pharmacological Preclinical screening of antidiarrheal and Laxatives

pharmacological Preclinical screening of antidiarrheal and Laxatives

1. PHARMACOLOGICAL AND
TOXICOLOGICAL SCREENING
METHODS-I
Submitted By, Submitted To,
P.Aswin Dr.G.Venkatesh M.Pharm.,Ph.D
M.Pharm First year Department of pharmacology
Department of pharmacology KMCH College of pharmacy
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2. Pharmacological preclinical
screening of Laxatives &
Antidiarrheal

CECUM
DESCENDING
COLON
TRANSVERSE
COLON
ASCENDING
COLON
RECTUM
ANUS10-Mar-20 DEPARTMENT OF PHARMACOLOGY 2

3. Constipation
Constipation refers to infrequent or difficult defecation caused by
decreased motility of the intestines. Because the feces remain in the
colon for prolonged periods, excessive water absorption occurs, and
the feces become dry and hard.
Constipation may be caused by poor habits (delaying defecation),
spasms of the colon, insufficient fiber in the diet, inadequate fluid
intake, lack of exercise, emotional stress, and certain drugs.
A common treatment is a mild laxative, such as milk of magnesia,
which induces defecation.
However, many physicians maintain that laxatives are habit-forming,
and that adding fiber to the diet, increasing the amount of exercise, and
increasing fluid intake are safer ways of controlling this common
problem.
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4. Diarrhea
Diarrhea is an increase in the frequency, volume, and fluid content of
the feces caused by increased motility of and decreased absorption by
the intestines.
When chyme passes too quickly through the small intestine and feces
pass too quickly through the large intestine, there is not enough time
for absorption.
Frequent diarrhea can result in dehydration and electrolyte
imbalances. Excessive motility may be caused by lactose intolerance,
stress, and microbes that irritate the gastrointestinal mucosa.
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5. Absorption and Feces Formation
in the Large Intestine

By the time chyme has remained in the large intestine 3–10 hours, it
has become solid or semisolid because of water absorption and is now
called feces.
Chemically, feces consist of water, inorganic salts, sloughed-off
epithelial cells from the mucosa of the gastrointestinal tract, bacteria,
products of bacterial decomposition, unabsorbed digested materials,
and indigestible parts of food.
Although 90% of all water absorption occurs in the small intestine, the
large intestine absorbs enough to make it an important organ in
maintaining the body’s water balance.
Of the 0.5–1.0 liter of water that enters the large intestine, all but about
100–200 mL is normally absorbed via osmosis. The large intestine also
absorbs ions, including sodium and chloride, and some vitamins.
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6. Bulk forming
Dietary fibre:
Bran,
Psyllium (Plantago)
Ispaghula,
Methylcellulose

Laxatives
Stool softener
Docusates (DOSS),
Liquid paraffin
Osmotic purgatives
Magnesium salts: sulfate,
hydroxide Sodium salts:
sulfate, phosphate
Sod. pot. tartrate
Lactulose
Anthraquinones
Senna,
Cascara sagrada
5-HT4 agonist
Prucalopride
Fixed oil
Castor oil
Diphenylmethanes
Phenolphthalein,
Bisacodyl,
Sodium picosulfate
Stimulant purgatives
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7. Anti diahrrhoeals

Non antimicrobial anti diahrrhoeals
I. Antimotility agents:
Diphenoxylate, loperamide, codeine.
II. Anticholinergic agents:
Atropine, scopolamine
Specific anti infective agents
I. Antimicrobials:
Co-trimaxozole, norfloxacin, doxycycline, erythromycin,
metronidazole
II. Antisecretary agents:
Sulfasalazine, mesalazine
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8. Screening models
Intestinal Functions
Laxatives
Laxative Activity in Rats
Enteropooling Test
Inhibition of Chloride Secretion in Rabbit Colon
Antidiarrheal Effect
Castor Oil Induced Diarrhea
Antidiarrheal Effect in Cecectomized Rats
Evaluation of Antidiarrheal Effect in Cold-Restrained Rats

