L20 Clinical Pharmacology Drug treatment of IBD

Overview

This lecture covers the rationale for drug treatment of inflammatory bowel disease and the mechanisms of action of the main drug classes used. It begins with what IBD is, how it differs from irritable bowel syndrome, its epidemiology, the hypotheses for its cause, and the two main pathologies (ulcerative colitis and Crohn’s disease) with their clinical course, risks and extra-intestinal complications. Because the therapeutic target is immune system function, the middle of the lecture works through the immune and inflammatory processes that the drugs act on: chemotaxis, leukocyte extravasation, and the proteases and reactive oxygen species released from immune cell granules that cause tissue damage. The final part maps three drug classes onto those targets: anti-inflammatory drugs (aminosalicylates and glucocorticoids), immunosuppressants, and monoclonal antibodies, ending with a worked example of stepped therapy in ulcerative colitis.

What IBD is, and what it is not

• IBD is chronic inflammation of the gastrointestinal tract. It is a generic term for chronic inflammatory disorders of the G.I.T. of unknown etiology.
• IBD is NOT the same as irritable bowel syndrome (IBS). IBS is multifactorial, and the two require therapeutically different approaches to treatment.
• Two main pathologies: ulcerative colitis and Crohn’s disease.

Epidemiology

• Caucasian greater than African-American, Hispanic and Asian populations.
• High incidence and prevalence in North America, the UK and Northern Europe.
• Ulcerative colitis incidence 10 to 12 per 100,000 in North America.
• Crohn’s disease incidence 6 per 100,000 in North America.
• Peak age 15 to 35.

Hypotheses for the cause

Three candidate factors are presented, converging on abnormal immune activation with raised cytokines:

1. Infection: mycobacteria, viruses.
2. Abnormal immune system activation, hereditary with a possible genetic predisposition, leading to increased inflammatory mediators (cytokines).
3. Diet: antigens in food sourced from industrialized countries, urban pollution.

Crohn’s disease versus ulcerative colitis

Crohn’s disease (CD)

• Can affect any part of the gastrointestinal tract, with distribution: small bowel 30 percent, small bowel and colon 50 percent, colon 20 percent.
• Not restricted to the GI tract. It is also a systemic disease, with lesions found throughout the body including skin, stomach and mouth.
• On the comparative colon diagram, CD shows patchy, segmental involvement including a narrowed or strictured segment.

Ulcerative colitis (UC)

• The inflammatory reaction is restricted to the colon.
• From the rectum, inflammation is uniform and continuous, shown as continuous diffuse inflammation of the colon.

Clinical course, risks and complications

Clinical course

• Episodes of abdominal pain, fever and diarrhoea.
• Time course of days to weeks.
• Symptoms remit after the initial attack.
• Relapses occur over unpredictable intervals, months to years.

Risks

• Extensive bleeding.
• Fissure formation.
• Toxic colon dilation.
• Carcinoma, shown as a further consequence of extensive bleeding, fissure formation and toxic colon dilation.
• Untreated progression cascade: severe pain, then dehydration, then fever, then tachycardia, then shock, then death.

Other complications (extra-intestinal)

• Joint manifestation: arthritis.
• Skin manifestations: eruption of painful red nodules of the legs; ulcerating lesions, often on the legs.
• Ulcers resembling canker sores of the mouth.
• Ocular manifestations, whose activity parallels the course of the bowel disease.
• Liver function abnormalities.
• Gall bladder problems.

Therapeutic goals and targets

Therapeutic goals:

1. Induce and maintain remission.
2. Limit drug toxicity.
3. Limit complications, which may require surgery.

Therapeutic target: immune system function.

The immune and inflammatory processes targeted

Innate versus adaptive immunity

Feature	Innate (non-specific)	Adaptive (specific)
Molecular target	Generic structural features	Antigens
Recognition	Generic	Specific, high affinity (molecular library)
Memory	No	Yes
Blood proteins	Cytokines	Antibodies, cytokines
Cell types	Phagocytes (neutrophils, macrophages)	Lymphocytes

How the inflammatory process begins

• Immune cells need to move towards the site of infection, which is chemotaxis.
• Chemotaxis is regulated via the concentration gradient of inflammatory mediators, with the cell moving from low toward high mediator concentration and becoming activated at the peak of the gradient.

