screening of reproductive pharmacology .pptx

REPRODUCTIVE PHARMACOLOGY
SCREENING MODELS FOR
ANTI-FERTILITYAGENTS
PRAVEEN KUMAR.S
DEPARTMENT OF PHARMACOLOGY
M.PHARM 1ST SEMESTER
PSG COLLEGE OF PHARMACY.
1
SCREENING METHODS IN REPRODUCTIVE
PHARMACOLOGY

CONTENTS
Anti-fertility agents:
• Introduction
• Endocrine control of reproduction
• Types of contraceptive preparation
• Pre clinical screening of anti fertility drugs:
* In vitro
* In vivo
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PHARMACOLOGY

ANTI FERTILITY AGENTS
INTRODUCTION:
Anti Fertility drugs :
 Antifertility drugs are chemical substances which suppress the
action of hormones that promote pregnancy. These drugs actually
reduce the chances of pregnancy and act as a protection.
 Antifertility drugs are made up of derivatives of synthetic
progesterone or a combination of derivatives of estrogen and
progesterone .
 These are also known as contraceptive agents.
 Contraception is the method of preventing normal process of
ovulation, fertilization and ovum implantation , nothing but
pregnancy.
 These are classified into two types.
(1) Female contraceptive agents.
(2) Male contraceptive agents .
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PHARMACOLOGY

ENDOCRINE CONTROL OF
REPRODUCTION
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PHARMACOLOGY

PHARMACOLOGY
5

ESTROGEN AND PROGESTIN
Mechanism of action:
 • Estrogens bind to specific nuclear receptors in target cells and produce
effects by regulating protein synthesis.Estrogen receptors (ERs) have been
demonstrated in female sex organs, breast, pituitary, liver, bone,
bloodvessels, heart, CNS and in certain hormone responsive breast
carcinoma cells.
 • Two ERs designated ERα and ERβ have been identified, cloned and
structurally characterized.
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Luteinizing hormone
• In females, an acute rise of LH
("LH surge")triggers ovulation
and development of the corpus
luteum.
• In males, where LH had also been
called interstitial cell–
stimulating hormone (ICSH), it
stimulates leydig cell production
of Testesterone.
FOLLICLE STIMULATING
HORMONE
• In both males and females, FSH
stimulates the maturation of
primordial germ cells.
• FSH sustains spermatogenesis
in Males.
• FSH stimulates the growth and
development of immature
ovarian follicles in the ovary.
LH & FSH act synergistically with
each other .
7
PHARMACOLOGY

ORAL
1.COMBINED
PILL.
2.PHASED
REGIMEN.
3.MINI
PILL(PROGESTI
N ONLY.
4.POSTCOITAL
INJECTABLE1.LONG
ACTING
PROGESTIN
ONLY
2.LONG
ACTING
PROGESTIN+O
ESTROGEN
IMPLANTS
1.BIODEGRADABLE
IMPLANTS.
2.NON
BIODEGRADABLE
IMPLANTS
8
PHARMACOLOGY

1. Combined pill :
 • Oestrogen + progesterone containing preparation.
 • 2nd gen contain less Oestrogen & progestin to reduce side effects
 • 3rd gen contain new progestin like Desogestrel along with anovulatory
progestin like 19 nor-Testesterone.
 • Taken daily for 21 days, 5th menstrual day ----
 7 day gap after 2nd menstruation ----
2. Phased regimen :
 • These have been introduced to permit reduction in total steroid dose
without compromising efficacy. These are
 biphasic or triphasic.
 • Oestrogen level constant with low progestin ------ ist regimen
 • Oestrogen level constant with progestin ------ 2nd regimen
 • Oestrogen level constant with progestin ------- 3rd regimen
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PHARMACOLOGY

3) Minipill (progestin only pill) :
• A low-dose progestin only pill is taken daily continuously without any gap.
The menstrual cycle tends to become irregular
• The efficacy is lower (96–98%) compared to 98–99.9% with combined pill.
4) Postcoital (emergency) contraception :
• Currently 3 regimens are available :
• a) Levonorgestrel 0.5 mg + ethinylestradiol 0.1 mg taken as early as possible
but within 72 hours of unprotected intercourse and repeated after 12 hours.
• (b) Levonorgestrel alone 0.75 mg taken twice with 12 hour gap within 72
hours of intercourse.
• (c) Mifepristone 600 mg single dose taken within 72 hours of intercourse
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PHARMACOLOGY