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9. Gut Motility
Isolated Ileum (MAGNUS Technique)
Cascade Superfusion Technique
In Vivo Evaluation of Spasmolytic activity in Rats
Colon Motility in Anesthetized Rats
Continuous Recording of Electrical and Mechanical Activity in the Gut of
the Conscious Rat
Propulsive Gut Motility in Mice
Nerve-Jejunum Preparation of the Rabbit
Motility of Gastrointestinal Tract in Dogs
Thiry–Vella Fistula
Continuous Recording of Mechanical and Electrical
Activity in the Intestine Colitis of Conscious Dogs

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10. Laxative Activity in Rats
PURPOSE AND RATIONALE
Laxatives of the sennoside type act mainly by acceleration
of large intestine transit and inhibition of fluid absorption
in the colon
Requirements:

Animal : Female Wistar rats
Weight : 200g
Chemicals used : Ether, Carmine Red
Drug : Test drug
Materials : PVC catheter
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11. Procedure
1.For measurement of large intestinal transit time,
female Wistar rats weighing approximately 200 g are
anesthetized with ether.
2.A PVC catheter is implanted into the caecum with the
distal end fixed on the animal’s neck.
3.The animals are allowed to recover and are placed
individually in a wire meshed cage to enable the feces to
fall through onto blotting paper
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12. 4.Carmine red (10 mg in 0.4 ml distilled water per
animal) is injected through the catheter immediately
after administration of the test substance
5.The time until appearance of the
first colored feces is registered


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13. A Catheter
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14. 10-Mar-20 DEPARTMENT OF PHARMACOLOGY 14

15. Fluid absorption
1.For measurement of fluid absorption in the colon,
female Wistar rats weighing approximately 200 g are
anesthetized with 50mg/kg pentobarbitone sodium.
2.The colon is ligated and cannulated distal to the
caecocolic junction (PE-tube, i.d. 1 mm) and, after a
thorough rinse with 50ml physiological saline to
remove all contents, a second cannula (silicone, i.d. 3
mm) is inserted proximal to the rectum for fluid
outflow.
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16. 3.Four and 6 h after oral administration of the test compounds an
open perfusion with an electrolyte solution (NaCl 6.72 g/l, KCl 0.37
g/l, NaHCO3 2.1 g/l, polyethylene glycol (PEG, mol wt 4000) 2.0 g/l,
[14C]PEG 5 µCi/l; pH 6.5, osmolality 275 milliosmol/kg) is started
at a rate of 12 ml/h for two consecutive 2 h periods.
4.[14C]PEG activity is measured by liquid scintillation counting,
Na+ and K+ by flame photometry, Cl- by coulometric titration,
osmolality by freezing point depression and mucus as
protein-bound total hexoses by the orcinol-sulphuric acid method.
5.Net H2O, Na+, K+ and Cl- transport are calculated and expressed
as ml or µmol/h and per 10 cm colon length.

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17. •EVALUATION
All values are expressed as mean ± standard deviation.
Statistical significance is assessed with Student’s t-test.
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18. Enteropooling Test
PURPOSE AND RATIONALE
The enteropooling assay in rats is to test the diarrheogenic
property of prostaglandins for prediction of this clinically
relevant side effect of several synthetic prostaglandins

Requirements:

AnimalSprague Dawley rats
Sex Female
Weight190–215 g
Test Prostaglandins
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19. Procedure
The animals are fasted overnight having free access to water. The
test compounds are administered orally, and the animals, 12 per
group, are sacrificed one hour later.
The fluid accumulation occurs in the small intestine which is cut at
the pylorus and the ileocecal junction, and its contents, consisting
of a thick fluid (in controls) and a very watery fluid (in
prostaglandin treated animals) are collected into a graduated test
tube by milking the whole length of the small intestine with the
fingers.
The volume of fluid is recorded

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20. EVALUATION
Using various doses, dose-response curves can be
established and potency ratios calculated.
16,16-dimethyl PGE2 was found to be the most active
compound

