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  1. MELANOCORTIN PHARMACOLOGY Dr Stephen J. Getting Department of Biochemical Pharmacology William Harvey Research Institute Charterhouse Square Barts & the London Queen Mary, University of London

  2. Melanocortin • Learning Objectives • POMC Gene • Melanocortin receptors • Physiological/Pharmacological actions

  3. POMC gene and ACTH-derived fragments POMC Product - lipotropin ACTH b 1-39 g MSH - g -lipotropin ACTH 1-13 ( a-MSH) CLIP b- MSH -endorphin b ACTH 4-10 (MEHFRWG) ACTH 4-10 (MEHFRWG)

  4. Neuro-peptides 209-240 amino acid sequence ACTH and LPH are derived from POMC In the pars distalis of the anterior pituitary. In the pars intermedia of the anterior pituitary, ACTH is further cleaved to produce a-MSH. Stimulated by CRH from the Hypothalamus. Its then glycosolated (sugars added) and then cleaved Better drug design Find agonists and antagonists of its Peptide products. Other peptides POMC gene Endorphins which make you feel good Wonder Drug? Altering POMC levels Will alter ACTH, MSH Levels and endorphins So side effects would Be immense Peripheral synthesis Features Synthesised in GI tract, immune cells, Placenta and gonads. Protein that tans skin Makes you feel good Makes you thin Features of the POMC gene

  5. Peptide Amino acid sequence (single letter code) Amino acid sequence for peptide fragments SYSMEHFRWGKPVKKRRPVKVYPNGAEDESAEAFPLEF ACTH ACTH4-10 MEHFRWG a-MSH SYSMEHFRWGKPV ß-MSH AEKKDEGPYRMEHFRWGSPPKD g-MSH YVMGHFRWDRFG

  6. melanocortins extracellular NH2 plasma membrane II III IV V VI VII I intracellular HOOC Melanocortin Receptors (MC-Rs) • Seven transmembrane GPCRs positively coupled to adenylate cyclase • Activation leads to elevation of cAMP • Diverse physiological role by melanocortins are mediated via different receptors. • To date five MC-Rs have been identified and cloned and are termed as MC1-5R • MC-Rs share a sequence homology of 40 - 60%

  7. a-MSH . . . out NH2 plasma membrane II III IV V VI VII I in HOOC MC1-R-MELANOCORTIN TYPE 1 RECEPTOR Agonist profile: a-MSH > ACTH > g-MSH • Identified in 1992 in Melanoma tumours • 1996 in testis, pituitary • 1995-1997 in cell lines of different lineage (macrophages, monocytes, endothelial, fibroblasts, Keratinocytes) • External receptor expressed on the cellular cell-surface although reports of internalisation upon exposure to peptides. RECEPTOR INVOVLVED IN SKIN PIGMENTATION

  8. Recessive yellow e/e Wild type MC1-R involved in skin pigmentation.

  9. ACTH . . . out NH2 plasma membrane II III IV V VI VII I in HOOC MC2-R-MELANOCORTIN TYPE 2 RECEPTOR Agonist profile: ACTH Expressed on the adrenal cortex (Zona fasiculata) and adipose tissue RECEPTOR INVOLVED IN STEROIDGENESIS

  10. Circadian Variation in plasma cortisol

  11. Endogenous corticosteroids 11b hydroxysteroid dehydrogenase OH 1 0.5 Cortisol Cortisone Glucocorticoid activity Synthetic corticosteroids F CH3 6 30 Methylprednisolone Dexamethasone

  12. Edward C Kendall 1886-1972 Philip S Hench 1896-1965 Identified, isolated, purified and synthesised corticosteroids Discovered the clinical anti-inflammatory effects of steroids

  13. Corticosteroids are widely used in the treatment of inflammatory and autoimmune diseases. “…over 250 000 people in the United Kingdom are taking continuous oral steroids…” (Walsh LJ et al. BMJ 1996:313,344-6) Prednisone 20mg tabs

  14. Immune system Cardiovascular Immunosuppressive - Hypertensive - leukocyte heightened pressor redistribution, responsiveness maintenance of lymphoid tissues, feedback regulation of Carbohydrate metabolism the HPA axis. Hyperglycaemic due Kidney to inhibition of insulin Glucocorticoids and stimulation of Permissive action on glucagon secretion tubular function and glomerular filtration Lipid metabolism Redistribution of Skeletal system CNS body fat - increased Maintenance of lipolysis by Regulation of muscular tone adipocytes neuronal excitability Glucocorticoids exert effects on multiple organs and metabolic pathways

  15. Unwanted effects of systemic corticosteroids Oedema Weight gain Myopathy Glycosuria Hypertension Hyperlipidaemia Thrombosis GI bleeding Peptic ulcers Pancreatitis Colonic perforation Glaucoma Cataracts Insomnia Depression / Psychosis HPA axis suppression Increased infection risk Osteoporosis Avascular / aseptic necrosis Juvenile growth retardation

  16. Corticosteroid-induced peptic ulceration Steroid -associated peptic ulcers of the stomach and oesophagus (left) are usually small, superficial and multiple.

