A Seminar on Immunomodulated Molecules Keyur Vasava…

IMMUNE SYSTEM The immune system is designed to protect the host from invading pathogens and growing neoplastic cells through interaction of a wide variety of cell types and secreted factors to eliminate disease. There are now many agents with different mechanisms of actions, targets and side effect profiles that can be used in treatment. They are of two categories:

1. Immunosuppressive/Inflammatory 2. Immunostimulantory.

Rational for Immunomodulation

1. To increase specificity for the immune system and minimize the toxicity. 2. To minimize non-specific immune suppression or enhance select components to obtain

the desired effect while avoiding decreases in host resistance or autoimmune diseases.

Immune Response: The role of immune system is to recognize and remove invading microorganisms and tumor cells through innate and acquired immune responses. When exposed to an invading organism two types of immune responses observed as follows: Innate immune response – first line of defense against an antigenic insult. Includes defenses like physical (skin), biochemical (complement,lysozyme, interferons) and cellular components (neutrophils, monocytes, macrophages).

Adaptive immune response a) Humoral immunity - Antibody production – killing extracellular organisms. b) Cell mediated immunity – cytotoxic / killer T cells – killing virus and tumour cells. When re exposed to invading organism, antigen-specific cells of the acquired immune response are called back into action for more rapid and effective response.

Comparison of Innate and Acquired immunity: Characteristics Innate Immunity Acquired Immunity Onset of action Immediate Days to week Mechanism of

action recognized

Receptors that recognize Common molecules on

microbes And viruses

Unique antigen specific (T-cell receptor, B cell receptor)

Cell types/factors

Macrophages, neutrophils, mast cells, natural killer

cells, complement, interferon

Antigen presenting cells, T-lymphocytes, B-lymphocytes

Mechanisms of Action

1. Innate Immunity 2. Acquired Immunity

Cell-mediated Immunity Humoral Immunity.

1. Innate Immunity: With bacterial infection, complement and phagocytic cells play a vital role in producing a local inflammatory response that leads to killing and removal of the invading microbes. Complement can be activated by antigen-antibody complexes or by natural substances such as bacterial cell wall components to produce split products that activate and recruit macrophages and neutrophils. Macrophages are activated and phagocytized the microbes, producing a variety of cytokines and chemokines. The cytokines activate neighboring cells and recruit neutrophils that kill and remove the invading microbe. 2. Acquired Immunity:

Acquired Immunity is commonly divided in to Humoral and cell-mediated responses. Initiation of acquired immunity first involves antigen-specific activation of naïve T-cells (CD4+, T-helper cells). This requires participation of antigen presenting cells (APC) that take up and process antigens into peptide fragments. The naïve T-cells requires two signals to be fully activated. The first is provided by the peptide binding to the T-cell receptor, and the second comes from the interaction of costimulatory molecules of the APC and the T-cell. “Signal One” in the absence of “Signal Two” leads to tolerance, or functional silencing, of the T-cells. In general, there are two types of effector T-helper cells: Th-1 and Th-2 cells. Cytokines produced by Th-1 cells stimulate generation of cell-mediated immune responses whereas Th-2 cells produce cytokines that drive formation of an antibody response.

Cell mediated Immunity: Cell mediated immune responses involve Th1-mediated activation of macrophages and generation of CD8+ cytotoxic T-lymphocytes. Th1 cells secrete cytokines, which recruit and activate macrophages. Macrophages are then capable of killing intracellular bacteria and produce a localized inflammatory response. This also occurs with chemicals such as urushiol from poison ivy. Humoral Immunity: Th2 cells secrete cytokines that stimulate proliferation and differentiation of B cells to antibody-secreting plasma cells or to long-lived memory cells. Specific antibodies can remove harmful foreign antigens by binding to and neutralizing their effects. Antigen-antibody immune complexes can activate complement to elicit a local inflammatory reaction for further antigen removal by phagocytes.

ABNORMAL IMMUNE RESPONSE Hypersensitivity reactions Type 1 – Anaphylactic shock Type 2 – Mismatched blood transfusion Type 3 – Serum Sickness, glomerulonephritis and arthritis. Type 4 – TB, leishmaniasis. Autoimmunity: Autoimmune diseases arise when the body mounts an immune response against itself as a result of failure to distinguish self tissues and cells from foreign antigens. Rheumatoid Arthritis, Type 1 Diabetes Mellitus, Multiple Sclerosis etc…. Immunodeficiency Diseases: Immunodeficiency diseases results from inadequite function in the immune system. Immuno deficiency syndromes are either Congentially acquired or arise from extrinsic factors such as bacterial or viral infection or drug treatement. Examples a) Congenital – Di George’s syndrome, Severe combined Immunodeficiency Diseases

(SCID) due to adenosine deamination (ADA) deficiency. b) Extrinsic – HIV causing AIDS.

