This document describes methods for testing anti-diarrheal and laxative drugs. It discusses causes of diarrhea and mechanisms of laxatives. Key animal models described include castor oil-induced diarrhea testing inhibition of diarrhea and stool properties. Gastrointestinal motility is assessed using charcoal meals. Castor oil-induced fluid accumulation in the intestines is also measured. Cold restraint in rats can evaluate anti-diarrheal effects by measuring fecal pellet output and fluid content. In vitro methods are also mentioned.
3. DIARRHOEA (CONT'D)
• ACUTE DIARRHOEA:
• SUDDEN ONSET IN A PREVIOUSLY HEALTHY PERSON
• LASTS FROM 3 DAYS TO 2 WEEKS
• SELF-LIMITING
• CHRONIC DIARRHOEA:
• LASTS FOR MORE THAN 3 WEEKS
• ASSOCIATED WITH RECURRING PASSAGE OF DIARRHEAL STOOLS, FEVER, LOSS
OF APPETITE, NAUSEA, VOMITING, WEIGHT LOSS, AND CHRONIC WEAKNESS.
7. LAXATIVES:
• THESE ARE THE DRUGS THAT PROMOTE EVACUATION OF BOWEL.
• ACCORDING TO INTENSITY OF ACTION, A DISTINCTION IS MADE :-
1) LAXATIVE OR APERIENT : MILDER ACTION, ELIMINATION OF SOFT BUT
FORMED STOOL.
2) PURGATIVE OR CATHARTIC : STRONGER ACTION RESULTING IN MORE FLUID
EVACUATION.
9. • BULK FORMING:
• HIGH FIBER
• ABSORBS WATER TO INCREASE BULK IN THE INTESTINE
• DISTENDS BOWEL TO INITIATE REFLEX BOWEL ACTIVITY
• EXAMPLES : PSYLLIUM (METAMUCIL), METHYLCELLULOSE
(CITRUCEL), EMOLLIENT.
• STOOL SOFTENERS AND LUBRICANTS
• PROMOTE MORE WATER AND FAT IN THE STOOLS
• LUBRICATE THE FECAL MATERIAL AND INTESTINAL WALLS
• EXAMPLES :
• STOOL SOFTENERS : DOCUSATE SALTS (COLACE, SURFAK)
• LUBRICANTS : MINERAL OIL
10. • HYPEROSMOTIC:
• INCREASE FECAL WATER CONTENT
• RESULT : BOWEL DISTENTION, INCREASED PERISTALSIS, AND EVACUATION
• EXAMPLES : POLYETHYLENE GLYCOL
(GOLYTELY), SORBITOL, GLYCERIN, LACTULOSE .
• SALINE:
• INCREASE OSMOTIC PRESSURE WITHIN THE INTESTINAL TRACT, CAUSING MORE
WATER TO ENTER THE INTESTINES
• RESULT : BOWEL DISTENTION, INCREASED PERISTALSIS, AND EVACUATION.
• STIMULANT
• INCREASES PERISTALSIS VIA INTESTINAL NERVE STIMULATION.
• EXAMPLES : CASTOR OIL , SENNA , CASCARA
11. ANIMAL SCREENING MODEL
IN VIVO MODELS:
• CASTOR OIL-INDUCED DIARRHEA
• GASTROINTESTINAL MOTILITY TEST
• CASTOR OIL-INDUCED ENTER POOLING
• EVALUATION OF ANTI-DIARRHEAL EFFECT IN COLD-RESTRAINED RATS
IN VITRO MODEL
12. RATIONALE & PURPOSE
• THE INDUCTION OF DIARRHEA WITH CASTOR OIL RESULTS FROM THE ACTION
OF RICINOLEIC ACID FORMED BY HYDROLYSIS OF THE OIL.
• RICINOLEIC ACID PRODUCES CHANGES IN THE TRANSPORT OF WATER AND
ELECTROLYTES RESULTING IN A HYPER SECRETORY RESPONSE IN ADDITION TO
HYPER SECRETION , RICINOLEIC ACID SENSITIZES THE INTRAMURAL NEURONS OF
THE GUT.