Leukocyte extravasation across the vessel wall

Sequential steps, with flow along the vessel lumen and endothelium:

1. Tether
2. Trigger
3. Strong adhesion
4. Migration

The molecules involved at each step in sequence are selectins, then chemokines, then integrins, then chemokines. These are labelled as drug targets. Structures labelled in the diagram: vessel lumen, cytokine, glycocalyx, endothelial cell, basement membrane, tissue, inflammatory cell.

What causes inflammation

• Immunological weapons: proteases and reactive oxygen species, both labelled as drug targets.
• These are contained in cytoplasmic granules of immune cells.
• Granule contents can be released inside or outside of the cell.

The belly of the phagocyte

Steps shown for a neutrophil phagocytosing a bacterium:

1. NADPH oxidase converts electrons (4e−) plus O2 (4O2) into superoxide (4O2−).
2. Ionic changes accompany this (4H+ / 4K+), with pH rising in the cytosol and falling at the granule.
3. Superoxide is converted to O2 and H2O2 (2H2O2).
4. Myeloperoxidase (MPO) with Cl−, and catalase, act on H2O2 to produce either hypochlorite and hydroxide (2HOCl + 2OH−) or water and oxygen (2H2O + O2).
5. Proteases are bound to an anionic sulfated proteoglycan matrix within the granule fused with the phagosome, positioned to attack the engulfed bacterium.

Damage done by reactive species

Lipid peroxidation: a free radical (R•) abstracts a hydrogen atom from a polyunsaturated lipid chain (releasing RH), generating a carbon-centred lipid radical. That radical reacts with O2 at either of two possible positions along the chain to form two different lipid peroxyl radical products (each ending in -O-O•), so the radical propagates along the fatty acid chain.

DNA / RNA oxidation: the bases shown are the pyrimidines thymine, cytosine, 5-methylcytosine and uracil, and the purines adenine and guanine. Hot spots for free radical attack occur at multiple positions on the base and on the sugar-phosphate backbone.

The drug classes

Three classes are used to treat IBD:

• Anti-inflammatory drugs: aminosalicylates (mesalazine) and glucocorticosteroids (prednisone).
• Immunosuppressants: purine synthesis inhibitor (azathioprine) and thymidine synthesis inhibitor (methotrexate).
• Monoclonal antibodies: infliximab.

(1) Anti-inflammatory drugs: 5-aminosalicylates (mesalazine)

• Administered by oral or rectal formulations.
• Hypothetical mechanisms of action:
  1. Inhibit COX and lipoxygenase pathways (chemical mediators).
  2. Scavenge free radicals.

Sulfasalazine as a prodrug

• Bacteria in the colon convert sulfasalazine, an azo-linked molecule, into two products: mesalazine (5-aminosalicylate), which is the active product, and sulfapyridine, which is inactive or of unknown activity.
• Dose: 1 to 2 g four times a day to induce remission; 2 g/day for maintenance.

As cyclooxygenase inhibitors: similar to aspirin?

• 5-aminosalicylate and aspirin both have an aromatic ring with a carboxylic acid group; 5-aminosalicylate additionally bears OH and NH2 substituents.
• Doses compared: 5-aminosalicylate 1 to 2 g four times a day for remission and 2 g/day maintenance; aspirin 300 to 900 mg every 4 to 6 hours for pain or pyrexia.

Arachidonic acid pathway shown:

1. Membrane phospholipids are converted by phospholipase A2 into arachidonic acid.
2. Arachidonic acid is then metabolised down two branches. The 12/15-LOX branch produces 12/15-HPETE. The 5-LOX branch produces 5-HPETE, then LTA4, which forms LTB4, LTC4, LTD4 and LTE4.
3. Centrally, arachidonic acid is also converted by COX-1 and COX-2 into a cyclic prostaglandin endoperoxide intermediate.
4. Aspirin, indomethacin and ibuprofen inhibit both COX-1 and COX-2. Celecoxib and rofecoxib are listed as COX-2 inhibitors.

As radical scavengers: similar to vitamin E?

5-aminosalicylate is shown alongside vitamin E (alpha-tocopherol). A free radical (R•) reacts with the phenolic (chromanol) ring to form a stable product (RH) plus a resonance-stabilized phenoxyl radical, shown as two resonance forms. Scavenging therefore works by donation of a hydrogen atom.

