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Screening of Anti-ulcer drugs

Dr. Bushra Hasan KhanJ R-1,Department Of Pharmacology,JNMC, AMU, Aligarh.

Definition of ulcer

A disruption of the mucosal integrity of the stomach and/or duodenum leading to a local defect or excavation due to active inflammation. Harrisons (2015), 19th ed.More common in middle-age to older ageSite : duodenum /stomach, in a ratio of about 4:1.Male/female ratio is 3:1 Lifetime prevalence of PUD - 12% in men and 10% in women.

An estimated 1 5,000 deaths per year occur as a consequence of complicated PUD.

An estimated burden on direct and indirect health care costs of $6 billion per year in the United States, with $3 billion spent on hospitalizations$2 billion on physician office visits $ 1 billion in decreased productivity and days lost from work

Harrisons (2015), 19th ed.PUD significantly affects quality of life by impairing overall patient well-being and contributing substantially to work absenteeism. 4

Pathophysiology The pathophysiology of peptic ulcer disease is best viewed as an imbalance between mucosal defense factors (bicarbonate, mucin, prostaglandin, NO, and other peptides and growth factors) and injurious factors (acid and pepsin). Gastric Defenses Against AcidThe extremely high concentration of H+ in the gastric lumen requires robust defense mechanisms to protect the esophagus and the stomach. The primary esophageal defense is the lower esophageal sphincter, which prevents reflux of acidic gastric contents into the esophagus. The stomach protects itself from acid damage by a number of mechanisms that require adequate mucosal blood flow, perhaps because of the high metabolic activity and oxygen requirements of the gastric mucosa. One key defense is the secretion of a mucus layer that helps to protect gastric epithelial cells by trapping secreted bicarbonate at the cell surface. Gastric mucus is soluble when secreted but quickly forms an insoluble gel that coats the mucosal surface of the stomach, slows ion diffusion, and prevents mucosal damage by macromolecules such as pepsin. Mucus production is stimulated by prostaglandins E2 and I2, which also directly inhibit gastric acid secretion by parietal cells. Thus, drugs that inhibit prostaglandin formation (e.g., NSAIDs, ethanol) decrease mucus secretion and predispose to the development of acid-peptic disease.

5PATHOPHYSIOLOGY

A keyenzne that controls the rate-limiting step in prostaglandin synthesisis cycloorgenase (COX) which is present in two isoforms (COX- 1 COX-2) each having distinct characteristics regarding structure tissuedistribution and expression. COX- 1 is expressed in a host of tissuesincluding the stomach platelets kidneys and endothelial cells. Thisisoform is expressed in a constitutive manner and plays an importantrole in maintaining the integrity of renal function platelet aggregationand gastrointestinal (GI) mucosal integrity. In contrast the expressionof COX-2 is inducible by inflammatory stimuli and it is expressed inmacrophages leukocytes fibroblasts and synovial cells. The beneficialeffects of nonsteroidal anti-inflammatory drugs (NSAIDs) on tissueinflammation are due to inhibition of COX-2; the toxicity of thesedrugs (e.g. GI mucosal ulceration and renal dysfunction) is related toinhibition of the COX - 1 isoform. Thhighly COX-2-selective NSAIDshave the potential to provide the beneficial effect of decreasing tissue inflammation while minimizing toxicity in the GI tract. SelectiveCOX-2 inhibitors have had adverse effects on the cardiovascular systemleading to incrased risk of myocardial infarction. Therefore theU.S. Food and Drug Administration (FDA) has removed two of theseagents (valdecoxib and rofecoxib) from the market6PHYSIOLOGICAL REGULATION OF GASTRIC ACID SECRETION