Injectables :
• These have been developed to obviate need for daily ingestion of
pills. They are given i.m. as oily solution; are highly effective.
• Menstrual irregularities, excessive bleeding or amenorrhoea are very
common.
• Two types of preparations have been tested:
• (i) Long acting progestin alone : injected once in 2–3 months
depending on the steroid. Two compounds have been marketed:
• (a) Depot medroxyprogesterone acetate.
• (b) Norethindrone (Norethisterone) enanthate.
• ii) Long acting progestin + long acting estrogen : —
• Once a month. These have been tested to a more limited extent, but
a combination of Medroxyprogesterone + estradiol cypionate has
been approved by US-FDA for i.m. injection every month. Main
advantage is that they allow a reasonable menstrual bleeding pattern
in most cases.
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PHARMACOLOGY

IMPLANTS
• Implants These are drug delivery systems implanted under the skin,
from which the steroid is released slowly
over a period of 1–5 years. They consist of either—
• (a) Biodegradable polymeric matrices—do not need to be removed on
expiry.
• (b) Non-biodegradable rubber membranes—have to be removed on
expiry.
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Tests used for screening :
Anti ovulatory activity :
:1) HCG – Induced ovulation in rats
Rationale :
• Immature female albino rats do not ovulate sponataneously and do not
show cyclic changes of the vaginal epithelium .
• Priming with HCG induces follicular maturation , followed by
sponataneous ovulation 2 days later .
• Injection of anti ovulatory drugs, prior to induction procedure will prevent
ovulation .
• This principle is used for the screening of anti-ovulatory agents
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PHARMACOLOGY

Procedure :
Immature female albino rats 24-26 days of age are used for the experiment .
The animals are treated with various test drugs in a different dose levels .
After the administration of the test drug ,HCG is given exogenously for
ovulation.
After 2 days ,animals are sacrificed ,ovaries are dissected out ,preserved in
10% buffered formalin and subjected to histopathological evaluation .
The results are compared with the control group. 15

2) Cupric acetate-induced ovulation in rabbits :
Rationale :
 The rabbits are reflex ovulators .They ovulate within a few hours after
mating or after mechanical stimulation of vagina or sometimes even
prescence of males ,or administration of certain chemicals like cupric
acetate .
 The rabbit ovulates within a few hours after injection of cupric acetate
(0.3mg/kg i.v of 1 %cupric acetate in 0.9 saline ) .
 Injection of anti ovulatory drugs ,24 hours before the induction procedure
prevents ovulation
Procedure :
Sexually mature female rabbits, weighing 3-4 kg are used for the study .
Animals are kept in isolation for at least 21 days to ensure ,they are not
pregnant and to prevent the induction of ovulation by mating .
They are treated with test drug and 24 hours later cupric acetate is given .The
rabbits are sacrificed and ovaries are examined 18-24 hrs later .
The total number of ovulation points on both ovaries are recorded for each
animal the ovaries and uterus are excised out and preserved in 10% buffered
formalin and subjected to histopathological evaluation .
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PHARMACOLOGY

Oestrogenic activity
In vivo Methods :
1) vaginal opening :
 This assay is based on the principal that vaginal opening occurs in
immature female albino mice & rats by treating with Oestrogenic
compounds . The sign of complete vaginal opening is observed as a sign of
Oestrogenic activity.
Procedure :
Immature female animals ( 18 day old mice , 21 day old mice rats) are used for
the study.
The test & standard drugs are administered to the animals intramuscularly in
cotton seed oil . The time of complete vaginal opening can be observed as a
sign of Oestrogenic activity
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PHARMACOLOGY

Ovariectomy
• The animals are slightly anesthetized with ether.
• A single transverse incision is made in the skin of the back.
• That incision can be shifted readily from one side to the other, so as to lie
over each ovary in turn.
• A small puncture is then made over the site of the ovary, which can be seen
through the abdominal wall, embedded in a pad of fat.
• The top of a pair of fine forceps is introduced and the fat around the ovary
was grasped, care being taken not to rupture the capsule around the ovary
itself.
• The tip of the uterine horn is then crushed with a pair of artery forceps and
the ovary together with the fallopian tube is removed with a single cut by a
pair of fine scissors.
• Usually no bleeding is observed. The muscular wound is closed by
absorbable sutures and outer skin wound is closed by nylon suture.
PHARMACOLOGY
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Ovariectomy
PHARMACOLOGY
19

