This presentation is all about the screening method used in anticancer and antiulcer & Introduction to OECD GUIDELINES.

1. PHARMACOLOGICAL SCREENING
(Anticancer, Antiulcer & OECD
guidelines)
PRESENTED BY
KANEEZ FATIMA
M.PHARM 1st YEAR

2. Pharmacological Screening
• Screening of drugs: It means thorough
investigations measure the pharmacological activity
of new or chemically undefined substances investigate
the function of endogenous mediators measure drug
toxicity and unwanted effects.
• The main purposes of screening are to determine
whether the new substance are worthy for further
attention and to indicate which among them have the
most interesting pharmacological properties. Types of
Screening:
A. Simple screening
B. Blind screening
C. Programmed screening.

4. PHARMACOLOGICAL SCREENING FOR
ANTICANCER
• ‘Cancer’ a fatal disease of uncontrolled proliferation of
genetically altered cells.
• They can be of benign tumors and malignant.
• Cancer of almost all tissues is known today. Such as:
carcinomas ,sarcomas ,leukemia ,lymphoma,myeloma,
gliomas and astrocytomas.
• Chemotherapy, surgery and radiotherapy have
remained the main attempts of cancer treatment.
• Usually, a combination of all the three treatment
regimes is used against any given cancer for maximum
benefit.

5. Causes
• External factor: chemicals, radiation, viruses,
and lifestyle.
• Internal factors: – hormones, immune
conditions, inherited mutations
• Theories: Cellular change/mutation theories ,
Carcinogens , Oncogenes / protooncogenes.

6. Need for Novel Anticancer Agents
• Development of multidrug resistance in patients.
• Long-term treatment with cancer drugs is also
associated with severe side effects.
• Cytotoxic drugs have the potential to be very harmful
to the body unless they are very specific to cancer
cells.
• New drugs that will be more selective for cancer cells.

7. ANTITUMOR MODELS
• In-vitro method
o Tetrazolium salt assay.
o Sulphorhodamine B assay.
o 3H-Thymidine uptake.
o Dye exclusion test.
o Clonogenic test.
o Cell counting assay.
o Morphological assay.
• In-vivo method:
o Carcinogen induced models.
o Viral infection models.
o Transplantation models.
o Genetically Engineered Mouse
models.
o In vivo hollow fibre assay.

9. 1.TETRAZOLIUM SALT ASSAY
• The assay is based on the capacity of mitochondrial
dehydrogenase enzymes in living cells to convert the
yellow water-soluble substrate 3-(4,5-dimethylthiazol-
2- yl)-2,5-diphenyl tetrazolium bromide (MTT) into a
dark blue formazan product which is insoluble in
water. The amount of formazan produced is directly
proportional to the cell number in range of cell lines

10. 2.Morphological assay
• Large-scale, morphological changes that occur at the
cell surface, or in the cytoskeleton, can be followed
and related to cell viability.
A) normal human skin keratinocyte B)Differentiated human skin keratinocyte
differentiated human skin keratinocyte.differentiated human skin keratinocyte.differentiated human skin keratinocyte.differentiated human skin keratinocyte.

11. 3.HEMOCYTOMETER CELL COUNTS
• Hemocytometer: The most common routine
method for cell counting which is efficient and
accurate is with the use of a hemocytometer.
4.SULPHORHODAMINE B ASSAY
• The Sulphorhodamine B assay measures whole-
culture protein content, which should be
proportional to the cell number.

12. • Advantages of in-vitro methods:
1. Reduce the usage of animals.
2. Less time consuming, cost effective & easy to
manage
3. Able to process a larger number of compounds
quickly with minimum quantity.
• Disadvantages of in-vitro methods:
1. Difficulty in Maintaining of cultures.
2. Show Negative results for the compounds which gets
activated after body metabolism and vice versa.
3. Impossible to ascertain the Pharmacokinetics.

14. 1.CHEMICAL CARCINOGEN MODEL:
• DMBA induced mouse skin papillomas
-Two stage experimental carcinogenesis:
• Initiator – DMBA (dimethylbenz[a]anthracene),
• Promotor – TPA (12-O-tetradecanoyl-phorbol-
13- acetate)
• Mice : Single dose – 2.5 µg of DMBA , 5 to 10 μg
of TPA in 0.2 ml of acetone twice weekly.
• Papilloma begins to appear after 8 to 10 wks –
Tumor incidence & multiplicity of treatment
group is compared with DMBA control group.

15. 2. TRANSPLANTATION MODELS
• Tumor cells or tissues (mouse or human)
transplanted into a host mouse.
• Ectopic – Implanted into a different organ than
the original (typically subcutaneous or kidney
capsule)
• Orthotopic – Implanted into the analogous
organ of the original tumor.
• Advantages :
• Typically cheap, fast & easy to use.
• Not covered by patents.

