screening methods

1. PRECLINICAL SCREENING
OF ANTIFERTILITY
AGENTS.
BY- KAHKESHA SAMSHAD
1600100558
M.PHARM 1ST YEAR(PHARMACOLOGY)
FACULTY OF PHARMACY,INTEGRAL UNIVERSITY
LUCKNOW

2. CONTENTS:
• Introduction.
• Screening methods for female
• In-vivo models
• In-vitro models
• Screening methods for male
• In-vivo models
• Androgenic activity
• Anti-androgenic activity

3. INTRODUCTION
• Antifertility agents are the agents, which prevent the fertility by interfering with various normal
reproductive mechanism, in both males and females.
• Antifertility agents are also called as contraceptive agents. Basic aim of this agents is to prevent
conception or fertilization.
• These are made up of derivatives of synthetic progesterone or a combination of derivatives of
estrogen and progesterone which suppress the action of harmone that promote pregnancy.

4. SCREENING METHODS FOR FEMALES
• In-vivo methods
• Anti-ovulatory Activity:
• HCG-Induced ovulation in rats.
• Cupric acetate-induced ovulation in rabbits.
• Estrogenic Activity:
• Vaginal opening
• Assay for water uptake
• IN- VITRO METHODS
• Estrogen Receptor-binding Assay

5. IN-VIVO MODELS
• Antiovulatory Activity:
• HCG-induced ovulation in Rats.
Purpose and Rationale
Method used for the screening of anti-ovulatory agents.
Immature female albino rats do not ovulate spontaneously and do not show cyclic changes of vaginal
epithelium.
Priming with human chorionic gonadotropin (HCG) induces follicular maturation, followed by spontaneous
ovulation 2days later.
Injection of anti-ovulatory drugs, prior to the induction procedure will prevent ovulation .

6. PROCEDURE:
Immature female rats 24-26 days of age are used for the experiment.
The animals are treated with various test drugs in different dose levels.
After administration of test drugs, HCG is given exogenously for ovulation.
After 2 days, animals are sacrificed; ovaries are dissected out, preserved in 10% formalin and subjected to
histopathological evaluation.
The results are compared with the control group.

7. Estrogenic Activity
• vaginal opening.
• Purpose and Rationale
This assay is based on the principle that vaginal opening occurs in immature female albino rats and mice by
treating with estrogenic compounds.
The sign of complete vaginal opening is observed as a sign of estrogenic activity.
• procedure:
Immature female animals (18 days old mice, 21days old rats) are used for study.
The test and standard drugs are administered to the animals intramascularly in cotton seed oil.
The time for complete vaginal opening can be observed as a sign of estrogenic activity.

8. IN- VITRO MODELS
Estrogenic Receptor-binding Assay.
• Purpose and Rationale
Estrogenic receptor binding assay uses the principle for competitive binding of labelled and
unlabelled estrogen on the estrogen receptor.
Estrogenic compounds displaces the labelled estrogen in a concentration dependent manner
from the estrogen receptor.

9. PROCEDURE
• cytosol preparation.
Uteri from 18days old female albino mice are removed and homogenized at 0ºc in 1:50 (w/v) of
tris-sucrose buffer in a conical homogenizer
Human endometrium from menopausal women is frozen with 2h of hysterectomy and stored in
liquid nitrogen.
The frozen endometrium is pulverized and homogenized in 1:5 (w/v) of Tris-sucrose buffer.
Homogenates are centrifuged for 1hr, determination of specific binding in mouse uterus cytosol as a
function of steroid concentration incubation time and temperature.

10. Triplicate aliquots of 125ml of cytosol are incubated with 5 or 25nm labelled steroid either for 24hr
at 0ºc for 2 or 24hr at 25ºc in the absence (total binding) or presence (non-specific binding) of a 100
folded excess of radio inert steroid
Bound steroids is measured by Dextran coated charcoal (DDC) adsorption.

11. Dextran coated charcoal (DDC) adsorption technique
• 100 µl aliquot of incubated cytosol is stirred for 10 minutes at 0°c in a micro titer plate with 100 μl
of DDC suspension (0.625% dextran 80,000, 1.25% charcoal norit A) and then centrifuged for 10
minutes at 800 g
• The concentration of bound steroid is determined by measuring the radioactivity in a 100 μl
aliquot of supernatant
• For calculation of relative binding affinity, the percentage of radioligand bound in the presence of
competitor compared to that bound in its absence is plotted against the concentration of
unlabelled competing steroid.