Four-day Uterine Weight Assay
• This assay is based on the observation that estrogens cause an increase in
protein synthesis and thus bring about an increase in uterine weight. A peak
in uterine weight is observed about 40 hours.
Procedure:
• Immature or adult ovariectomized albino mice or rats can be given test drug
intramuscularly in cotton seed oil for three consecutive days.
• On the fourth day animals are killed by cervical fracture, the uteri are
rapidly excised, and the uterine contents are gently squeezed out (results
are unreliable if the uterine contents are not removed).
• The uteri are weighed immediately in the wet state.
• The uteri may be dehydrated in an oven at 100 C for 24 h and reweighed to
obtain the dry weight increase.
• The log dose is plotted against the wet weight, produces a sigmoid curve,
and the ED, can be determined for comparison of the test compound with
estradiol.
PHARMACOLOGY
20

Vaginal cornification
Rationale :
 This assay is based on the fact that rats and mice exhibit a cyclical
ovulation with associated changes in the
secretion of hormones ,this lead to the changes in the vaginal epithelial
cells.The oestrus cycle is classified into
1. Proestrus.
2. Estrus.
3. Metestrus.
4. Diestrus .
 Drugs with Oestrogenic activity change the animal into estrus stage
21
PHARMACOLOGY

Procedure
Adult female albino rats having regular estrus cycle are used for the study.
Animals are treated with various test and standard drugs.
Changes in the vagina can be observed by taking vaginal smears and
examining these for cornified cells, leukocytes and epithelial cells in the
normal animals and treated animals twice daily over a period of 4 days.
The drug, which changes the animals into estrus stage skipping other stages, is
considered to have estrogenic activity.
PHARMACOLOGY
22

Stage of estrus cycle in rat
•Post ovulatory
stage.(21hrs)
•Declinine(57hrs)
•Period of heat or sexual
receptivity(12hrs)
•Beginning
cycle(12hrs)
proestrus
estrus
metestrus
diestrus
23
PHARMACOLOGY

PHARMACOLOGY
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In vitro methods
• Estrogenic Receptor-binding Assay
• Estrogenic receptor binding assay uses the principle of competitive binding of
labeled and unlabeled estrogen on the estrogenic receptors.
Procedure: Cytosol preparation:
Uteri from 18-day old female albino mice are removed and homogenized at 0°C in 1:50
(w/v) of Tris-sucrose buffer in a conical homogenizer.
Human endometrium from menopausal women is frozen within 2 hours of
hysterectomy and stored in liquid nitrogen until use also can be used.
The frozen endometrium is pulverized and homogenized in 1:5 (w/v) of Tris-Sucrose
buffer. Homogenates are centrifuged for 1 h at 1,05,000 g.
Determination of specific binding in mouse uterus cytosol as a function of steroid
concentration incubation time and temperature.
Triplicate aliquots of 125 ml of cytosol are incubated with 5 or 25 nM labeled steroid
either for 2 or 24 h at 0°C or for 2 or 5 hours at 25°C in the absence (total binding) or
presence (non-specific binding) of a 100 fold excess of radio inert steroid. Bound
steroid is measured by Dextran coated charcoal (DCC) adsorption.
PHARMACOLOGY
25

Dextron-coated charcoal (DCC) adsorption
technique
• A 100 µl aliquot of incubated cytosol is stirred for 10 minutes at 0°C
in a micro titer plate 100 ul of DCC 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 ul Aliquot of supernatant.
• Potency Assay:
This assay determines the affinity of the test compound for estrogen
receptor sites in the uterus (rats, rabbits, mice).
PHARMACOLOGY
26

Procedure:
Four immature female mice (20 days old) are killed. The uteri are quickly
excised and are placed in Krebs'-Ringer phosphate buffer.
Pieces of diaphragm are taken from each animal to serve as control tissue for
nonspecific uptake of estradiol.
The uteri are dividedthe cervix into two horns; in this way one horn is used as
the control and the other for testing the compound.
The tissues are placed in vials containing 5.0 ml of Krebs-Ringer phosphate
buffer, incubated, and shaken at 37°C with 95% oxygen and 5% carbon
dioxide is bubbled through.
The radiochemical purity of the H-estradial can be checked chromato
graphically. Buffer solution of radioactive estradiol is made up so that each
5ml of buffer contains 0.0016 ug of radioactive estradiol (0.25 µCi).
A stock solution can be made and kept refrigerated for up to 6 weeks.
PHARMACOLOGY
27