17. SCREENING FOR ANTIULCER
ULCER
“A disruption of the mucosal integrity of the
stomach and/or duodenum leading to a local
defect or excavation due to active inflammation.”

18. Pathophysiology
• Aggressive factors:
1. Acid
2. Pepsin
3. NSAIDs
4. H.pylori
5. Alcohol
6. Smoking
7. Stress
8. Spicy foods
• Protective factors
1. Mucus
2. Prostaglandins
3. Mucosal blood flow
4. Bicarbonate

20. PHARMACOLOGICAL TREATMENT
• ACID SUPPRESSANTS
1. Proton pump inhibitors : Omeprazole ,
Pantoprazole, Lansoprazole
2. H2 receptor blockers : Ranitidine, Famotidine,
Nizatidine
• CYTOPROTECTANTS
1. Prostaglandin analogues : Misoprostol
2. Ulcer protectants : Sucralfate
3. Antacids : (replaced by more effective and
convenient drugs)

22. Antiulcer Screening Models
• In vivo models
• Pylorus Ligation in Rats
• Stress ulcer Model
-Restraint- induced ulcers
-Cold water immersion induced
ulcer
• 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
• In vitro models
• [I125] Gastrin Binding Assay
• Tiotidine Binding Assay
• H+ / K+ -ATPase Inhibition
Assay

23. IN-VIVO METHOD
1.PYLORUS LIGATION METHOD:
Principle:
• Pylorus is ligated over a certain period of time
• Accumulation of gastric acid causes ulceration
Procedure:

24. Procedure:
• Wistar rats weighing 150-200 grams
• Fasting : 48 hours ; water ad libitum.
• Housed singly in cages with raised bottoms of wide wire mesh .
• Under anaesthesia, a one-inch midline abdominal incision is
given.
• Pylorus is ligated without damaging its blood supply.
• Stomach is replaced and abdominal wall closed with sutures.
• Test compounds are given either orally or injected s.c.
• About 17-19 hours after pyloric ligation, rats are sacrificed and
stomach are dissected out.
• Contents of the stomach are drained into a graduated centrifuge
tube and subjected to analysis for volume, pH, free and total
acidity, mucin, prostaglandin, total carbohydrate:protein ratio
etc.
• Stomach is opened along the greater curvature, pinned on a
cork plate. Its inner surface is examined for ulceration with a
binocular microscope.
• The ulcer index is calculated and the Ulcer severity graded.

25. EVALUATION OF THE TEST
 Ulcer severity:
• 0 = No ulcer
• 1 = Superficial ulcer
• 2 = Deep ulcer
• 3 = Perforation
 Ulcer index (UI ) : UI = (UN + US + UP )× 10-1
Where,
• UN = Average number of ulcers per animal
• US = Average of severity scores
• UP = Percentage of animals with ulcers
 Advantage:
• Evaluates anti-ulcer drugs with various mechanisms of action and different doses.
 Disadvantage:
• The ulcers localized in antrum of the stomach.
 Inference:
• Ulcer index of test drug compared with control group to detect anti-ulcer effect of
test drug.
• Other parameters help to infer the mechanism of ulcer protection
- Decrease in volume, free & total acidity: antisecretory action
- Rise in pH: acid neutralising action
- Increase in mucin, PGs: cytoprotective effect.

26. 2.Stress Induced Ulcer

27. A)Restraint- induced ulcers (Hanson and Brodie
,1960)
• Principle: Stress plays a significant role in the
pathogenesis of gastric ulcers.
• Procedure:
• Albino rats weighing 150-200 grams are taken.
• Fasted for 36 hours before experiment.
• Drug is administered orally or subcutaneously
• 30 min later animals are subjected to restraint
• For restraint, the rats were placed in a piece of galvanized
steel window screen of appropriate size.
• Screen was moulded around the animal and held in place
with wire staples.
• To restrain the rats, the limbs were put together in pair
and tightened with adhesive tape.
• Rats were kept under restraint for 24 hours
• Rats were then sacrificed & their stomachs dissected out.
• Ulcer index and ulcer severity were determined.

28. B) Cold water immersion induced ulcer (Takagi et al,
1964)
• Principle: Exposure of cold conditions to restrained animals
accelerates the occurrence of gastric ulcers and Shortens the
immobilization time.
• Procedure: Wistar rats weighing 150-200 grams are used.
• After fasting the animals for 16 hours, the test compound is
administered orally.
• Rats are then placed individually in restraint cages vertically,
and then immersed in water upto the xiphoid process, at 22°C
for 1 hour.
• Then rats are removed from the cages, dried Evan’s blue
(30mg/kg) injected i.v. via the tail vein 10 min later, they are
sacrificed
• The stomach is removed & ligated at both ends.
• It is filled with Formol saline & kept overnight.
• On the next day, the stomach is opened along the greater
curvature and examined for ulcerative lesions.