(1) Anti-inflammatory drugs: glucocorticoids (prednisone)

• Prednisone is the major steroid for both UC and CD.
• Mechanisms of action:
  1. Modulate cytokine levels.
  2. Inhibit the transcription factor NF-κB, which prevents cytokine gene expression.
  3. Inhibit the function of the enzyme phospholipase A2, and so prevent formation of inflammatory messenger molecules.

Mechanism of glucocorticoid receptor action, in order:

1. Glucocorticoid (GC) crosses the cell membrane.
2. GC binds the cytoplasmic glucocorticoid receptor (GR), which is held inactive by hsp90 chaperones.
3. GC binding causes hsp90 to dissociate, exposing zinc finger regions.
4. The GC-GR complex translocates into the nucleus.
5. In the nucleus it either binds a glucocorticoid response element (GRE) on DNA to increase transcription, or binds a negative GRE (nGRE) to decrease transcription.

(2) Immunosuppressants

• Prevent immune cell proliferation by inhibiting DNA synthesis.
• Examples: azathioprine (purine bases) and methotrexate (thymine bases).
• Used in refractory CD, or in patients intolerant to steroids or mesalazine.
• Adverse side effects: susceptibility to infection.

Why blocking proliferation works: clonal expansion

A variety of B cells carry different antigen receptors. One B cell binds matching antigens and undergoes cell proliferation, dividing repeatedly to form a clone of plasma cells, which secrete antibodies into the circulation, and a clone of memory cells.

(3) Monoclonal antibodies

Therapeutic modulation of cytokine signalling

• Therapeutic tools available to manipulate cytokine networks: recombinant cytokines, monoclonal antibodies, receptor agonists and antagonists, and soluble receptors.
• Despite cytokine redundancy this can still be therapeutically useful, because the outcome of cytokine administration is determined by the balance of activating versus inhibiting factors.
• It is therefore possible to out-compete the natural cytokine network.

TNF-α as the target

Tumour necrosis factor (TNF-α) is a cytokine regulator of innate immunity:

• Principal mediator of the acute inflammatory response to gram negative bacteria.
• Major source: mononuclear phagocytes.
• Stimulus: LPS (lipopolysaccharide, bacterial endotoxin).
• Function: recruit and stimulate neutrophils and monocytes.
• Effects:
  1. Vascular endothelial cells secrete adhesion proteins that bind circulating neutrophils.
  2. Vascular endothelial cells secrete chemokines, which induce chemotaxis.
  3. Macrophages are caused to secrete other cytokines (IL-1).
  4. Apoptosis is caused in some cell types.

Infliximab

• Monoclonal antibodies are antibodies or proteins that block inflammatory cascades of cytokines such as TNF-α.
• Infliximab is a monoclonal antibody to TNF-α.
• Used in both UC and CD to induce and maintain remission, as a 2nd or 3rd line of therapy.
• Only administered by i.v. infusion or s.c. injection.
• Many adverse effects due to infection.

Worked example: stepped drug treatment of ulcerative colitis

To induce remission, therapy escalates with increasing severity:

1. Oral aminosalicylate or oral steroid (immediate response).
2. Topical aminosalicylate OR topical steroid (suppository and enema), plus oral aminosalicylate or steroid.
3. Intravenous OR rectal steroids.
4. Monoclonal antibody (most severe).

For maintenance:

1. Aminosalicylates.
2. Immunosuppressants.
3. Nutritional supplementation.

Important

Infliximab is reserved as a 2nd or 3rd line therapy and sits at the most severe step of the induction ladder, while aminosalicylates are both a first step for induction and the first-line maintenance option.