The excised tissues are treated as follows:
. Control: Four pieces of diaphragm are incubated and shaken with 5 ml of
buffer solution for 15 minutes at 37°C and are then shaken for 1h with 5 ml of
buffer containing the radioactive estradiol and 2% w/v bovine albumin.
Experimental: Four uterine horns are incubated and are shaken in 5 ml of
buffer at 37-C for 15 minutes. Then they are incubated and shaken with 5 ml
of buffer containing 2% of albumin and radioactive estradiol at 37°C for 1
hour. Both control and experimental tissue are removed and washed with
buffer at 37°C for 5 minutes, kept in damped filter paper, and weighed. The
tissues are then prepared for counting. Samples of 100 µl of the incubation
solution are also taken for counting.
PHARMACOLOGY
28

Treatment of tissue for counting
PHARMACOLOGY
29
• The tissues are dried for constant weight and the dry weight is recorded.
Each piece of tissue is placed in a glass counting vial and incubated at 60°C
in a shaking water bath with 0.5 ml of hyamine hydrochloride 10x until the
tissue has completely dissolved.
• If the solution is discolored 50 µl of 20% hydrogen peroxide may be added
50 µl of concentrated HCl and 15 ml of phosphor solution are added to each
vial.
• The vials are allowed to equilibrate in the packed liquid scintillation counter
and counts are taken. Counting efficiency is determined by the addition of
an internal standard.
• The results are expressed as disintegrations per minute per unit of wet
weight (dpm/mg).

In vitro methods
Aromatase Inhibition-This assay is based on the principle
that compounds which inhibit aromatase (estrogen synthase)
possess anti-estrogenic activity
Procedure:
Ovarian tissue from adult Golden hamsters is used.
Estrus cycle is monitored for at least 3 consecutive 4 days estrus cycle prior to
the experiment.
The experiments for evaluating inhibitor effects are performed with ovaries
obtained from animals sacrificed on day 4 (pro-estrus).
The ovaries are excised freed from adhering fat tissue and quartered. The
quarters are transferred into plastic incubation flasks with 2 ml of Kreb's
Ringer bicarbonate salt (KBR) solution pH 7.6 containing 8.4 mM glucose.
The flasks are gassed with O₂/CO₂ (95%/5%) tightly closed and placed in a
shaker/water bath (37°C) for incubation of the fragments.
PHARMACOLOGY
30

PHARMACOLOGY
31
The incubation media are replaced with fresh KBR after pre incubation for 1
hour. The ovaries are further incubated for 4 h in the presence or absence of
inhibitors.
4 OH androstendione is used as standard in concentrations between 0.33 and
330 µM/L. At the end of the experiment the incubation media are removed
and centrifuged.
In the supernatant estrogen, progesterone and testosterone are determined by
radioimmunoassays. The data of control and test groups are compared with
suitable statistical analysis

Progestational Activity :
In vivo methods :
Proliferation of uterine endometrium in oestrogen primed rabbits
(Clauberg Mcphail test)
Principle :
• Female rabbits weighing between 800-1000g are primed with
estradiol and followed by the administration of progestational
compound leading to the proliferation of the endometrium and
converted into secretory phase .
32
PHARMACOLOGY

Procedure :
Female rabbits weighing 800- 1000g are primed with injection of estradiol
0.5mcg/ml in aqueous solution daily on day 7 treatment is begun .
The total dose is given in 5 equally divided fraction daily over 5 days .
24hrs after the last injection , animals are killed and uteri are dissected out and
frozen sections of segment of middle portion of one horn is prepared and
examined for histological interpretation .
For interpretation of progestational proliferation of endometrium , beginning
of glandular development may be graded 1 & endometrium consisting only
of glandular tissue may be graded 4
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PHARMACOLOGY

Progesterone Receptor-binding Assay
progesterone receptor binding assay uses the principle of competitive
binding of labeled and unlabeled progesterone on the progesteronic receptors.
Procedure:
Human uteri obtained after hysterectomy is frozen in liquid nitrogen and
stored at-80°C until use.
For cytosol preparation uterine tissues are minced and homogenized with a
homogenizer at 0-4°C in ice-cold buffer composed of 10 mM KH.PO, 10 mM
KHPO, 1.5 M EDTA, 3 mM NaN, 10% glycerol, pH 7.5 (PENG buffer).
The homogenates are then centrifuged at 10,500 g at 4°C for 30 minutes. The
supernatant is taken as cytosol.
PHARMACOLOGY
34