29. 3.Histamine-induced Gastric Ulcer
• Principle: Gastric acid secretion is increased when histamine is
administered intraperitoneally.
• Procedure:
• Guinea pig weighing 300-400 grams are taken
• Fasted for 36 hours before experiment; water ad libitum
• 1 ml of histamine acid phosphate (50 mg base) was
administered i.p.
• Promethazine hydrochloride 5 mg was injected i.p.
• 15 min before and 15 min after histamine to protect the animals
against histamine toxicity.
• The standard/test drugs were administered p.o. or s.c. 45
minutes before histamine injection.
• 4 hours after histamine injection, guinea pigs were sacrificed
and stomach dissected out.
• The gastric contents were subjected to analysis
• Stomach was opened along the greater curvature, ulcers were
identified.

30. ULCER SCORING : (Barrett et al, 1955)
• Type 0 : No visible ulcers
• Type 1 : 10 or less small ulcers, 1-3 mm in
diameter
• Type 2 : 11 or more ulcers, 1-3 mm in diameter
• Type 3 : 1 or more ulcers, 4-6 mm in diameter
• Type 4 : 1 or more ulcers, 7 mm or more in
diameter
• Type 5 : Perforation of the gastric wall
• Advantages:
• Produces 100% gastric ulceration.
• Increased volume of gastric acid secretion.
• Marked enhancement of free and total acidity.

31. 4.Ethanol-induced Mucosal damage
• 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; water ad
libitum.
• 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 stomachs
dissected out.
• Severity score and ulcer index are calculated

32. 5. Indomethacin-induced gastric
lesions (Schoen & Vender et al.,1989)
Principle: NSAID induced gastric damage by blocking
COX enzyme, endogenous prostaglandin production
inhibited.
Procedure:
• Rats fasted for 36 hours before Indomethacin
administration (20 mg/kg, orally)
• 30 min prior to the administration of the Indomethacin,
standard/test drug is administered.
• Indomethacin-induced gastric lesions (Schoen & Vender
et al.,1989) 1 hour after Indomethacin administration,
• Rats are sacrificed, their stomach dissected out and
examined for the number of lesions under the
microscope.
• Ulcer index and ulcer severity are determined

33. 6. Acetic Acid-induced gastric Ulcer
Takagi et al. (1969)
• A model for inducing chronic gastric ulcer in rats by
means of submucosal injection of acetic acid.
Principle:
• Acetic acid enhances the ulceration in stomach by
increasing the acidity of stomach contents.
Procedure: (Takagi et al. (1969)
• Albino rats used 0.05 ml of Acetic acid (1-30%)
injected in the submucosal layer of the stomach
• Penetrating peptic ulcers : adhered to Liver
• Chronic ulcers with repeated healing and re-
aggravation
• Effect of test drug given twice daily for 10-15 days is
noted.

34. Okabe et al,( 1972)
• New method which involves temporary instillation of acetic acid
solution.
Procedure: (Okabe and Pfeiffer, 1972)
• Wistar rats weighing 150-200 grams are taken
• Fasted for 24 hours before experiment.
• Pentobarbital anaesthesia were done.
• A cylindrical glass tube of 6 mm diameter tightly placed upon the
anterior serosal surface of stomach 1 cm away from the pyloric end.
• 50% Acetic acid (0.06 ml per animal) was instilled into the tube and
allowed to remain for 1 minute on the gastric wall .
• After removal of Acetic acid solution, the abdomen was closed.
• Animals were caged and fed normally.
• Test drugs were given orally on Day 1 twice daily, 4 hours after
application of acetic acid and continued upto 10 days after induction
of ulcer
• Animals were sacrificed after 18 hours of the last dose to assess ulcer
size and healing.
• Ulcer index and Severity score calculated.

35. IN VITRO METHOD

36. 1. [ I125] Gastrin binding assay
Principle:
• Gastrin ( G cells of gastric antrum)
• Bind to CCK2 receptors on parietal cells --> release HCl
• Bind 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 50μl [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 37°C
• 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.

37. 2. Tiotidine Binding Assay
Principle:
• H2 receptor blocker
• Assay 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)
• 5 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 buffer
• Filters 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.

