be indicative of more severe RA disease. 77 RA Factor RA hemagglutination test for presence of RA Factor Sheep red cells coated with rabbit IgG and placed into wells. Add patient serum (dilutions) If patient serum has anti-IgG antibodies (IgM RA Factor) then red cells will agglutinate (diffuse appearance) If patient is Neg the sheep cells fall into button on bottom of the well. 78  Immunodeficiency Reduced ability to produce antibodies due to genetic (many different defects); bacterial, viral and drug induced. Tests of Immunocompetence: Serum Immunoglobulins Total Lymphocyte Count Lymphocyte Subsets (CD receptors) Various WBC function assays 79  Immunosuppressive Drugs Glucocorticoids (prednisone, dexamethasone) Most inhibit B cell proliferation They inhibit production of inflammatory mediators (prostaglandins and cytokines). Antibody concentrations decrease. IgG RI 565-1756 mg/dL IgM RI 50-300 mg/dL IgE RI 0.01 - 0.04 mg/dL 80  Immunosuppressive Drugs Cyclosporine (Sandimmune®) Used to prevent transplant rejection, hematopoietic stem cell transplantation and some autoimmune disorders. Drug blocks B cell proliferation. Drug considered to have made organ transplants possible. 81 Cyclosporine Polypeptide made of 11 amino acids derived from fungus Trichoderma polysporum. Given po (wide variation in absorption) and iv. Whole blood levels correlate with immunosuppression. For kidney 100- 300 ng/mL whole blood. 82 Tacrolmus (Prograf®) Macrolide antibiotic isolated from Streptomyces tsukubaensis. Blocks B cell proliferation similar to cysclosporin but 10 -100 times more potent. Given po or iv with blood levels correlating with suppression and toxicity. Used for kidney transplant. 83  Thalidomide The sedative that was used to treat nausea of pregnancy and refused FDA approval in 1962 due to reported birth defects. Currently in active clinical trials for use in > 40 different conditions. Drugs has significant immunomodulatory actions. 84 Thalidomide Currently in use to treat leprosy and a few immune disorders (lupus, myeloma) Mechanism not clear. 85  Cytotoxic Agents Azathiopurine, a derivative of mercaptopurine, acts as an immunosuppressive agent (drug), an antimetabolite. Drug is metabolized to mercaptopurine, which inhibits purine (DNA) synthesis and lymphoid cell proliferation. Drug use in RA, lupus, Crohn’s disease, transplants, multiple sclerosis etc. 86  Cytotoxic Agents Cyclophosphamide, an alkylating agents (intercalates with DNA) Drug inhibits proliferation of lymphoid cells. Used in transplants and numerous autoimmune disorders. 87  Immunomodulating Drugs Cytokines: used to increase the response of the immune system in patients with compromised immune system Heterogeneous group of proteins with diverse functions. Among the cytokines are TNF, interleukins, interferons etc 88  Immunomodulating Drugs Cytokines Drug applications of the cytokines vary greatly from cancer to immune disorders. Most cytokines regulate the proliferation of immune response lymphoid cells. Drugs used as adjunct therapy in a wide variety of diseases. 89  Immunotherapy for MS Multiple Sclerosis: demyelinating inflammatory disease of the CNS (with no PNS involvement). Disease may be autoimmune, treat with immune suppresion. Cortocosteroids (1g/day iv for 3-5 days) Beta-1 Interferons to suppress T lymphocyte proliferation. Alkylating agents (cyclophosphamide) 90  Drugs Available 5 “cept” drugs 22 “mab” drugs 3 globulins 5 interferons 1 interleukin A few misc others 91