13. CASTOR OIL-INDUCED DIARRHOEA
• PROCEDURE
• ANIMALS SWISS ALBINO RATS (150 – 180 G) OF EITHER SEX WERE SELECTED FOR THE
EXPERIMENTS.
• RATS WERE FASTED FOR 18 H AND DIVIDED INTO FIVE GROUPS OF SIX ANIMALS PER
GROUP.
• CASTOR OIL AT A DOSE OF 1 ML/ANIMAL ORALLY, WAS GIVEN TO ALL GROUPS OF
ANIMALS FOR THE INDUCTION OF DIARRHOEA .
• THIRTY MINUTES AFTER CASTOR OIL ADMINISTRATION, THE FIRST GROUP (CONTROL
GROUP) RECEIVED VEHICLE (0.5% V/V TWEEN 80 IN DISTILLED WATER)
• WHILE THE SECOND, THIRD AND FOURTH GROUPS WERE GIVEN PETROLEUM ETHER
EXTRACT AT DOSES OF 25, 50 AND 100 MG/KG BODY WEIGHT RESPECTIVELY BY
ORAL ROUTE.
14. CONTINUE…
• THE FIFTH GROUP RECEIVED THE REFERENCE DRUG, DIPHENOXYLATE (50 MG/KG
BODY WEIGHT).
• ANIMALS OF ALL GROUPS WERE PLACED SEPARATELY IN INDIVIDUAL CAGES
LINED WITH FILTER PAPER.
• THE FILTER PAPERS WERE CHANGED EVERY HOUR AND THE SEVERITY OF
DIARRHOEA WAS ASSESSED HOURLY FOR SIX HOURS.
• THE TOTAL NUMBER OF FAECES EXCRETED AND THE TOTAL WEIGHT OF FAECES
WERE RECORDED WITHIN A PERIOD OF SIX HOUR AND COMPARED WITH THE
CONTROL GROUP.
15. CONTINUE
…
• THE TOTAL NUMBER OF DIARRHOEAL FAECES OF THE CONTROL GROUP WAS
CONSIDERED 100%.
• THE RESULTS WERE EXPRESSED AS PERCENTAGE OF INHIBITION OF DIARRHOEA .
16. GASTROINTESTINAL MOTILITY TEST
• THIS EXPERIMENT WAS DONE BY USING CHARCOAL MEAL AS A DIET MARKER.
• THE RATS WERE DIVIDED INTO FIVE GROUPS OF SIX ANIMALS EACH AND FASTED
FOR EIGHTEEN HOURS BEFORE THE EXPERIMENT.
• THE FIRST GROUP (THE CONTROL GROUP) WAS ORALLY ADMINISTERED THE
VEHICLE (0.5% TWEEN 80 IN DISTILLED WATER).
• THE SECOND, THIRD AND FOURTH GROUPS ORALLY RECEIVED PETROLEUM
ETHER EXTRACT AT DOSES OF 25, 50 AND 100 MG/KG BODY WEIGHT
RESPECTIVELY.
• THE FIFTH GROUP RECEIVED THE STANDARD DRUG, ATROPINE SULPHATE (0.1
MG/KG BODY WEIGHT INTRAPERITONEAL ).
17. CONTINUE…
• THIRTY MINUTES LATER EACH ANIMAL WAS GIVEN 1 ML OF CHARCOAL MEAL
(10% ACTIVATED CHARCOAL IN 5% GUM ACACIA) ORALLY.
• EACH ANIMAL WAS SACRIFICED THIRTY MINUTES AFTER ADMINISTRATION OF
CHARCOAL MEAL.
• THE DISTANCE COVERED BY THE CHARCOAL MEAL IN THE INTESTINE WAS
EXPRESSED AS A PERCENTAGE OF THE TOTAL DISTANCE TRAVELED FROM THE
PYLORUS TO THE CAECUM .
18. CASTOR OIL-INDUCED ENTEROPOOLING
PROCEDURE:
• OVER NIGHT FASTED RATS WERE DIVIDED INTO FIVE GROUPS OF SIX ANIMALS
EACH.
