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Screening methods for Anti Ulcer Drugs

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FoziyaKhan

This document discusses peptic ulcer disease and various animal models used to screen for potential anti-ulcer drugs. It describes the pylorus ligation model in rats which induces ulcers by accumulating gastric acid in the stomach. Various parameters analyzed from rat stomach contents provide information on a test drug's mechanism of action, such as antisecretory or cytoprotective effects. In vitro binding assays like the [I125] gastrin binding assay and H+/K+-ATPase inhibition assay also help identify potential anti-ulcer compounds.

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BY:- FOZIYA KHAN PHARMACOLOGY BRANCH SEM I
Introduction Approaches to treatment Requirements of an ideal screening model Ideal animal for screening Animal models
Peptic ulcer is one of the most prevalent chronic gastrointestinal disorder. It is a sore that develops on the lining of the oesophagus, stomach or small intestine. More common in middle- age to older age Site : 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
AGGRESSIVE FACTORS Acid Pepsin NSAIDs H. Pylori Alcohol Smoking Stress Spicy food PROTECTIVE FACTORS Mucus Prostaglandins Mucosal blood flow Bicarbonate
1. Reduction of gastric acid secretion 2. Neutralization of gastric acid 3. Ulcer protectives 4. Anti H. pylori drugs
Should be simple, reproducible & allow for easy quantification of results Should induce characteristics ulceration in specific locations Should involve different mechanism by which ulceration is produced Ulcers produced should not spontaneously heal during observation period
RATS because.. Continuous secretion of acid Glandular portion of rat stomach analogous to body of stomach in man both anatomically & functionally Being omnivorous resembles man nutritionally *** Guinea pigs are used when histamine is used to induce ulcers and in in vitro assays.
IN VITRO METHODS:- [I125] Gastrin Binding Assay Tiotidine Binding Assay H / K- ATPase Inhibition Assay
IN VIVO METHODS:- Pylorus ligation in rats Stress ulcer Model 1. Restraint- induced ulcers 2. Cold water immersion induced ulcers Histamine- induced Gastric Ulcer Ethanol- induced mucosal damage NSAIDs- induced Gastric lesions Acetic acid- induced Gastric Ulcer Cysteamine- induced Duodenal Ulcer Dimaprit- induced Duodenal Ulcer
Screening methods for Anti Ulcer Drugs
PRINCIPLE:- Pyloric ligation in rats leads to accumulation of gastric acid in the stomach leading to acute gastric ulceration REQUIREMENTS:- Albino Wistar Rats (150-200g) Drugs: Ether (Anesthetic) Saline (control) Omeprazole (standard) Test drug (3 different concentrations x, 2x, 4x)
Reagents: NaOH (0.01N) Topfers reagent (dimethyl amino azo benzene) Phenolphthalein Dissecting microscope (10x magnification) pH meter Burette Surgical instruments
PROCEDURE:- Rats fasted for 24 hrs prior to pyloric ligation. Randomly divided into 5 groups of 3 animals each. Group I : Control vehicle Group II : Standard drug (Omeprazole0 Group III : x concentration of test drug Group IV : 2x concentration of test drug Group V : 4x concentration of test drug Drugs administered once for 2 days and 30 mins prior to ligation
Screening methods for Anti Ulcer Drugs
Rats anesthetized with ether Pyloric ligation procedure done Rats placed in separate cages & allowed to recover 19 hrs after pyloric ligation, animals sacrificed by decapitation
Abdomen opened and stomach dissected out Contents of the stomach collected in a centrifuge tube Stomach opened along greater curvature & ulcers observed under 10x magnification Ulcer index is calculated : 10/X ( X= Total mucosal area/ total ulcerated area) Intensity of ulcers is scored as below:- 0- normal stomach 1- superficial mucosal action 2- deep ulcer 3- penetrated or perforated ulcer