Physiology of Gastric SecretionGastric acid secretion is a complex, continuous process in which multiple central and peripheral factors contribute to a common end point: the secretion of H+ by parietal cells. Neuronal (acetylcholine, ACh), paracrine (histamine), and endocrine (gastrin) factors all regulate acid secretion (Figure 451). Their specific receptors (M3, H2, and CCK2, respectively) are on the basolateral membrane of parietal cells in the body and fundus of the stomach. Some of these receptors are also present on enterochromaffin-like cells (ECL), where they regulate the release of histamine. The H2 receptor is a GPCR (Chapters 3 and 32) that activates the Gsadenylyl cyclasecyclic AMPPKA pathway. ACh and gastrin signal through GPCRs that couple to the GqPLC-IP3Ca2+ pathway in parietal cells. In parietal cells, the cyclic AMP and the Ca2+-dependent pathways activate H+, K+-ATPase (the proton pump), which exchanges hydrogen and potassium ions across the parietal cell membrane. This pump generates the largest ion gradient known in vertebrates, with an intracellular pH of ~7.3 and an intracanalicular pH of ~0.8.

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PHARMACOLOGICAL TREATMENT:

ACID SUPPRESSANTSProton pump inhibitors : Omeprazole , Pantoprazole, Lansoprazole H2 receptor blockers : Ranitidine, Famotidine, Nizatidine

CYTOPROTECTANTSProstaglandin analogues : MisoprostolUlcer protectants : SucralfateAntacids : (replaced by more effective and convenient drugs)

The proton pump inhibitors are used most commonly, followed by the histamine H2 receptor antagonists.

The most important structures for CNS stimulation of gastric acid secretion are the dorsal motor nucleus of the vagal nerve, the hypothalamus, and the solitary tract nucleus. Efferent fibers originating in the dorsal motor nuclei descend to the stomach via the vagus nerve and synapse with ganglion cells of the enteric nervous system. ACh release from postganglionic vagal fibers directly stimulates gastric acid secretion through muscarinic M3 receptors on the basolateral membrane of parietal cells. The CNS predominantly modulates the activity of the enteric nervous system via ACh, stimulating gastric acid secretion in response to the sight, smell, taste, or anticipation of food (the "cephalic" phase of acid secretion). ACh also indirectly affects parietal cells by increasing the release of histamine from the ECL cells in the fundus of the stomach and of gastrin from G cells in the gastric antrum.The relative effectiveness of antacid preparations is expressed as milliequivalents of acid-neutralizing capacity (defined as the quantity of 1N HCl, expressed in milliequivalents, that can be brought to pH 3.5 within 15 minutes); according to FDA requirements, antacids must have a neutralizing capacity of at least 5 mEq per dose. In general, antacids should be administered in suspension form because this probably has a greater neutralizing capacity than powder or tablet dosage forms. If tablets are used, they should be thoroughly chewed for maximum effect.

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PHARMACOLOGICAL REGULATION OF GASTRIC ACID SECRETIONIn the hope of providing more rapid onset of action and sustained acid suppression, reversible inhibitors of the gastric H+, K+-ATPase (e.g., the pyrrolopyridazine derivative AKU517) are being developed for clinical use. Antagonists of the CCK2 gastrin receptor on parietal cells also are under study. Physiological and pharmacological regulation of gastric secretion: the basis for therapy of acid-peptic disorders. Shown are the interactions among an enterochromaffin-like (ECL) cell that secretes histamine, a ganglion cell of the enteric nervous system (ENS), a parietal cell that secretes acid, and a superficial epithelial cell that secretes mucus and bicarbonate. Physiological pathways, shown in solid black, may be stimulatory (+) or inhibitory (). 1 and 3 indicate possible inputs from postganglionic cholinergic fibers; 2 shows neural input from the vagus nerve. Physiological agonists and their respective membrane receptors include acetylcholine (ACh), muscarinic (M), and nicotinic (N) receptors; gastrin, cholecystokinin receptor 2 (CCK2); histamine (HIST), H2 receptor; and prostaglandin E2 (PGE2), EP3 receptor. A red indicates targets of pharmacological antagonism. A light blue dashed arrow indicates a drug action that mimics or enhances a physiological pathway. Shown in red are drugs used to treat acid-peptic disorders. NSAIDs are nonsteroidal anti-inflammatory drugs, which can induce ulcers via inhibition of cyclooxygenase.prostanoids bind to the EP3 receptor on parietal cells (Chapters 33 and 34) and stimulate the Gi pathway, thereby decreasing intracellular cyclic AMP and gastric acid secretion. PGE2 also can prevent gastric injury by cytoprotective effects that include stimulation of mucin and bicarbonate secretion and increased mucosal blood flow. The degree of inhibition of gastric acid secretion by misoprostol is directly related to dose; oral doses of 100-200 g significantly inhibit basal acid secretion (up to 85-95% inhibition) or food-stimulated acid secretion (up to 75-85% inhibition). The usual recommended dose for ulcer prophylaxis is 200 g four times a day. Misoprostol is FDA-approved to prevent NSAID-induced mucosal injury. However, it rarely is used because of its adverse effects and the inconvenience of four-times-daily dosing.

