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Pharmacology of Inflammation and Immune Disorders SCIBIOM-301 Pharmacology Project Immunopharmacology

› Individual projects: › Make an attractive brochure for your selected – Study the selected drug – What is it used for? (pathophysiology) – Treatment modalities (type of drug, potential targets) – (, ) – (, , distribution, , , etc.) – Contra-indications, adverse events, drug interactions – Discussion of usefulness › Make use of different sources (including scientific research papers!) Innate and adaptive immunity

Non-specific Specific Memory Specificity Recognition of non-self Types of adaptive immunity

Humoral -mediated immunity immunity

• •T-lymphocytes •B-lymphocytes •Intracellular •Extracellular microbes microbes Primary and secondary immune response What will happen with ? Cytokines as mediators

› Cell division › Inflammation › Immunity › Differentiation › Migration › Repair structure

› The arms of the (Fab) recognize and bind antigen

› The Fab arms consist of regions of variable sequences and give each Fab antibody its unique specificity. Fc Antibody structure › The stem (Fc) interacts with effector (complement and Fc receptors) leading to elimination of the antibody-coated foreign material.

Fab › The Fc stem of the molecule has a more conserved sequence. Fc › Differences in the Fc regions lead to different classes and subclasses of antibodies. Polyclonal antibodies

› Polyclonal antibodies are antibodies that are secreted by different B-cell lineages.

› The antibodies in the serum react against the same antigen but recognize different antigenic determinants or epitopes on the antigen. Monoclonal antibodies

› Monoclonal antibodies are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell.

› Given any substance, it is possible to create monoclonal antibodies that specifically bind to that substance.

› This has become an important tool in , molecular and . Monoclonal antibodies Monoclonal antibody production

› The myeloma cells lack the HGPRT and in the presence of aminopterin, they cannot produce tetrahydrofolate, resulting in a defect in de novo purine synthesis.

› Hybrid cells receive HGPRT from the splenocytes and have the capacity for uncontrolled proliferation from the myeloma partner; as a result, only hybrid cells survive in HAT medium. Monoclonal antibody production

› Spleen cells from an immunized mouse are fused with a myeloma cell line, with use of polyethylene glycol that facilitates fusion of plasma membranes and the formation of hybrid cells.

› The hybrid cells are placed in a selection medium that permits the survival of only immortalized hybrids.

› The hybrid cells are grown as single cell clones and tested for the of antibody. Methodologies commonly used to isolate human monoclonal antibodies Methodologies commonly used to isolate human monoclonal antibodies Methodologies commonly used to isolate human monoclonal antibodies Therapeutic antibodies: Strengths

› Availability of several well-established and broadly applicable methods for antibody generation and optimization. › A growing repertoire to redesign antibodies in order to enhance their clinical potential (example: terminal half-life can be tuned from minutes up to weeks). › High success rate compared to other : 17% for humanized antibodies from the first human trial to regulatory approval. › Well-established and broadly applicable production technologies. › Often well tolerated by patients. › Broad experience and expanding knowledge of antibody . Therapeutic antibodies: Limitations

› Expensive, reflecting high production costs and commonly large doses, potentially limiting patient access or clinical applications. › Clinical applications currently limited to cell surface or extracellular targets. › Cannot be orally administered. › The large size, particularly of the IgG format (∼150 kDa), may limit penetration. › There is limited penetration of the central by IgG owing to inefficient penetration of the –brain barrier. Therapeutic antibodies: Future opportunities

› Greater efficacy, higher affinity or potency, greater safety, reversibility of antibody action. › Enhanced effector functions — antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement- dependent cytotoxicity (CDC). › Target multiple disease mediators from distinct signaling pathways or using bispecific antibodies. › Greater selectivity for target, and avoid activation of the target. › Improved delivery, increased bioavailability, ability to cross the BBB, more facile ocular delivery, and oral delivery. IgG structure Mechanisms of action Mechanisms of action IgG architecture supports bispecific formats Inflammation › Inflammation is mediated in part by prostaglandins produced by the cyclooxygenase pathway.

› Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit this pathway and serve as combined anti-inflammatory, antipyretics, and .

› Because NSAIDs are generally nonspecific and exert numerous side effects, there is great interest in more specific therapeutics such as selective COX-2 inhibitors and anti-cytokine agents. Pharmacology of eicosanoids: Role of arachidonic acid Pharmacology of eicosanoids: Lipoxygenase pathway

› Lipoxygenases (LOX) are that catalyze the insertion of molecular oxygen into arachidonic acid, using non-heme iron.

› Major forms: 5-LOX, 12-LOX, and 15- LOX Pharmacology of eicosanoids: Lipoxygenase pathway

› Immediate products are hydroperoxyeicosatetraenoic acids (HPETEs).

› HPETEs are reduced to hydroxyeicosatetraenoic acids (HETEs) by glutathione peroxidase- dependent enzymes.

› LTC4, LTD4, LTE4 and LTF4 represent the cysteinyl leukotrienes. Lipoxygenase pathway › 5-lipoxygenase is not present in all tissues but is limited to neutrophils, eosinophils, monocytes, and certain mast cell .

› Lipoxygenases produce leukotrienes (e.g. LTB4, LTD4), which are potent bronchoconstrictors and chemotactic agents.

› Leukotrienes have important roles in asthma, glomerulonephritis and inflammatory bowel disease. Prostaglandins › All cells in the body have the capacity to synthesize prostaglandins.

› In response to inflammatory stimuli arachidonic acid (AA) is separated from plasma phospholipids by phospholipase A2.

› Cyclooxygenase metabolizes AA to the cycloendoperoxide prostaglandin H2 (PGH2), which is then converted to either PGD2, PGE2, PGF2α, PGI2 (prostacyclin) or TxA2 (thromboxane) by appropriate enzymes (i.e. thromboxane synthase in , prostacyclin synthase in endothelial cells). Prostaglandins and their functions › The prostaglandins exert numerous physiologic and pathophysiologic functions :

– Physiologic: temperature , bronchial tone, cytoprotection (gastric and renal mucosa), intestinal mobility, myometrial tone, semen viability (some prostaglandins like PGE1 have anti-inflammatory effects), renin secretion

– Pathologic: fever (aberrant hypothalamic thermoregulation), asthma (airway responsiveness and immune hyperreactivity), ulcers (loss of cytoprotection), diarrhea (intestinal mobility), dysmenorrhea (myometrial tone), inflammation, erosion, pain (thought to be caused by PGD2) Prostaglandins and inflammation

› PGI2: inhibits aggregation, vasodilatation, vascular permeability (edema)

› PGE2: pain, hyperalgesia, heat, vasodilatation, bronchoconstriction, synergistically act with other pro-inflammatory mediators (, complement, LTB4)

› TxA2: promotes platelet aggregation, vasoconstriction, bronchoconstriction Pharmacology of eicosanoids: Cyclooxygenase pathway

› Two cyclooxygenase isoforms (COX-1 and COX-2) are found in humans.

› Though COX-1 and COX-2 catalyze the same reaction, their expression, functions and properties are markedly different. COX-1 vs COX-2

COX-1 COX-2 Expression Constitutive (activated by Inducible by pro-inflammatory stimuli physiologic stimuli) Tissue localization Ubiquitous Inflammmatory and neoplastic sites (small amounts in kidney, uterus, ovary, CNS) Role Housekeeping and maintenance Pro-inflammatory and mitogenic functions and plays a role in stress and pregnancy Goal of anti-inflammatory therapy

› The goal of pharmacologic anti-inflammatory therapy has been to inhibit COX-2 produced prostaglandins.

› Non-specific inhibition of COX-1 results in gastrointestinal and platelet side effects.