Contents of the stomach analyzed for :- Volume pH : pH of it is noted with pH meter Free acidity & total acidity : Titration of the solution against 0.01N NaOH done using Topfers reagent and phenolphthalein as indicators. Volume of NaOH which turns the solution to yellowish orange corresponds to free acidity. Titration is continued till solution turns pink. The total volume of NaOH used up corresponds to total acidity. Acidity(meq/I/100g) = (Vol of NaOH X Normality X 100)/ 0.1 Mucin & prostaglandin levels can be estimated to detect cytoprotective effects
INFERENCE:- Ulcer index of test drug compared with control group to detect anti- ulcer effect of test drug. If present, it is compared with that of standard group. Other parameters help to infer the mechanism of ulcer protection. e.g. Decrease in vol, free & total acidity : antisecretory action Rise in pH : acid neutralising action Increase in mucin, PGs : cytoprotective effect
Gastric ulceration is produced in rats by certain drugs. The ability of the test drug to protect against the ulceration is observed. NSAIDs like Aspirin, Indomethacin, Ibuprofen PRINCIPLE:- Inhibition of endogenous prostaglandin production and consequent loss of gastric mucosal defence. Important model for identifying drugs that could be effective in NSAID induced gastropathy
PROCEDURE:- Rats fasted for 24 hrs in separate cages & randomly divided into 5 groups The control, standard, test drug are administered once daily for 2 days and 30 mins prior to administration of ulcerogenic agent Ulcerogens administered by oral gavage Rats sacrificed 4- 6 hrs later Ulcer index, analysis of stomach contents done.
PRINCIPLE:- Ethanol, being a necrotizing agent, damages the superficial epithelial layers & inhibits the release of mucosal prostaglandins. PROCEDURE:- Wistar rats weighing 150- 200 grams are taken Fasted for 18 hours before experiment; Rats are given test drugs or standard drug orally
30 mins later 1 ml/ 200gm of 99.80% alcohol is administered orally After 1 hour, rats are sacrificed and stomach dissected out Severity score and ulcer index are calculated ADVANTAGES:- Gastric lesions are observed an hour of administration of ethanol Reproducible method to produce gastric lesions in experimental animals
Screening methods for Anti Ulcer Drugs
PRINCIPLE:- Gastrin (G cells of gastric antrum) 1. Binds to CCK2 receptors on parietal cells release HCl 2. Binds 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
PROCEDURE:- Fundic gland suspension (Guinea pig stomach) Incubated with 50亮l [I125] 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 mins at 37C Ice cold buffer, in Micro centrifuge tubes, is layered with incubated mixture Centrifuged for 5 mins at 10,000g Radioactivity is quantified in pellet after discarding the supernatant
EVALUATION:- Total binding, non- specific binding & specific binding are determined Percentage of specifically bound [I125] Gastrin displaced by a given concentration of the test compound calculated The higher the displaced [I125] Gastrin, more is the gastrin antagonistic activity of test compound
H/K - ATPase or proton pump final step in the synthesis of acid by parietal cells PROCEDURE:- Homogenous of 80 ng Microsomal gastric H/K - ATPase (pig gastric mucosa) incubated with 100亮l buffer, 1mM ATP and test compound in microtitre plate for 30 mins at 37C Reaction is stopped by adding Malachite green (colorimetric agent) After 10 secs, 15% sodium citrate is added for 45 mins Release of orthophosphate from ATP quantified by colorimeter at 570 nm
EVALUATION:- Percentage inhibition of H/K - ATPase is calculated Lesser the orthophosphate release, more is the inhibition of H/K - ATPase by test compound
Screening methods for Anti Ulcer Drugs