SUCRALFATEIn the presence of acid-induced damage, pepsin-mediated hydrolysis of mucosal proteins contributes to mucosal erosion and ulcerations. This process can be inhibited by sulfated polysaccharides. Sucralfate (CARAFATE, others) consists of the octasulfate of sucrose to which Al(OH)3 has been added. In an acid environment (pH animals sacrificed after 28 hours of 1st dose

2 times at 4 hours interval--> animals sacrificed after 40 hours of 1st dose39Perforating duodenal ulcers are produced ;Located 2-4 mm from the pylorus, mainly on the anterior wall of duodenumNecrotic material and acute inflammatory response is present at ulcer craterAdvantages:Ulcerogenesis is seen with one full dose of cysteamine. Easily reproducible

Disadvantage:Ulcers, located on the anterior wall, frequently perforate, resulting in peritonitis, or penetrate into the liver. A small ulcer is usually present on the posterior wall (kissing ulcer) of the duodenum , penetrates the pancreas.

Dimaprit-induced Duodenal Ulcer del Soldato P (1982)Principle:H2 receptor agonistInduced gastric erosion in rats after single i.v. dose.Duodenal ulcer in guinea pigs after repeated s.c. dose.Procedure:Wistar rats weighing 150-200 g or guinea pigs 250-300 g are takenFasted for 24 hours before experiment; free access to water Test drug or standard drug is given orally 60 min before injecting Dimaprit in rats and 30 min before injecting it in guinea pig.

This model is specially useful for screening of H2- blockers.

On the basis of universally recognised hypothesisabout the involvement of HP receptors in thepathogenesis of peptic ulcer an appropriate ratmodel has been designed with dimaprit as specificHP receptor agonist. Dimaprit was administered i.p.or i.v. to 24 hour fasted rats and the animals weresacrificed 4 hours after the injection. The drugs forstudying their gastroprotective effects were given 30min before dimaprit. The procedure is extremelysimple and rapid. Its feasibility and specificity areadded advantages. It is very useful for evaluatingnot only the absolute potency of a drug given by anyroute but also of other pharmacodynamic parametersparticularly the duration of action whichseems to be an important criterion in selecting newpotentially H2-antagonistic drugs.42Dimaprit is given in a dose 100 mg/kg i.v. in rats and 2 mg/kg s.c. every hour for 6 hours in guinea pig.After 1 hour of Dimaprit injection, Animal is sacrificed and stomach dissected out. Stomach is opened along the greater curvature and examined for ulceration.Dimaprit 3- dimethylaminopropylsulfanylmet hanimidamide

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In-vitro methods [125I] Gastrin binding assayPrinciple: Gastrin ( G cells of gastric antrum)Bind to CCK2 receptors on parietal cells --> release HClBind to CCK2 receptors on ECL cells -->Histamine --> act on H2 receptors of parietal cells --> release HCl

Compounds with gastrin receptor antagonistic activity --> can be potential antiulcer agents

Gastrinis apeptide hormonethat stimulates secretion ofgastric acid(HCl) by theparietal cellsof thestomachand aids in gastric motility. It is released byG cellsin thepyloric antrumof the stomach,duodenum, and thepancreas.After release from gastric antrum, it stimulates acid secretion by binding to its receptors on parietal cells as well as by releasing histamine from ECL cells. Compounds with gastrin receptor antagonistic activity can prove to be useful antiulcer drugs.[125I] Gastrin is used for radioligand binding assay for gastrin receptors.