  17. Corticosteroid-induced glaucoma Photo of normal optic nerve on left and optic nerve with glaucoma damage on the right (note the central white cupping or depression representing nerve fiber  loss)

  18. response to stress stressor physiological 'tone' Glucocorticoid hormones prevent overzealous rebound responses to stress.

  19. Cushing’s Syndrome • Caused by too much ACTH which results in overproduction of cortisol • Caused by benign pituitary adenoma (70% of endogenous cases) • Lung tumours (15% of endogenous cases) • Benign and malignant adrenal tumours (15% of endogenous cases) The most common cause is exogenous ingestion of glucorticoids.

  20. Symptoms of Cushing's syndrome Hypertension Atherosclerosis Congestive heart failure Oedema Menstrual irregularities Psychological disturbances Osteoporosis Increased infections

  21. The classical ‘moon face’ associated with corticosteroid use

  22. Buffalo hump in a 34yr old woman taking corticosteroids

  23. g-MSH . . . out NH2 plasma membrane II III IV V VI VII I in HOOC MC3-R-MELANOCORTIN TYPE 3 RECEPTOR Agonist profile: g-MSH = ACTH > a-MSH • Identified in 1993 in Brain, Gut and Placenta • 1996 in Heart • 1999 in leucocytes (macrophages) • Found to have a wide distribution within the brain, including the Hippocampus, thalamus and midbrain. • It has been reported that it may function as an auto-receptor, regulating the release of MSH peptides from POMC neurones. Receptor proposed to be involved in modulating inflammation

  24. a-MSH . . . out NH2 plasma membrane II III IV V VI VII I in HOOC MC4-R-MELANOCORTIN TYPE 4 RECEPTOR Agonist profile: a-MSH = ACTH > g-MSH • Expressed in multiple sites within the brain (cotex, thalamus, hypothalamus, brain stem) • Not detected in any other organ in human, although one study has proposed message on adipose tissue. • Distribution much wider than MC3-R RECEPTOR INVOLVED IN CONTROLLING FOOD INTAKE

  25. a-MSH . . . out NH2 plasma membrane II III IV V VI VII I in HOOC MC5-R-MELANOCORTIN TYPE 5 RECEPTOR Agonist profile: a-MSH > ACTH > g-MSH • MC5-R is ubiquitously expressed in many peripheral tissues including adrenal glands, fat cells, kidneys, lung , liver etc • It has been proposed that the seborrhoea observed in Parkinson’s • Disease has been attributed to an increased activity of MSH and that receptor blocking might lead to a treatment. RECEPTOR INVOLVED IN SEBORRHOEA

  26. Cardinal signs of inflammation The accumulation and subsequent activation of leukocytes are central events in the pathogenesis of virtually all forms of inflammation Inflammation: Basic Principles and Clinical Correlates 2nd Ed. Gallin JI, Goldstein IM and Snyderman R, 1992

  27. % emigration adhesion rolling detachment %

  28. Experimental Murine Colitis • Reduces Faecal Blood • Inhibits weight loss • Reduces TNF-a and Nitric Oxide In lower colon [Rajora, N. et al.,Peptides 1997, 18, 381-385]

  29. ileitis Control a-MSH-treated Endotoxin-induced intestinal inflammation • reducing neutrophil migration • reduce severity of the lesions macroscopically and microscopically in the distal ileum • [San, T. et al.,Peptides 2001, 22(12), 2077-2082.]

  30. Mesenteric I/R Injury • a-MSH reduced depressed intestinal transit • reduced myeloperoxidase activity in ileal cytoplasmic extracts • reduced NF-kB activation in ileal nuclear extracts following I/R [Hassoun, H.T. et al., Am. J. Physiol. 2002, 282, G1059-1068]

  31. OVA Control a-MSH/OVA Lung Inflammation • a-MSH inhibits eosinophil migration • and mucus secretion (Rapp et al., J. Immunol, 2003, 171(1): 353-359.)

