BPS 401 EXAM 2 Eicosanoids, NSAIDs & DMARDs

What causes arachidonic acid to release from the cell membrane?

Stimulation of phospholipase A2 (PLA2)

What are the major PLA2 families?

1. Cytosolic (c) PLA2 (Ca2+ dependent)

2. Secretory (s) PLA2 (Ca2+ dependent)

3. Calcium-independent (i) PLA2

What happens to arachidonic acid under non-stimulated conditions?

Still freed from cell membrane by iPLA2, but is reincorporated into membrane. minimal biosynthesis/reoccurring feedback loop

What causes phospholipases to release arachidonic acid from the cell membrane?

Stimulation; tissue trauma, immune response

What are eicosanoids derived from?

arachidonic acid oxygenated by cyclooxygenase (COX), lipoxygenase, P450 epoxygenase, and isoecosanoid.

All but isoecosanoid are enzymatic pathways

What factors determine eicosanoid synthesis (what pathway is generated)?

1. Substrate lipid species

2. Type of cell

3. Manner in which the cell is stimulated 

What are the two COX isozymes?

• COX-1

  • Most cells, normal physiology, “constitutive”

  • Ongoing process @ baseline

  • Responsible for naturally occurring activity

• COX-2

  • Inflammatory cells, inducible (upregulated during inflammatory/stress responses)

    • Downstream generation of mediators promotes inflammatory response

How do prostaglandins differ?

Substituents on pentane ring: PGE, PGF

Number of double bonds on side chains, PGE1, PGE2

What do COX isozymes oxygenate arachidonic acid into?

Endoperoxidase with a 15-hydroxyl group

What is the half-life of eicosanoids?

Seconds. Drug doesn’t circulate/isn’t a systemic agent

What are the eicosanoid receptors?

PGI2 (IP), PGFα2 (FP), TXA2 (TP)

PGE2 (EP1-4); PGD2 (DP1,2)

What do EP3 eicosanoid receptors do?

Increase intracellular Ca2+

Alter cAMP activity

What do DP2 eicosanoid receptors do?

Inhibit cAMP

Increase intracellular Ca2+

What do EP1, FP, and TP eicosanoid receptors do?

Activate metabolism. Mobilizes Ca2+ and increases free intracellular Ca2+

What do EP2, EP4, IP, and DP1 eicosanoid receptors do?

Activate adenylyl cyclase and increase cAMP. Activates protein kinases

How do eicosanoid receptors promote vasodilation?

Upregulation of cAMP

How do eicosanoid receptors promote contraction?

Upregulation of Ca2+

What are the effects of TXA2 (TF) and PGF2α (FP) on the vascular system

Vasoconstriction → upregulation of intracellular Ca2+

What are the effects of PGI2 (IP) and PGE2 (EP1-4) on the vascular system?

Vasodilation → inhibition of smooth muscle cell proliferation

What are the effects of PGI2 (IP) and PGE2 (EP1-4) on the respiratory system?

Vasodilation → cAMP upregulation

What are the effects of PDG2 (DP1, DP2), TXA2 (TP), and PGF2α (FP) on the respiratory system?

Vasoconstriction → Upregulation of intracellular Ca2+

How does PGE2 (EP1-4) affect platelet aggregation?

Low concentration, increase aggregation (EP3)

High concentration, decrease aggregation (IP)

How does PGD and PGI2 affect platelet aggregation?

Inhibit aggregation via cAMP

How does TXA2 affect platelet aggregation?

Amplifies effects of platelet agonists

• ex: thrombin

  • Major product of COX-1

What drugs inhibit eicosanoid synthesis?

Corticosteroid injections. Inhibit PLA2 → less AA

• Shuts down inflammatory pathway

NSAIDs, Inhibit COX → block PG & TX 

• Decreases generation of inflammatory mediators

• AA still produced

Difference between COX-1 and COX-2

COX-1

• Constitutive, non-inflammatory cells

COX-2

• Induced, activated immune cells

What are the 3 phases of the inflammatory response

1. Acute

2. Immune

3. Chronic

How is the immune response produced?

Cell damage associated with inflammation releases AA → eicosanoids synthesized

What are the 2 inflammatory treatment goals?

1. Relief of inflammation symptoms/pain relief

2. Slow the tissue-damaging process

What is the chronic phase of inflammation mediated by?

Proinflammatory cytokines

What can NSAIDs be used for/what are they suitable for?