PROCEDURE :The assay is done using fundic gland suspension obtained from Guinea pig stomach.For the binding and competition assays, the gland suspension is incubated with 50l of [125I] Gastrin in the presence of either buffer alone (for total binding) OR in the presence of unlabeled gastrin (for non-specific binding), OR in the presence of test compound for 90 mins at 37 degrees C.Subsequently, ice cold buffer , in microcentrifuge tubes, is layered with incubated mixture and centrifuged for 5 mins at 10,000 g.Radioactivity is quantified in pellet after discarding the supernatant.

45Procedure: Fundic gland suspension (Guinea pig stomach)Incubated with 50l [125I] Gastrin --> 1) In buffer alone (for total binding)2) In presence of unlabeled gastrin (for non-specific binding)3) In presence of test compound (for competition assay) For 90 minutes at 37C

Ice cold buffer, in Microcentrifuge tubes, is layered with incubated mixture

Centrifuged for 5 minutes at 10,000 g

Radioactivity is quantified in pellet after discarding the supernatant.Test compounds (at various concentrations, to determine their inhibition of specific binding)

PROCEDURE :The assay is done using fundic gland suspension obtained from Guinea pig stomach.For the binding and competition assays, the gland suspension is incubated with 50l of [125I] Gastrin in the presence of either buffer alone (for total binding) OR in the presence of unlabeled gastrin (for non-specific binding), OR in the presence of test compound for 90 mins at 37 degrees C.Subsequently, ice cold buffer , in microcentrifuge tubes, is layered with incubated mixture and centrifuged for 5 mins at 10,000 g.Radioactivity is quantified in pellet after discarding the supernatant.

Suppose that the same mixture is split into two test-tubes. One test-tube is placed into a rack and the mixture in it is left to freely sediment. The other tube is centrifuged at 10000g. It means that particles in the centrifuged tube re influenced by ten thousand times bigger acceleration compared to the freely standing tube.It can be easily derived that the relative centrifugal force depends on the radius of the rotor and on the number of revolutions per minute:R = 1.12 n2 r 10-5where R is the relative centrifugation force, n is the number of revolutions per minute and r is the radius of the rotor in centimeters.

46Evaluation: Total binding, non-specific binding and specific binding are determined.

Percentage of specifically bound [125I] Gastrin displaced by a given concentration of the test compound calculated.

The higher the displaced [125 I] Gastrin , more is the gastrin antagonistic activity of test compound.

I.C.50 AND DISSOCIATION CONSTANT (Ki) values are calculated.Thehalf maximal inhibitory concentration (IC50)is a measure of the effectiveness of a substance in inhibiting a specific biological or biochemical function.This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological process (or component of a process, i.e. anenzyme,cell,cell receptorormicroorganism) by half. It is commonly used as a measure ofantagonist drugpotencyin pharmacological research. According to theFDA, IC50represents the concentration of a drug that is required for 50% inhibitionin vitro

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Tiotidine Binding Assay

H2 receptor blockerAssay is done using cerebral cortex homogenate obtained from guinea pigs.Procedure:Cerebral cortex homogenate is incubated with Tiotidine for 90 min at 40C in the presence of :Na2HPO4/ KH2PO4 buffer alone (to determine total binding)Unlabeled Ranitidine and buffer (to determine non-specific binding)Test compound in buffer (for competition assay)