38. 3.H+/K+ - ATPase inhibition assay
Principle:
• H+/K+ - ATPase or proton pump --> final step in the synthesis
of acid by parietal cells
Procedure:
• Homogenate of 80 ng Microsomal gastric H+/K+-ATPase
incubated with 100µl buffer, 1mM ATP and(pig gastric
mucosa)
• Test compound in microlitre plate for 30 mins at 37°C
• Reaction is stopped by adding Malachite green (colorimetric
agent)
• After 10 seconds, 15% sodium citrate is added for 45 minutes
• Release of orthophosphate from ATP quantified by colorimeter
at 570 nm
• Evaluation:
• Percentage inhibition of H+/K+ - ATPase is calculated.
• Lesser the orthophosphate released, more is the inhibition of
H+/K+ - ATPase by test compound.

39. TOXICITY STUDIES AS PER
OECD GUIDELINES

40. TOXICITY
• Toxicity:
It is the study of adverse effects of chemical and
physical agents and the degree to which a substance
can harm human or animals.
• TYPES OF TOXICITY:
-Acute toxicity: Harmfull in single or short term
exposure.
-Sub-chronic toxicity: Causes effect by multiple doses
or long exposure.
-Chronic toxicity: Upon repeated dosage or life time
exposure.

41. ACUTE TOXICITY STUDIES
• The Globally Harmonized System (GHS),defines it as
"those adverse effects occurring following oral
or dermal administration of a single dose of a
substance, or multiple doses given within 24
hours, or an inhalation exposure of 4 hours" .
• The preferred species for oral and inhalation testing is
the rat, and for dermal testing, the rat or rabbit.

42. Five OECD Test Guidelines (TGs 402, 403,
420, 423, and 425) describe acute
systemic testing:
• Fixed Dose Procedure (OECDTG 420) ,
• Acute Toxic Class method (OECDTG 423) ,
• Up‐and‐Down Procedure (OECDTG 425),
• Acute Dermal Toxicity OECDTG 402,
• Acute inhalation toxicity OECDTG 403.

43. DESCRIPTION OF THE METHOD
• Selection of animal species:
• Housing and feeding conditions
• Preparation of animals
• Procedure:
i. Administration of doses,
ii. Limit test at 2000mg/kg,
iii. Limit test at 5000mg/kg,
iv. Main test,
v. Observations,
vi. Body weight,
vii. Pathology .
• Data and reporting

44. CHRONIC TOXICITY STUDIES
• The Globally Harmonized System (GHS) defines it as
"specific target organ/systemic toxicity arising
from a repeated exposure".
• Chronic toxicity testing consists of oral, dermal, and
inhalation subacute repeated dose studies (28‐day)
and subchronic repeated dose studies (90‐day) in
rodents.
• It consist of an evaluation of clinical observations,
blood analysis, whole body gross necropsy, and
microscopic examination of all organs and tissues
(histopathology).

45. OBJECTIVES:
• Chronic Toxicity Test: To determine the toxicity
observing changes in the function, shape, of a living
organism.
• On Reproductive Potential and Future Generations
• Teratogenicity Test: To determine damage to the birth
of the fetus.
• Mutagenicity Test: To test carcinogenicity and genetic
impact on the next generation.
• Identification of the hazardous properties of a chemical.
• Identification of target organs.
• Characterization of the dose:response relationship.
• Identification of a no-observed-adverse-effect level
(NOAEL).
• Prediction of chronic toxicity effects at human exposure
levels.

46. SEVEN OECD Test Guidelines (TGs 402,
403, 420, 423, and 425) describe acute
systemic testing
• Six OECD Test Guidelines describe short‐term repeat‐dose
toxicity testing
• Repeated Dose 28‐day OralToxicity Study in Rodents
(TG407)
• Repeated Dose 90‐Day OralToxicity Study in Rodents (TG
408)
• Repeated Dose DermalToxicity: 21/28‐day Study (TG 410)
• Subchronic DermalToxicity: 90‐day Study (TG 411)
• Repeated Dose InhalationToxicity: 28‐day or 14‐day Study
(TG 412)
• Subchronic InhalationToxicity: 90‐day Study (TG 413).

47. PRINCIPLE OF THE TEST
• The study design consists of two parallel phases:
1) A chronic phase, normally of one year
duration ,
2)A carcinogenicity phase, normally of two
years duration

48. DESCRIPTION OF THE METHOD
• Selection of animal species
• Housing and feeding conditions
• Preparation of animals
• Procedure
I. Number and sex of animals
II. Provision for interim kills and satellite groups
III. Dose groups and dosage
IV. Preparation of doses and administration of test
substance
V. Duration of study
• OBSERVATIONS: -Chronic toxicity phase
-Carcinogenecity phase
• Data and reporting
• Results
• Conclusions