1. To decrease pain and inflammation

2. Suitable for acute or chronic inflammation

What are the classifications of therapeutic agents

1. NSAIDs (non-steroidal anti-inflammatory drugs)

   • Inhibition of prostaglandins

     • Non-selective COX inhibitors

     • COX-2 selective inhibitors

2. Acetaminophen

3. Salicylates

   • E.g. asprin

What are the general pharmacokinetic properties of NSAIDs?

• Well absorbed

• Lower GI irritation

• Hepatic metabolism

• Renal excretion

What are the general pharmacodynamic properties of NSAIDs?

• Anti-inflammatory activity via prostaglandin inhibition

  • Aspirin irreversibly blocks platelet COX (COX-1, can cause platelet aggregation)

  • Other non-selective NSAIDs reversibly inhibit COX

What are some common side effects of NSAIDs (good and bad)?

• Anti-inflammatory due to decreased mediator release

• Analgesic

• Antipyretic

• Inhibit platelet aggregation (except COX-2 specific)

• Gastric irritation

• Adverse renal and hepatic effects

DO NOT ALTER DISEASE COURSE.

ONLY RELIEVES PAIN FROM SAID DISEASE!!

Give examples of drugs in the following NSAID categories

1. COX-1 inhibitors

2. COX-2 selective inhibitors

3. Non-selective inhibitors

Aspirin is a COX-1 inhibitor

Celecoxib is a COX-2 selective inhibitor

Ibuprofen is a non-selective inhibitor

Aspirin should not be used in what patient population?

Patients with hemophilia

Where does PG synthesis happen in COX-2 selective inhibitors? Why is this important?

Happens in vascular endothelium

Results in no cardioprotection 

What is acetaminophen’s mode of action (MOA)?

Weak inhibition of peripheral COX

What are the proinflammatory cytokines?

IL-1, IL-2, IL-3, GM-CSF, TNFα, IFNs

What are the key features of DMARDs (disease-modifying anti-rheumatic drugs)?

1. Slow-acting anti-rheumatic

2. May slow bone damage, but might be more toxic than other options

Can DMARDs slow or reverse progression of joint damage?

Yes

What are the two main classifications of DMARDs

Non-biologic

Biologic

In general, what does the suffix “-mab” mean?

Example: rituximab, tocilizumab

Monoclonal antibody

Refers to biologic DMARDs

In general, what does the suffix “-cept” refer to?

Ex: Abatacept, Etanercept

Fusion proteins, including monoclonal antibodies, seen in biologic DMARDs

What is the first medication people are given for treatment of RA? What does it do?

Methotrexate

• Non-specific drug

  • Taken as an immunosuppressive, anti-cancer, and anti-proliferative drug

• Increases extracellular adenosine and inhibits inflammation

What are the key features of azathioprine?

Mode of action: active metabolite 6-thioguanine (6-TG) → that decreases B&T cell function, immunoglobulin production, and IL-2 secretion

• prodrug

  • Dependent on thiopurine methyltransferase for metabolism

What are the proposed MOAs for chloroquine & hydroxychloroquine?

Suppression of T-lymphocytes in response to mitogen, decrease leukocyte chemotaxis, inhibition of DNA/RNA synthesis, traps free radicals

• Decreasing T cell function decreases the release of inflammatory cytokines

When are patients treated with chloroquine & hydroxychloroquine?

When previous anti-RA therapy hasn’t worked (methotrexase)

What is the mode of action of cyclosporine?

Inhibition of IL-1 and IL-2 macrophage-T-cell interactions and T-cell responsiveness

• T-cell-dependent B-cell function is also affected

CYP3A metabolism

• Lots of drug interactions

What is the mode of action of mycophenolate mofetil?

Drug is converted to active mycophenolic acid (MPA) once metabolized. Inhibits inosine monophosphate dehydrogenase. Suppress T- and B-lymphocyte proliferation

• Inhibits leukocyte adhesion to endothelial cells/ E- and P-selectin/ ICAM-1

• Is a prodrug

What is the mode of action of sulfasalazine?

Metabolized to sulfapyridine and 5-aminosaliylic acid

• Inhibits pro-inflammatory cytokines

List details about leflunomide

Inhibits dihydroorotate dehydrogenase → decrease ribonucleotide synthesis

• Decreased T cell proliferation

• Decrease NF-kB activation

• Increase in IL-10

As effective as MTX but could cause hepatotoxicity when used alongside MTX

What does “fab” refer to in an antibody?

Fragment antigen-binding region

• Region of antibody that binds to antigens

• Has one constant and one variable domain

• Is the top part of the “Y” / where the Y starts to curve

What does “fc” refer to in an antibody?