485 ml of ice cold phosphate buffer is added to terminate the incubation.Subsequently reaction mixture is filtered under vacuum through glass fiber filters that are presoaked with bufferFilters are then washed with 5ml of ice cold buffer twice.Radioactivity measured by liquid scintillation counting.Evaluation:Specific binding is measured.Specific binding = Total binding Non specific binding Liquid scintillation countingis the measurement of activity of a sample of radioactive material which uses the technique of mixing the active material with a liquid scintillator, and counting the resultant photon emissions. The purpose is to allow more efficient counting due to the intimate contact of the activity with the scintillator. It is generally used for alpha and beta particle detection.Samples are dissolved or suspended in a "cocktail" containing asolvent(historicallyaromaticorganics such asbenzeneortoluene, but more recently less hazardous solvents are used), typically some form of asurfactant, and small amounts of other additives known as "fluors" orscintillators. Scintillators can be divided into primary and secondaryphosphors, differing in their luminescence properties.Beta particles emitted from the isotopic sample transfer energy to the solvent molecules: the cloudof the aromatic ring absorbs the energy of the emitted particle. The energized solvent molecules typically transfer the captured energy back and forth with other solvent molecules until the energy is finally transferred to a primary scintillator. The primary phosphor will emitphotonsfollowing absorption of the transferred energy. Because that light emission may be at awavelengththat does not allow efficient detection, many cocktails contain secondary phosphors that absorb the fluorescence energy of the primary phosphor and re-emit at a longer wavelength.The radioactive samples and cocktail are placed in smalltransparentortranslucent(oftenglassorplastic) vials that are loaded into an instrument known as a liquid scintillation counter. Newer machines may use 96-well plates with individual filters in each well. Many counters have twophotomultipliertubes connected in acoincidence circuit. The coincidence circuit assures that genuine light pulses, which reach both photomultiplier tubes, are counted, while spurious pulses (due toline noise, for example), which would only affect one of the tubes, are ignored.Counting efficiencies under ideal conditions range from about 30% fortritium(a low-energy beta emitter) to nearly 100% forphosphorus-32, a high-energy beta emitter. Some chemical compounds (notablychlorinecompounds) and highly colored samples can interfere with the counting process. This interference, known as "quenching", can be overcome through data correction or through careful sample preparation.High-energy beta emitters, such asphosphorus-32, can also be counted in a scintillation counter without the cocktail, instead using an aqueous solution. This technique, known asCherenkov counting, relies on theCherenkov radiationbeing detected directly by the photomultiplier tubes. Cherenkov counting in this experimental context is normally used for quick, rough measurements, since the geometry of the sample can create variations in the output.

49H+/K+ - ATPase inhibition assayH+/K+ - ATPase or proton pump --> final step in the synthesis of acid by parietal cellsProcedure:Homogenate of 80 ng Microsomal gastric H+/K+ - ATPase (pig gastric mucosa) incubated with 100l buffer, 1mM ATP and Test compound in microtitre plate for 30 mins at 37CReaction is stopped by adding Malachite green (colorimetric agent)After 10 seconds, 15% sodium citrate is added for 45 minutesRelease of orthophosphate from ATP quantified by colorimeter at 570 nmProton pump exchanges intracellular H with extracellular k in the canaliculi of parietal cells in response to stimulation by all gastric acid secretagogues, i.e. histamine, acetylcholine and gastrin. PPIs like OMEPRAZOLE, LANSOPRAZOLE etc are now well established antiulcer drugs.

50Evaluation:

Percentage inhibition of H+/K+ - ATPase is calculated.Lesser the orthophosphate released, more is the inhibition of H+/K+ - ATPase by test compound.

THANK YOUPathogenesisAn imbalance between offensive or injurious (acid, pepsin andHelicobacter pylori) and defensive mucosal factors (mucin, prostaglandin, bicarbonate, nitric oxide and growth factors).Factors such as stress, smoking and ingestion of NSAIDs all can increase the incidence of gastric ulcers.Prolonged anxiety, emotional stress, haemorrhagic surgical shock, burns and trauma are known to cause severe gastric irritation.Regulation of acid secretion by parietal cell is especially important in the pathogenesis of peptic ulcer.Secretion of the parietal cell is an isotonic solution of HCl with a pH less than 1.Cl- is actively transported into canaliculi in the cell that communicate with the lumen of the gastric gland thus with the stomach itself.Cl- secretion accompanied by K+, which is then exchanged for H+ from within the cell by H+ / K+-ATPase.

Principle stimuli acting on parietal cell

Gastrin Acetylcholine HistamineProstaglandins E2 & I2 . Every year 4 million people are diagnosed with this disease. An estimated 6000 people die every year because of the complications associated with gastric ulcer. 40, 000 people undergo surgery in order to get relief from the persistent symptoms of ulcer annually. An estimated 15,000 deaths occur annually as a consequence of PUD. (Sandhya et al, 2013)