Self-test

1. Distinguish inflammatory bowel disease from irritable bowel syndrome, and say why the distinction matters therapeutically.
2. State the incidence of ulcerative colitis and of Crohn’s disease in North America, and the peak age of onset.
3. List the three hypothesised causes of IBD given in the lecture, with the specific examples named under each.
4. Distinguish Crohn’s disease from ulcerative colitis by anatomical distribution and pattern of inflammation, and give the percentage distribution of Crohn’s disease sites.
5. Describe the clinical course of IBD, including time course and relapse pattern.
6. List the four risks of IBD, and describe the cascade of untreated progression.
7. List the extra-intestinal complications of IBD.
8. State the three therapeutic goals of IBD drug treatment and the therapeutic target.
9. Distinguish innate from adaptive immunity across the five features compared in the lecture.
10. Describe the steps of leukocyte extravasation and name the molecule class acting at each step.
11. What are the two immunological weapons named as drug targets, and where are they stored?
12. Describe the steps by which a phagocyte generates reactive oxygen species after engulfing a bacterium.
13. Explain how lipid peroxidation propagates along a fatty acid chain.
14. List the three drug classes used in IBD with the example drugs given for each.
15. Describe what colonic bacteria do to sulfasalazine and name both products, saying which is active.
16. What is the dose of 5-aminosalicylate for inducing remission, and for maintenance?
17. Describe the two hypothetical mechanisms of action of aminosalicylates.
18. Describe the arachidonic acid pathway from membrane phospholipids to the leukotrienes, and name the drugs that inhibit COX-1 and COX-2.
19. Explain how a vitamin E-like structure scavenges a free radical.
20. Describe the three mechanisms of action of prednisone.
21. Describe the steps of glucocorticoid receptor activation from drug entry to change in transcription. What would you predict if hsp90 could not dissociate from the receptor?
22. Explain how immunosuppressants work in IBD, name the two examples with their targets, and state when they are used.
23. Explain why blocking DNA synthesis is an effective way to suppress an adaptive immune response.
24. List the source, stimulus, function and four effects of TNF-α.
25. A patient with ulcerative colitis has failed oral and topical aminosalicylates and steroids and intravenous steroids. What is the next step, by what route is it given, and what is its main adverse effect concern?
26. Explain why manipulating a cytokine network with a monoclonal antibody can be therapeutically useful despite cytokine redundancy.
27. Integrative: trace how the inflammatory processes described in the lecture map onto the mechanism of each of the three drug classes.