Crystallizable fragment

• Part of antibody that interacts with cell surface

• Links antibody with other parts of immune system

What part of the antibody is involved in specificity and amino acid binding?

Hypervariable regions

What part of the antibody illicits the immune response?

The constant regions; receptors bind to Fc to activate immune response like NK cells

What biologic RA treatment is missing Fc?

What are the mechanisms of action for monoclonal antibodies (MAB)?

• Direct modulation of target antigen

  • Apoptosis

  • Inhibition of cell signaling

• Complement-dependent cytotoxicity (CDC)

• Antibody dependent cellular cytotoxicity (ADCC)

• Delivery of cytotoxic agent

Describe the following MAB mechanism of action: direct modulation of target. Give examples of drugs.

Inhibition of cell signaling: Neutralize signaling factors → Decrease surface receptor expression

Blocking action/removal of target: Bind to receptor/antigen → prevent activation

ex: anti-TNF-a, anti-CD20, abatacept (inhibition), tocilizumab (blockage)

Describe the following MAB mechanism of action: apoptosis. Give examples of drugs.

Antigen cross-linking targets the growth factor receptor

• Antagonize ligand-receptor signaling

• Inhibit growth-factor signaling mediated by tyrosine kinase

  • Ex: EGF, VEGF

• Induction of apoptotic pathways

ex: Rituximab (RTX)

What happens during antibody-dependent cell-mediated cytotoxicity (ADCC)? Does it happen in a tumor cell or an effector cell?

Cells that are coated in antibodies are recognized by NK cells, macrophages, and monocytes, inducing cell lysis.

Happens in an effector cell

What happens during complement dependent cytotoxicity (CDC)? Does it happen in a tumor cell or an effector cell?

Monoclonal antibodies are bound to complement, resulting in activation of complement cascade/immune system, generating membrane attack complexes and causing cell lysis.

Happens in a tumor cell

What is the role of CTLA-4 in T cell activation? (LOOK INTO THIS MORE BC WTF DOES THIS EVEN MEAN)

CTLA-4 inhibits the reaction of an antigen and blunts CD28 binding, mimicking the activation of 2nd T cell activation.

What is the mechanism of action for abatacept? Can it be combined with other meds?

Binds to CD80/86 antigen → inhibits CD28 binding, preventing T-cell activation (immune response)

Can be combo (MTX or NSAIDs) or mono therapy. CANNOT combo w TNF-α antagonists

What is the mechanism of action for rituximab? Can it be combined with other meds?

Depletes B lymphocytes through complement-dependent cytotoxicity and stimulation of apoptosis

Causes reduction in pro-inflammatory cytokine secretion

Can be combined with MTX if there is incomplete response to anti-TNFα therapy. Glucocorticoid typically given 30 min prior to injection; chill out immune response

What is the mechanism of action for tocilizumab? Can it be combined with other meds?

Direct modulation of target, blocks action

anti-IL-6 receptor antibody; inhibits B- and T-cell signaling, fibroblasts, synovial cells, and endothelial cells. can also suppress CYP450.

Can be mono or combined w non biologic DMARDs for incomplete responses to anti-TNFα agents

How does TNF-α affect cellular function?

Activation of membrane-bound TNF receptors. Promotes an anti-inflammatory response

What biologic DMARDs interfere with TNF-α? (TNF-α antagonists)

Adalimumab, Certolizumab, Etanercept, Golimumab, Infliximab

Which anti-TNF-α agent (TNF-α antagonist) DOES NOT involve the immune system? Why?

Certolizumab does not involve the immune system because there is no Fc (fragment crystallizable receptor) present. Fc helps immune system identify cell.

What is the mechanism of action for the following TNF-α antagonist: Adalimumab?

Prevents interaction with p55 and p75 cell surface receptors → down-regulating macrophage and T-cell function (decreased immune response)

What is the mechanism of action for the following TNF-α antagonist: Infliximab?

Forms antibodies that inhibit membrane bound TNF

What is the mechanism of action for the following TNF-α antagonist: Etanercept?

Fusion protein: two TNF-α (p75) moieties linked to Fc segment of human IgG1. Inhibits binding of TNF-α and TNF-β to surface receptors

What is the mechanism of action for the following TNF-α antagonist: Golimumab?

Neutralizes inflammatory effects in TNF-α

What is the mechanism of action for the following TNF-α antagonist: Certolizumab? What makes it unique?

Neutralizes membrane-bound/soluble TNF-α in dose-dependent manner. Fab fragment is conjugated to PEG and has no Fc region

• PEG insertion increases MW, decrease CL, decrease administration rate

Unique because of no Fc and is a co-stimulating drug