An immunomodulator is a substance (e. g. a drug) which has an effect on the immune system. There are two types of effects - immunostimulation and immunosuppression.

Immunosuppressants primarily have the suppressant effect. Immunostimulants primarily have the stimulant effect.

Most drugs however do not have effects on only one receptor, so an immunomodulator may be at the same time an immunosuppressant and an immunostimulant, on different targets within the immune system.

Products that are not single chemical entities, such as herbal extracts and impure products, may have even greater plurality of effect. Many species of plants, depending on the specific extraction conditions used, have immunomodulatory effects.

Immunosuppressant is any substance that performs immunosuppression of the immune system. They may be either exogenous, as immunosuppressive drugs, or endogenous, as, e. g., testosterone. When the immune system function is suppressed, there is an increased susceptibility to infectious diseases and cancers.

The term immunotoxin is also sometimes used (incorrectly) to label undesirable immunosuppressants, such as various pollutants. Polychlorinated-biphenyls (PCB) and the herbicide DDT are immunosuppressants.

Glucocorticoids - Prednisolone. Calcineurin inhibitors –Cyclosporine Tacrolimus. Cytotoxic agents – Azathioprine, Leflunomide, Cyclophosphamide, Methotrexate,

Dactinomycin, Vincristine. Antibodies – ALG, ATG like Munomonab CD3, IGIV, Monoclonal antibodies like

Daclizumab, Basiliximab. Others – Interferons, Thalidomide, Sirolimus, Mycophenolate Mofetil. GLUCOCORTICOIDS: Glucocorticoids were the first hormonal agents recognized as having lympholytic properties. Administration of any glucocorticoids reduce the size and lymphoid content of the lymph nodes and spleen. Glucocorticoids modulate allergic reaction and are useful in the treatement of diseases like asthma or premedication for other organs (e.g. blood products, chemotherapy) that might

cause undesirable immune response. Glucocorticoids are first line of immuno suppresive therapy for both solid organ and hematopoletic stem cell transplant reciepts.

CALCINEURIN INHIBITORS 1) Cyclosporine –

– Fat soluble peptide antibiotic.

– Binds to cyclophilin and inhibits a cytoplasmic phosphatase, calcineurin which is necessary for activation of a T cell specific transcription (NFAT) which causes synthesis of interlukins (IL-2) by activated T cells.

– Metabolized by Cyt P450 – drug interactions

Side Effect – nephrotoxicity, hypertension, hyperglycaemia, liver dysfunction and hirsuitism.

Very low incidence of bone marrow toxicity.

– Used in renal, pancreatic and liver transplantion. Also in Rheumatoid Arthritis, psoriasis, uveitis and asthma.

2)Tacrolimus ( FK 506 )

– Immunosupressant macrolide antibiotic. – Binds to immunophilin FK-binding protine. – It is 10-100 times more potent than cyclosporine in inhibiting the immune response. – Utilized for same indication as cyclosporine, particularly in organ and stem cell

transplantation. – Topical preparation available for use in atopic dermatitis and psoriasis.

CYTOTOXIC AGENTS

1) Azathioprine

- Azathioprine is a prodrug of mercaptopurine as an antimetabolite. - Azathioprine and mercaptopurine produce immunosuppression by interfering with purine

nucleic acid metabolism and destroy stimulated lymphoid cells. - Used in Systemic Lupus Erythematosus, Renal allograft, Rheumatoid Arthritis, Crohn’s

disease, glomerulonephritis.

Side Effect : bone marrow supression, skin rashes, diarrhea, hepatic disfunction

2) Leflunomide - It is a prodrug of an inhibitor of pyrimidine synthesis.

- Used in Rheumatoid Arthritis. Also have antiviral activity.

Side Effect : elevation of liver enzyme with some risk of liver damage, renal impairement and teratogenic effect.

Cyclophosphamide : - It is alkylating agent which destroys proliferating lymphoid cells.

- Used in Systemic Lupus Erythematosus, autoimmune haemolytic anaemia, multiple sclerosis, Wegener’s granulomatosis.