Answers

Reveal answers

1. IBD is chronic inflammation of the gastrointestinal tract; IBS is multifactorial. They are not the same condition and require therapeutically different approaches to treatment.
2. Ulcerative colitis 10 to 12 per 100,000; Crohn’s disease 6 per 100,000; peak age 15 to 35.
3. Infection (mycobacteria, viruses); abnormal immune system activation, hereditary with possible genetic predisposition, leading to increased inflammatory mediators (cytokines); diet (antigens in food sourced from industrialized countries, urban pollution). These converge on abnormal immune activation with raised cytokines.
4. Crohn’s disease can affect any part of the GI tract and is patchy and segmental, including strictured segments, and is also systemic with lesions in skin, stomach and mouth. Distribution: small bowel 30 percent, small bowel and colon 50 percent, colon 20 percent. Ulcerative colitis is restricted to the colon, with uniform continuous inflammation extending from the rectum.
5. Episodes of abdominal pain, fever and diarrhoea over days to weeks; symptoms remit after the initial attack; relapses occur over unpredictable intervals of months to years.
6. Extensive bleeding, fissure formation, toxic colon dilation and carcinoma. Untreated progression runs severe pain, dehydration, fever, tachycardia, shock, death; bleeding, fissure formation and toxic dilation also lead on to carcinoma.
7. Arthritis; eruption of painful red nodules of the legs; ulcerating lesions often on the legs; mouth ulcers resembling canker sores; ocular manifestations whose activity parallels the bowel disease; liver function abnormalities; gall bladder problems.
8. Goals: induce and maintain remission; limit drug toxicity; limit complications, which may require surgery. Target: immune system function.
9. Molecular target: generic structural features versus antigens. Recognition: generic versus specific and high affinity (molecular library). Memory: no versus yes. Blood proteins: cytokines versus antibodies and cytokines. Cell types: phagocytes (neutrophils, macrophages) versus lymphocytes.
10. Tether, trigger, strong adhesion, migration. The molecules in sequence are selectins, chemokines, integrins, chemokines.
11. Proteases and reactive oxygen species, contained in cytoplasmic granules of immune cells; granule contents can be released inside or outside the cell.
12. NADPH oxidase converts electrons (4e−) plus O2 into superoxide (4O2−), accompanied by ionic changes (4H+/4K+) with pH rising in the cytosol and falling at the granule; superoxide is converted to O2 and H2O2; myeloperoxidase with Cl−, and catalase, act on H2O2 to give either hypochlorite and hydroxide (2HOCl + 2OH−) or water and oxygen (2H2O + O2). Proteases bound to an anionic sulfated proteoglycan matrix in the granule fuse with the phagosome and attack the bacterium.
13. A free radical (R•) abstracts a hydrogen atom from a polyunsaturated lipid chain, releasing RH and creating a carbon-centred lipid radical. That radical reacts with O2 at either of two possible positions along the chain, forming two different lipid peroxyl radicals (-O-O•), so the radical propagates along the chain.
14. Anti-inflammatory drugs: aminosalicylates (mesalazine) and glucocorticosteroids (prednisone). Immunosuppressants: azathioprine (purine synthesis inhibitor) and methotrexate (thymidine synthesis inhibitor). Monoclonal antibodies: infliximab.
15. Colonic bacteria cleave the azo-linked sulfasalazine into mesalazine (5-aminosalicylate), which is the active product, and sulfapyridine, which is inactive or of unknown activity.
16. 1 to 2 g four times a day to induce remission; 2 g/day for maintenance.
17. Inhibition of the COX and lipoxygenase pathways (chemical mediators), and scavenging of free radicals. Both are described as hypothetical.
18. Phospholipase A2 converts membrane phospholipids to arachidonic acid. The 12/15-LOX branch gives 12/15-HPETE; the 5-LOX branch gives 5-HPETE then LTA4, which forms LTB4, LTC4, LTD4 and LTE4. COX-1 and COX-2 convert arachidonic acid to a cyclic prostaglandin endoperoxide intermediate. Aspirin, indomethacin and ibuprofen inhibit both COX-1 and COX-2; celecoxib and rofecoxib are listed as COX-2 inhibitors.
19. The free radical (R•) reacts with the phenolic (chromanol) ring, which donates a hydrogen atom to give the stable product RH plus a resonance-stabilized phenoxyl radical.
20. Modulates cytokine levels; inhibits the transcription factor NF-κB, preventing cytokine gene expression; inhibits phospholipase A2, preventing formation of inflammatory messenger molecules.
21. Glucocorticoid crosses the cell membrane, binds the cytoplasmic glucocorticoid receptor held inactive by hsp90, hsp90 dissociates and exposes zinc finger regions, the GC-GR complex translocates to the nucleus, and it binds either a GRE to increase transcription or an nGRE to decrease transcription. If hsp90 could not dissociate, the zinc finger regions would not be exposed and the complex could not translocate and bind DNA, so transcription would not be altered.
22. They prevent immune cell proliferation by inhibiting DNA synthesis. Azathioprine acts on purine bases and methotrexate on thymine bases. They are used in refractory Crohn’s disease or in patients intolerant to steroids or mesalazine, with susceptibility to infection as the adverse effect.
23. Adaptive responses depend on clonal expansion: a lymphocyte whose receptor matches the antigen proliferates repeatedly to form clones of plasma cells that secrete antibody and of memory cells. Blocking DNA synthesis blocks that proliferation, so the clone cannot expand.
24. Source: mononuclear phagocytes. Stimulus: LPS (bacterial endotoxin). Function: recruit and stimulate neutrophils and monocytes. Effects: vascular endothelial cells secrete adhesion proteins that bind circulating neutrophils; vascular endothelial cells secrete chemokines inducing chemotaxis; macrophages secrete other cytokines (IL-1); apoptosis in some cell types.
25. The next step is a monoclonal antibody, infliximab, an anti-TNF-α antibody used as 2nd or 3rd line therapy. It is only given by intravenous infusion or subcutaneous injection, and has many adverse effects due to infection.
26. Because the outcome of cytokine administration is determined by the balance of activating versus inhibiting factors, so it is possible to out-compete the natural cytokine network.
27. Aminosalicylates act on the mediator and radical arms: they inhibit COX and lipoxygenase pathways, cutting production of prostaglandin and leukotriene mediators, and scavenge the reactive oxygen species released from phagocyte granules. Glucocorticoids act upstream at the level of gene expression and mediator formation, inhibiting NF-κB so cytokine genes are not expressed and inhibiting phospholipase A2 so arachidonic acid is not liberated. Immunosuppressants block the clonal expansion of the lymphocytes driving the adaptive response by inhibiting DNA synthesis. Monoclonal antibodies neutralise TNF-α itself, removing the signal that drives endothelial adhesion protein and chemokine secretion, and therefore the tethering, adhesion and chemotaxis steps that bring inflammatory cells into the bowel wall.