• GROUP 1 WHICH RECEIVED NORMAL SALINE (2 ML/KG INTRAPERITONEAL )
SERVED AS THE CONTROL GROUP.
• GROUP 2 RECEIVED ATROPINE (3 MG/KG INTRAPERITONEAL )
• GROUPS 3, 4 AND 5 RECEIVED EXTRACT OF 25, 50 AND 100MG/KG
INTRAPERITONEALY, RESPECTIVELY, ONE HOUR BEFORE THE ORAL
ADMINISTRATION OF CASTOR OIL (1 ML).
• TWO HOURS LATER, THE RATS WERE SACRIFICED.
19. CONTINUE
…
• THE SMALL INTESTINE WAS REMOVED AFTER TYING THE ENDS WITH THREADS
AND WEIGHED.
• THE INTESTINAL CONTENT WAS COLLECTED INTO A GRADUATED CYLINDER
AND THEIR VOLUME WAS MEASURED.
• THE INTESTINE WAS REWEIGHED AND THE DIFFERENCE BETWEEN THE FULL AND
EMPTY WAS CALCULATED.
20. STATISTICAL ANALYSIS
• STATISTICAL ANALYSIS DATA WERE ANALYZED BY ONE-WAY ANOVA FOLLOWED
BY DUNNETT’S T-TEST USIN COMPUTERIZED GRAPHPAD INSTAT VERSION 3.05
(GRAPH PAD SOFTWARE , U.S.A .).
21. MODIFICATIONS OF THE METHOD
• INHIBITION OF CASTOR OIL-INDUCED DIARRHEA IN MICE WAS TESTED BY
BIANCHI AND GOI (1977)
• DAJANI ET AL. (1977) TESTED ANTIDIARRHEAL ACTIVITY IN CASTOR-OIL
TREATED MONKEYS .
• MANNITOL -INDUCED DIARRHEA WAS USED AS MODEL IN CALVES (
FIORAMONTI AND BUÉNO 1977) AND IN PIGS
22. EVALUATION OF ANTI-DIARRHEAL EFFECT IN
COLD-RESTRAINED RATS
• MALE SPRAGUE - DAWLEY RAT WEIGHING 260-310GMS & MAINTAIN ON FOOD &
WATER.
• SINCE GASTRIC ULCER ARE REDUCED IF COLD-RESTRAINED RAT ARE ALLOWED
FREE ACCESS TO FOOD AND WATER.
• SO FOR STUDY ON FECAL OUT PUT, FOOD WAS NOT WITHDRAWN PRIOR TO THE
EXPERIMENT.
• THE RAT ARE STUDY IN NORMAL LIVING CAGES AT ROOM TEMP. OR IN WIRE
MESH RESTRAINING CYLINDERS PLACED IN A COLD (4 C) ENVIRONMENT.
23. • TEST DRUG ARE ADMINISTERED BY APPROPRIATE ROUTES OVER OPTIMAL
EFFECTIVE DOSE-RANGES FOR THEIR ACTIVITIES AND AT OPTIMAL PRE
TREATMENT TIMES TO MAXIMIZE THEIR EFFECTS.
• THE NUMBER OF PELLETS EXPELLED BY EACH ANIMAL IS MEASURED AT 1 AND 3
HR. (FECAL PELLET OUTPUT).
• GENERALLY, THE FECAL PELLETS OF STRESSED ANIMALS ARE LESS FIRM.
• FECAL PELLET FLUID CONTENT ARE DETERMINED BY WEIGHING FECAL PELLETS ,
DRYING THEM IN OVEN AT 37 C, AND WEIGHING THEM AGAIN.
24. EVALUATION OF EVALUATION OF ANTI-
DIARRHEAL EFFECT IN COLD RESTRAINED
RATS
• THE DOSE IN MG/KG THAT INHIBITS THE COLD RESTRAINED STRESS INDUCED
INCREASE IN FECAL PELLET OUTPUT BY 50% (ID₅₀) IS DETERMINED USING LEAST-
SQUARES FIT ANALYSIS DIRECTLY FROM THE REGRESSION LINE.