  32. “The Gout” by James Gilray, 1799. Gout depicted as an evil demon attacking a toe. “By Royal Authority” by George Cruickshank. A gout sufferer helped onto his horse. 2.2 million cases of gout in the USA equivalent to 1:60 American males 37 million working days are lost in the USA every year to gout.

  33. GOUT-CLINICAL PICTURES After years of gouty attacks, patients develop a chronic arthritis resulting in bone and cartilage destruction and deformity. Uric acid crystals deposit within and surrounding the joint causing a chronic destructive inflammatory process. X-rays characteristically show “punched out” erosions. X-ray showing soft tissue swelling and erosion

  34. ACTH is effective in the clinical treatment of gout with an efficacy over and above that obtainable with conventional glucocorticoid therapy [Ritter et al., J. Rheumatol, 1994; 696-699] Must be another mechanism Action besides reflex stimulation Of the HPA axis

  35. in vivo Inflammation Time (h) -0.5 0 2 6 Agonists and Antagonists PMN accumulation MSU crystals (3 mg in 0.5 ml PBS) Chemokine levels by ELISAs [Getting et al., 1999; J. Immunol, 162; 7446-7453]

  36. # 10 100 8 75 * 6 * PMN Migration PMN (10 per mouse) 50 * (% of control) 6 * 4 * * ACTH4-10 -MSH -MSH 25 2 0 0 ACTH4-10- + - + - + Antagonist None SHU9119 SHU9119 [3 mg] [10 mg] 1 10 100 1000 0.1 Peptide (nmol s.c.) Similar observation with respect to cytokine/chemokine levels MELANOCORTIN AGONISTS INHIBIT PMN RECRUITMENT: EFFECT BLOCKED BY MC3-R ANTAGONISTS [Getting et al., 1999; J. Immunol, 162; 7446-7453]

  37. MC3-R mRNA AND FUNCTIONALITY [Getting et al., 1999; J. Immunol, 162; 7446-7453]

  38. Injection of MSU crystals (1 mg in 50 ml) 2-72 h knee joints were examined and inflammatory exudates collected and analysed for: 1 ] Arthritic Score 2 ] Size of knee joint 3 ] PMN accumulation by light microscopy 4 ] Cytokine contents by specific ELISAs [Getting et al., 2002; Arthritis & Rheumatism, 46; 2765-2775]

  39. A 2.4 1.8 Neutrophil influx (105per joint) * 1.2 * 0.6 0 g2-MSH PBS ACTH - + - + - + SHU B IL-1b IL-6 80 * 60 Cytokine release (pg per joint) 40 * 20 0 ACTH PBS PBS ACTH - + - + - + - + SHU MC3-R AGONISTS INHIBIT JOINT INFLAMMATIONEFFECT BLOCKED BY ANTAGONIST

  40. A 4.5 3.0 Neutrophil influx (105per joint) 1.5 * * 0 0 6 16 72 96 Time (h) B * * 60 40 Neutrophil influx (% of inhibition) # 20 0 # -20 g2-MSH PBS ACTH - + - + - + SHU REPEATED ADMINISTRATION OF MSU CRYSTALS

  41. Lipton et al News physiol. Sci, 15,192-195.

  42. Diverse Physiological Roles Mediated By MC-Rs AGONIST PROFILE TISSUE mRNA FUNCTION Pigmentation Steroidgenesis Feeding Control, Cardiovascular Function, Inflammation Obesity Control of the Sebaceous Gland MC1-R MC2-R MC3-R MC4-R MC5-R Melanocyte, Endothelial Cells, Fibroblast, Monocytes Adrenal Cortex, Adipocytes MØ, Brain, Gut, Placenta Brain Brain, Several Peripheral Tissues -MSH > ACTH > -MSH ACTH -MSH = ACTH =-MSH -MSH = ACTH > -MSH -MSH > ACTH > -MSH

  43. Immune system Anti-Pyretic Inhibit pro-inflammatory cytokine production Inhibit leukocyte migration Induce anti-inflammatory Cytokines. Inhibit cytokine induced pyresis Antiserum directed against a-MSH Prolongs fever induced by LPS. Cardiovascular Modulates disease pathologies Melanocortins Prevent ischaemic damage Arthritis, asthma, IBD, Dermatitis, Neuro- Inflammation, Brain and Kidney ischaemia Obesity and anorexia Agonists cause increase In food intake, whilst Antagonists inhibit food intake Endotoxemia HIV infection Plasma a-MSH increases after endotoxin injection. Plasma a-MSH increases In CDC III and IV patients And is a marker for 6 month Survival. Effects of melanocortins