Side Effect : pancytopenia and hemorrhagic cystitis, cardiac toxicity, electrolyte imbalances.

OTHERS 1) Sirolimus :

– Sirolimus binds immunophilins and inhibit calcinurin. It inhibits T cell response to Interlukin-2.

– It is a potent inhibitor of B-cell proliferation and immunoglobuline production. – Use to prevent rejection of solid organ allografts, dermatologic disorders, in management

of uveoretinitis.

Side effect : myelosupression, hepatotoxicity, diarrhea, hypertriglyceridemia and hadache.

2) Thalidomide :

– inhibits angiogenesis, reduces phagocytosis, enhances cell mediated immunity via intractin with T cells.

– Increases levels of IL-10. – Used in multiple myeloma, acute myelogenous leukemia, graft versus host disease,

myelodysplastic syndrome, Renal Cell Cancer, prostrate cancer, colon cancer.

Side effect : teratogenesis, peripheral neuropathy, constipation, hypothyroidism.

3) Mycophenolate Mofetil : – It is a semisynthetic derivative of mycophenolic acid. – It inhibits T- and B- lymphocyte responses, including mitogen and mixed lymphocyte

responses by denovo synthesis of purines.

– Mycophenolate Mofetil is hydrolyed to mycophenolic acid which is the active immunosupressive moiety and administered as MMF to enhance to increase bioavailibity.

– Used in steroid refractory graft versus host disease, Rheumatoid Arthritis, some dermatologic disorders.

ANTIBODIES 1) Antilymphocyte and Antithymocyte Antibodies

– Antilymphocyte globulin (ALG) and Antithymocyte Antibodies globulin (ATG) are in clinical use in transplantation program.

– ALG and ATG are useful for suppressing certain major compartments of immune system and play a definite role in the management of solid organ and bone marrow transplantation.

Side effect

– Local pain and erythema (type III hypersensitivity reaction). – Anaphylactic and serum reaction.

2) Muromonab – CD3

– T cell receptor complex. – It bloks killing killing by cytotoxic human T-cells and several oyher T-cell function.

Use

– In auto immune disorders. – In treatement of renel transplant rejection, renel allograft rejection crisis.

3) Monoclonal Antibodies used as immunosupressants

– Daclizumab, Basiliximab are the monoclonal antibodies used as monoclonal immunosupresssant.

– IL-2 receptor antagonist, blocks IL-2 mediated T cell activation and thus act as immunosupression.

Use

– Prophylaxis of acute organ rejection for renal transplantation and as a part of immunosupressive regimen.

CLINICAL USE OF IMMUNO SUPRESSIVE AGENTS Immunosuppressants may be prescribed when a normal immune response is undesirable, such as in:

1) Solid Organ and Bone Marrow Transplantation. After an organ transplantation, the body will nearly always reject the new organ(s) due to differences in human leukocyte antigen haplotypes between the donor and recipient. As a result, the immune system detects the new tissue as "hostile", and attempts to remove it by attacking it with recipient leukocytes, resulting in the death of the tissue. Immunosuppressants are applied as a countermeasure; the side-effect is that the body becomes more vulnerable to infections and malignancy, much like in an advanced HIV infection.

2) Autoimmune Disorders

Immunosuppressive drugs are widely used in autoimmune disorders. With immunosuppressive therapy, remission obtain in many intances of autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, type-1 diabetes, temporal arthritis, Rhematoid Arthritis, chroni severe asthma.

SOURCE IMMUNOSUPPRESSIVE AGENT USED.

Autoimmune Disorder

Idiopathic thrombocytopenic

purpura(ITP)

Prednisone, vincristine, occasionally cyclophosphamide,

Mercaptopurine, azathioprine.

Autoimmune hemolytic

Anemia

Prednisone, cyclophosphamide, chlorambucil, mercaptopurine, azathioprine.

Acute glomerulonephritis Prednisone, cyclophosphamide, mercaptopurine

Acquired factor XIII antibodies Cyclophosphamide plus factor XIII

Autoreactive tissue disorder Prednisone, cyclophosphamide, methotrexate, azathioprine, cyclosporine.

Isoimmune Disease

Hemolytic disease of the newborn

Immune globuline

Organ Transplantation

Renal and Heart azathioprine, cyclosporine, Prednisone, baciliximab, sirolimus

Liver azathioprine, cyclosporine, Prednisone, tacrolimus, sirolimus

Bone Marrow cyclosporine, Prednisone, cyclophosphamide, methotrexate.