› Recent data on the of COX-2 selective NSAIDs illustrate that this is an overly simplistic view. The magnitude of the COX-2 problem is still unclear. NSAIDs › Most NSAIDs are polycyclic carboxylic acid derivatives with relatively low pKa values. NSAIDs are often classified on the basis of their chemical structure. – Salicylates: ; diflunisal, 5-aminosalicylate, sodium salicylate, salicylate, sulfasalazine, olasalzine – Acetic acids: indomethacin, diclofenac, sulindac, etodolac, ketorolac, tolmetin – Propionic acids: ibuprofen, naproxen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin – Fenamic acids: meclofenamate, mefenamate – Enolic acids (oxicam class): piroxicam – Ketones: nabumetone (converted to 6-naphthylacetic acid in liver) NSAIDs › All NSAIDs (except aspirin) act as reversible, competitive cyclooxygenase inhibitors. They block the hydrophobic channel by which the substrate arachidonic acid accesses the enzyme active site. Aspirin covalently modifies and destroys the cyclooxygenase enzyme.

› The ultimate of the NSAID is to inhibit COX-2, preventing generation of proinflammatory eicosanoids, and thus limiting the extent of inflammation and adverse signs and symptoms.

› Despite the benefits of NSAIDs, they only provide symptomatic relief, as the underlying pathophysiology or injury generally is unaffected. NSAIDs › NSAIDs have three primary therapeutic effects: – Analgesia

– Anti-pyrexia (decreasing hypothalamic PGE2) – Anti-inflammatory

› NSAIDs are also used as anti-thrombotics. Since they impair platelet aggregation, they prolong bleeding time, and function as . The COX-2 specific inhibitors do not exert anti-thrombotic effects. › Other functions of NSAIDs include inhibition of superoxide generation, lymphocyte function, lysosomal enzyme release, cytokine release (IL-6), and neutrophil aggregation / adhesion COX-2 selective inhibitors (Coxibs)

› The coxibs represent a subset of NSAIDs that preferentially block the hydrophobic substrate channel in COX-2. › Currently approved coxibs include celecoxib and valdecoxib. › These drugs are approved for rheumatoid arthritis, osteoarthritis, pain, primary dysmenorrhea, and familial adenomatous polyposis (they decrease the number and size of adenomas in patients with history of FAP). COX-2 selective inhibitors (Coxibs)

› The potential therapeutic role of coxibs in Alzheimer’s disease is being studied (COX-2 is the predominant isoform in the neocortex and hippocampus).

› COX-2 is induced by LH prior to ovulation and at delivery. COX-2 selective inhibitors may have a role in preventing preterm labor and delivery. of coxibs › Rofecoxib was recently withdrawn from the market when an increased rate of myocardial infarction and stroke was seen in a -controlled trial for FAP.

› There is now concern that all COX-2 inhibitors may increase the risk of thrombotic events during chronic therapy. › Evidence for this has now appeared in one trial of valdecoxib and one study of celecoxib. › A possible explanation may be that coxibs can inhibit endothelial prostaglandin synthesis but lack a compensatory effect on platelet thromboxane synthesis. Toxicities of coxibs › The situation is currently unsettled, but it seems prudent to restrict the use of these drugs to patients for whom the potential benefits are clearly worth the risk.

› The risk vs. benefit may be difficult to characterize: COX-2 inhibitors were designed in part to limit the gastrotoxicity associated with NSAIDs. › Although the rate is lower, events still occur and symptoms are similar. However, events are less serious (i.e. less perforation) vs. conventional NSAIDs. Platelet function › Platelets only express COX-1, so COX-2 has no effect on platelet function or the production of TXA2. The implications of this are: – Patients on MI prophylaxis still need aspirin even if they are on a COX-2 selective inhibitor – Coxibs unlike NSAIDs may be administered safely with .

› COX-2 is present in the kidney and knock out mice show kidney inflammation and papillary changes. › This suggests that chronic treatment with coxibs may impair normal renal development and function. Some evidence suggests that inhibition of COX-2 may generate problems with wound healing and angiogenesis.