12. SCREENING METHODS FOR MALES
• IN VIVO METHODS
• Cohabitation test
• Fertility test
• Subsidiary test
• Androgenic activity
• Chicken womb method
• Weight of ventral prostate, seminal vesicles and musculus Levator Ani
• Nitrogen retention
• Anti-androgenic Activity
• Chicken womb method
• Antagonisim of effect of testosterone on weight of central prostate, seminal vesicle and musculus Levator Ani

13. IN-VIVO MODELS
Cohabitation test.
• Purpose and Rationale
This test determines the time interval for litter production after placing treated males with 2
females.
The date of mating is calculated from the date of parturition. this method is suitable for drugs
known to cause several weeks sterility.

14. PROCEDURE
Adult female and male albino rats of proven fertility are used for the study
They are kept for mating in the ratio of 2:1 till both females deliver the litters
The date of mating is calculated from the date of parturition
The time interval for litter production after placing treated males with two females is
calculated

15. Fertility test
• Purpose and Rationale:
Fertility test is based on the evaluation of average litter size. Anti-fertility agents negatively affect
the average litter size.
Procedure
Groups of 5-10 male rats of proven fertility are treated with drug and are paired with fertile
females in the ratio of 1:3 daily vaginal are examined for the presence of sperms
All females passed through 1 estrus cycle must have mated
The mated animals are kept separately till the completion of the gestational period

16. The litters are counted and using the following formula average litter size is calculated
total no. of litter
Average litter size= ---------------------------
no. of female mated
If vaginal smear shows leukocytes for 10-14days, pseudopregnancy is confirmed.
Fertility patterns can be obtained from changes in average litter size

17. Subsidiary test
• Purpose and Rationale:
This test determines the changes in spermatozoa count time. The ant-fertility drugs affect the
spermatozoa count negatively.
Procedure:
Adult male albino rats weighing between 150-250 g are used for the study
They are kept in a cage containing artificial or animal vagina artificial vagina
Artificial vagina is the cylindrical plastic jacket with the rubber liner, filled with water at 5°c. 0.5 ml of
ejaculate is diluted with saline containing traces of formalin
Resulting suspension counted on haemocytometer.

18. Androgenic activity:
• Weight of ventral prostate, Seminal Vesicles and Musculus levator Ani
Purpose and Rationale:
This assay is based on the principle of androgens affect the secondary sex organs in male
individual
In the rats, the growth of the ventral prostate, the seminal vesicle and the musculus levator ani
depend on the presence of male sexual hormones

19. PROCEDURE:
• Immature male rats weighing about 55g are orchidectomized
The animals are treated with the test compounds in various doses orally in 0.5 ml 0.5% carboxymethyl
cellulose or 0.2 ml sesame oil suspension daily over a period of 10 days
Testosterone given subcutaneously in doses of 0.02, 0.1 and 0.5 mg per animal, or methyl testosterone in
doses of 0.25, 1.5, and 5 mg per animal, serve as standard
Controls receive the vehicle only. on the eleventh day, the animals are sacrificed and the seminal vesicles,
the ventral prostate, and the musculus levator ani carefully dissected and weighed
The weight of control and test group is compared with suitable statistical analysis

20. Anti-androgenic activity:
• Antagonism of effect of testosterone on weight of ventral prostate,
seminal vesicles and musculus Levator Ani
Purpose and Rationale:
In the rats, the growth of the ventral prostate, the seminal vesicle and the musculus levator ani
stimulated by testosterone, anti-androgenic compound inhibits this effect.

21. PROCEDURE:
• Male rats weighing 50-70 g are castrated and one day after surgery, the rats are injected once
daily for 7 days with 0.15 mg testosterone propionate in 0.1ml sesame oil
• The test compound also dissolved or suspended in sesame oil at various doses and injected
subcutaneously daily at a separate site for 7 days
• Controls receive testosterone injections only. on 8th day, the animals are sacrificed and weights of
ventral prostate, seminal vesicles and musculus levator ani weighed.
• The weight of control and test group is compared with suitable statistical analysis

22. References:
• sciarra jj. the continuing need for contraceptive research. fertil steril 1981;36:697-8.
• Turner Ra. screening methods in pharmacology. new york: academic press 1971:85-118
• vogel hg, vogel wh, editors. drugs discovery and evaluation. berlin: springer 1997.
• ghosh r. modern concept on pharmacology and therapeutics. (24th ed). calcutta, hilton
and co, 1991. 30