Prevention of cell proliferation

Coronary stents Sirolimus

Immunostimulating Agents

Immunostimulants, also known as immunostimulators, are substances (drugs and nutrients) that stimulate the immune system by inducing activation or increasing activity of any of its components. One notable example is the granulocyte macrophage colony-stimulating factor.

Classification

There are two main categories of immunostimulants:

Specific immunostimulants provide antigenic specificity in immune response, such as vaccines or any antigen.

Non-specific immunostimulants act irrespective of antigenic specificity to augment immune response of other antigen or stimulate components of the immune system without antigenic specificity, such as adjuvants and non-specific immunostimulators.

Non-specific

Many endogenous substances are non-specific immunostimulators. For example, female sex hormones are known to stimulate both adaptive and innate immune responses. Some autoimmune diseases such as lupus erythematosus strike women preferentially, and their onset often coincides with puberty. Other hormones appear to regulate the immune system as well, most notably prolactin,growth hormone and vitamin D.

Some publications point towards the effect of DCA as an immunostimulant of the unspecific immune system, activating its main actors, the macrophages. According to these publications, a sufficient amount of DCA in the human body corresponds to a good immune reaction of the unspecific immune system.

1) Cytokines like INF alpha, INF beta, INF gamma, IL-2 – metastatic RCC and malignant melanoma. TNF alpha- malignant melanoma and soft tissue sarcoma of extremity.

2) Levamisole – anti helminthic. Used to treat immunodeficiency associated with Hodgkins disease. Causes severe agranulocytosis.

3) BCG – used as intravesical therapy for superficial bladder cancer. It causes activation of macrophages to make them more effective killer cells.

4) Other drugs – inosiplex, azimexon, imexon, thymosin, methylinosine monophosphate…….

Pharmacokinetics Azathioprine

• Usually given as loading dose on the day of transplantation, with subsequent oral maintenance dose

• Rapidly absorbed and converted to 6-mercaptopurine, which is active drug • Most metabolites are excreted through urine • Half life – 5-6hrs •

Mycophenolate mofetil • Rapidly metabolized to active metabolite, mycophenolic acid • Mofetil moiety increases bioavailability(BA) • Bound to serum albumin and excreted through kidney as glucuronide conjugation • Half life – 17hrs

Cyclosporine

• Poorly absorbed from small intestine (BA of 30%) and dependent on biliary flow of absorption

• Metabolized by Cyt P450 3A, which may cause drug interaction • Cause nephrotoxicity • Half life – 10-40hrs •

Tacrolimus • 10 to 100 times more potent than cyclosporine and does not rely on bile for absorption • Metabolized by Cyt P450 3A • Causes nephrotoxicity with similar frequency as cyclosporine • Half life – 12hrs

Antibodies

• Administered only through Parenteral route • Half life depend on is stability and clearance mechanism • Half life can be increased by addition of PEG • Conjugation with PEG increases size and modifies the overall charge to decrease

glomerular filtration

Drug Half Life Basiliximab

7-13 days

Daclizumab

11-38 days

Infliximab

8-10 days

Adanimumab

12-13 days

Marketed Preparations

Azathioprine (generic, Imural)

Oral: 50mg tablet Parenteral: 100mg/vial for IV injection

Basiliximab (Simulect)

Parentral: 20mg powder, reconstitute for IV injection

Leflunomide (Arava)

Oral: 10, 20, 100mg tablets

Cyclosporine (Sandimmune, Neoral, SangCya)

Oral: 25,50,100 mg capsules; 100mg/ml solution Parenteral: 50mg/ml for IV aadministration

Muromanab-CD3 (Orthoclone OKT3)

Parenteral: 5mg/ml ampule for injection

Sirolimus (Raoamune)

Oral: 1mg tablet; 1mg/ml solution REFERENCES 1. Brody’s Human Pharmacology Molecular to clinical 4th edition Minnerman,

wecker, Larner and Brody, page no. 681-699. 2. Principles of Pharmacology – The pathophysiologic basis of Drug therapy, 2nd

edition by David E.Golan, Armen H. Tashjian, Jr. Ehrin J. Armstrong, April W.Armstrong : Lippincott williams and Wilkins, page no. 908-933

3. Basic and clinical pharmacology Lange, 10th edition by Bertram G. Katzung, page no. 556-586.