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21. Castor oil induced diarrhea
PURPOSE AND RATIONALE
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 hypersecretory response. In addition to
hypersecretion, ricinoleic acid sensitizes the intramural neurons of the
gut.
Requirements
AnimalWistar rats
Sex Female
Weight210–230 g
Test antidiarrhoic agent
PromotorCastor oil
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22. Procedure
For the experiment, the rats are housed in individual
cages with no access to drinking water. The potential
antidiarrhoic agents are administered orally by gavage
in various doses.
Controls receive the solvent only. Each dose is given to
10 animals. One hour after dosage, 1 ml of castor oil is
administered orally.
Stools are collected on non-wetting paper sheets of
uniform weight up to 24 h after administration of the
castor oil.
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23. Every 15 min during the first 8 h, urine is drained off by
gravity, and the net stool weight, termed early diarrheal
excretion, is recorded
The diarrhea-free period is defined as the time in
minutes between castor oil administration and the
occurrence of the first diarrheal output.
The acute diarrheal phase is the time between the first
and the last diarrheal output of the 8-h observation
period. Stools occurring between 8 and 24 h after castor
oil administration are called late diarrheal excretion
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24. EVALUATION
With antidiarrheal agents dose-response curves are
obtained for decrease of hypersecretion (stool weight) and
for increase of the diarrhea-free period are obtained.
Inhibitors of prostaglandin biosynthesis increase the
diarrhea free period but do not affect early diarrheal
secretion.
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25. Evaluation of Antidiarrheal Effect
in Cold-Restrained Rats
PURPOSE AND RATIONALE
Barone et al. (1990) tested the effect of various antidiarrheal
and other drugs on increased fecal pellet output in
cold-restrained rats resembling clinical observations that
stressful situations can produce diarrhea in humans.
Requirements:


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AnimalRats
StrainSprague Dawley
Sex Male
Weight260-310
Test unknown

26. PROCEDURE

Male Sprague-Dawley rats weighing 260–310 g are
maintained on Purina lab chow and water. Since gastric
ulcers are reduced if cold-restrained rats are allowed
free access to food and water, for studies on fecal
output food was not withdrawn prior to the
experiment.
The rats are studied in normal living cages at room
temperature (control, non-stressed animals) or in wire
mesh restraining cylinders placed in a cold (4°C)
environment.
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27. Test drugs are administered by appropriate routes over
optimal effective dose-ranges for their activities and at
optimal pretreatment times to maximize their effects.
The number of pellets expelled by each animal is
measured at 1 and 3 h (fecal pellet output). Generally, the
fecal pellets of stressed animals are less firm. Fecal pellet
fluid content is determined by weighing fecal pellets,
drying them in an oven at 37°C, and weighing them again
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28. EVALUATION
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.
If fecal pellet output is decreased by a drug but no clear
dose-related effects occur, the maximum percent decrease
is determined.
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29. Propulsive Gut Motility in Mice

PURPOSE AND RATIONALE
The passage of a charcoal meal through the gastrointestinal
tract in mice is used as parameter for intestinal motility and
to study the effect of laxatives.

Requirements:
AnimalMice-NMRI Strain
Sex Female
Weight15g
MarkerCharcoal meal
Test Laxative
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30. PROCEDURE

Groups of 10 female mice (e. g., NMRI strain) weighing
15 g are fed an oat diet for 3 days. Eighteen hours prior
to the experiment food, but not water, is withdrawn.
The animals are treated either subcutaneously 15min
or orally 60min before administration of the charcoal
meal (0.2 ml of a 4% suspension of charcoal in 2%
carboxymethylcellulose solution).
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31. The mice are sacrificed after various time intervals,
20min, 40min, 60 min and 120 min. Ten animals serve
as controls for each time interval. The entire intestine is
immediately removed and immersed in 5% formalin to
halt peristalsis; then washed in running water.
The distance the meal has traveled through the
intestine as indicated by the charcoal is measured and
expressed as percent of the total distance from the
pylorus to the caecum.
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32. EVALUATION
Student’s t-test is used to compare the control and the
drug-treated group.
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33. Reference
Hans Gerhard vogel 3rd edition; drug discovery and
evaluation: drug effects on , pharmacological assay,
Pg no-1241-1265.
SK Gupta 1st edition; drug screening methods;drugs for
Laxatives and anti diarrheal.

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