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Screening methods for Anti Ulcer Drugs

  • 1. BY:- FOZIYA KHAN PHARMACOLOGY BRANCH SEM I
  • 2. Introduction Approaches to treatment Requirements of an ideal screening model Ideal animal for screening Animal models
  • 3. Peptic ulcer is one of the most prevalent chronic gastrointestinal disorder. It is a sore that develops on the lining of the oesophagus, stomach or small intestine. More common in middle- age to older age Site : 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
  • 4. AGGRESSIVE FACTORS Acid Pepsin NSAIDs H. Pylori Alcohol Smoking Stress Spicy food PROTECTIVE FACTORS Mucus Prostaglandins Mucosal blood flow Bicarbonate
  • 5. 1. Reduction of gastric acid secretion 2. Neutralization of gastric acid 3. Ulcer protectives 4. Anti H. pylori drugs
  • 6. Should be simple, reproducible & allow for easy quantification of results Should induce characteristics ulceration in specific locations Should involve different mechanism by which ulceration is produced Ulcers produced should not spontaneously heal during observation period
  • 7. RATS because.. Continuous secretion of acid Glandular portion of rat stomach analogous to body of stomach in man both anatomically & functionally Being omnivorous resembles man nutritionally *** Guinea pigs are used when histamine is used to induce ulcers and in in vitro assays.
  • 8. IN VITRO METHODS:- [I125] Gastrin Binding Assay Tiotidine Binding Assay H / K- ATPase Inhibition Assay
  • 9. IN VIVO METHODS:- Pylorus ligation in rats Stress ulcer Model 1. Restraint- induced ulcers 2. Cold water immersion induced ulcers Histamine- induced Gastric Ulcer Ethanol- induced mucosal damage NSAIDs- induced Gastric lesions Acetic acid- induced Gastric Ulcer Cysteamine- induced Duodenal Ulcer Dimaprit- induced Duodenal Ulcer
  • 11. PRINCIPLE:- Pyloric ligation in rats leads to accumulation of gastric acid in the stomach leading to acute gastric ulceration REQUIREMENTS:- Albino Wistar Rats (150-200g) Drugs: Ether (Anesthetic) Saline (control) Omeprazole (standard) Test drug (3 different concentrations x, 2x, 4x)
  • 12. Reagents: NaOH (0.01N) Topfers reagent (dimethyl amino azo benzene) Phenolphthalein Dissecting microscope (10x magnification) pH meter Burette Surgical instruments
  • 13. PROCEDURE:- Rats fasted for 24 hrs prior to pyloric ligation. Randomly divided into 5 groups of 3 animals each. Group I : Control vehicle Group II : Standard drug (Omeprazole0 Group III : x concentration of test drug Group IV : 2x concentration of test drug Group V : 4x concentration of test drug Drugs administered once for 2 days and 30 mins prior to ligation
  • 15. Rats anesthetized with ether Pyloric ligation procedure done Rats placed in separate cages & allowed to recover 19 hrs after pyloric ligation, animals sacrificed by decapitation
  • 16. Abdomen opened and stomach dissected out Contents of the stomach collected in a centrifuge tube Stomach opened along greater curvature & ulcers observed under 10x magnification Ulcer index is calculated : 10/X ( X= Total mucosal area/ total ulcerated area) Intensity of ulcers is scored as below:- 0- normal stomach 1- superficial mucosal action 2- deep ulcer 3- penetrated or perforated ulcer
  • 17. Contents of the stomach analyzed for :- Volume pH : pH of it is noted with pH meter Free acidity & total acidity : Titration of the solution against 0.01N NaOH done using Topfers reagent and phenolphthalein as indicators. Volume of NaOH which turns the solution to yellowish orange corresponds to free acidity. Titration is continued till solution turns pink. The total volume of NaOH used up corresponds to total acidity. Acidity(meq/I/100g) = (Vol of NaOH X Normality X 100)/ 0.1 Mucin & prostaglandin levels can be estimated to detect cytoprotective effects
  • 18. INFERENCE:- Ulcer index of test drug compared with control group to detect anti- ulcer effect of test drug. If present, it is compared with that of standard group. Other parameters help to infer the mechanism of ulcer protection. e.g. Decrease in vol, free & total acidity : antisecretory action Rise in pH : acid neutralising action Increase in mucin, PGs : cytoprotective effect
  • 19. Gastric ulceration is produced in rats by certain drugs. The ability of the test drug to protect against the ulceration is observed. NSAIDs like Aspirin, Indomethacin, Ibuprofen PRINCIPLE:- Inhibition of endogenous prostaglandin production and consequent loss of gastric mucosal defence. Important model for identifying drugs that could be effective in NSAID induced gastropathy
  • 20. PROCEDURE:- Rats fasted for 24 hrs in separate cages & randomly divided into 5 groups The control, standard, test drug are administered once daily for 2 days and 30 mins prior to administration of ulcerogenic agent Ulcerogens administered by oral gavage Rats sacrificed 4- 6 hrs later Ulcer index, analysis of stomach contents done.
  • 21. PRINCIPLE:- Ethanol, being a necrotizing agent, damages the superficial epithelial layers & inhibits the release of mucosal prostaglandins. PROCEDURE:- Wistar rats weighing 150- 200 grams are taken Fasted for 18 hours before experiment; Rats are given test drugs or standard drug orally
  • 22. 30 mins later 1 ml/ 200gm of 99.80% alcohol is administered orally After 1 hour, rats are sacrificed and stomach dissected out Severity score and ulcer index are calculated ADVANTAGES:- Gastric lesions are observed an hour of administration of ethanol Reproducible method to produce gastric lesions in experimental animals
  • 24. PRINCIPLE:- Gastrin (G cells of gastric antrum) 1. Binds to CCK2 receptors on parietal cells release HCl 2. Binds 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
  • 25. PROCEDURE:- Fundic gland suspension (Guinea pig stomach) Incubated with 50亮l [I125] 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 mins at 37C Ice cold buffer, in Micro centrifuge tubes, is layered with incubated mixture Centrifuged for 5 mins at 10,000g Radioactivity is quantified in pellet after discarding the supernatant
  • 26. EVALUATION:- Total binding, non- specific binding & specific binding are determined Percentage of specifically bound [I125] Gastrin displaced by a given concentration of the test compound calculated The higher the displaced [I125] Gastrin, more is the gastrin antagonistic activity of test compound
  • 27. H/K - ATPase or proton pump final step in the synthesis of acid by parietal cells PROCEDURE:- Homogenous of 80 ng Microsomal gastric H/K - ATPase (pig gastric mucosa) incubated with 100亮l buffer, 1mM ATP and test compound in microtitre plate for 30 mins at 37C Reaction is stopped by adding Malachite green (colorimetric agent) After 10 secs, 15% sodium citrate is added for 45 mins Release of orthophosphate from ATP quantified by colorimeter at 570 nm
  • 28. EVALUATION:- Percentage inhibition of H/K - ATPase is calculated Lesser the orthophosphate release, more is the inhibition of H/K - ATPase by test compound