PHARMACOLOGY
35
The cytosol preparations are incubated with 'H-R5020 as radioligand at a
concentration of 8 nmol/L and increased concentrations (1 x 10 to 1 x 10
mol/L) of the competitor steroid overnight at 4°C.
Then unbound steroids are adsorbed by incubating with 0.5 ml of DCC
(0.5% Norit A, 0.05% dextran T400 in PENG buffer) for 10 min at 4°C.
After centrifugation (10 min at 1,500 g at 4°C) 0.5 ml of the supernatant is
withdrawn and counted for radioactivity.
To calculate the 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 unlabeled competing steroid.

Anti-progestational activity
• The anti-progestational compound inhibits some or all the physiological
effect of progesterones. This principle is used to screen the anti-
progestational activity.
• The procedure for assay of Clauberg/McPhail and deciduoma formation is
followed except that the test compounds are given along with the
progesterone.
• Anti-progestational Activity in Immature Rabbits (Mc Ginty Test) Mc
Ginty method used to determine the local progestational activity of the test
drugs. It determines the degree of endometrial proliferation and
transformation in immature rabbits initially primed with estradiol and
subsequently treated with the test substances.
PHARMACOLOGY
36

Procedure:
Local progestational test involves a direct injection of progesterone into a
uterine segment.
The test is performed in immature rabbits (700-950 g) primed for 6 days with
estrogen.
Estradiol at a dose of 1 µg/kg/ rabbit is injected by subcutaneous route to the
rabbits of positive control and test groups, 16 The rabbits of negative control
group received only the vehicle (0.5% CMC) by subcutaneous route for 6
days.
On the 7th day, the rabbit is anesthetized by intramuscular injection of
ketamine hydrochloride (Dose 35 mg/kg).
PHARMACOLOGY
37

The abdomen is openest and the uterus is exposed by laparotomy
The upper middle segment of nach horn is ligated without disturbance of blood
circulation. In the positive control group.
0.1 ml of progesterone was injected alone in the right uterine horn (Conc. 0.1
mg/ mb." In the opposite horn, only 0.1 ml of the vehicle is injected (05 %
CMC).
In the test groups, solution of the progesterone and the test drugs in 0.5%
C.M.C is injected into the left lumen of segment through the lower ligature,
which is drawn tight after the injection. Progesterone is injected alone in the
right uterine horn In the negative control group, progesterone alone is injected
in the right uterine horn. In the opposite horn, only vehicle is injected.
After the injection, the lower ligature can be tightened
Three days later, the animals were sacrificed. The uteri were excised,
weighed, fixed in buffered formalin The sections of the horn are evaluated
histologically according to Mc Phail
PHARMACOLOGY
38

Scores
• Score 0-Ramification of the uterine mucosa but no proliferation (estrogen
treatment only)
• .Score 1-Slight proliferation of the uterine mucosa
• Score 2- Medium proliferation of the uterus mucosa, slight additional
ramification.
• Score 3- Pronounced proliferation of the uterine mucosa.
• Score 4- Very pronounced, proliferation of the uterus mucosa, pronounced
proliferation of the uterus mucosa, pronounced ramification.
• The scores from each dosage group are averaged.
PHARMACOLOGY
39

Method for Males:
• In vitro – Spermicidal activity.
• In vivo – fertility and Cohabitation test.
• Androgenic activity-
• Anti –androgenic activity-in vitro chicken comb method.
PHARMACOLOGY
40

In vivo methods
Cohabitation Test
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.
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.
PHARMACOLOGY
41

Fertility Test
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 smears are examined for the presence of sperms.
All females passed through lestrus cycle must have mated.
The mated animals are kept separately till the completion of the gestational
period.
The litters are counted and using the following formula average litter size is
calculated.
Average litter size =Total no. of litter
No. of females mated
PHARMACOLOGY
42

In vitro method
Spermicidal Activity
Procedure:
Spermicidal drugs are diluted with normal saline and serial dilutions are
made in 0.2 ml of human seminal fluid with 1ml of spermicidal solution.
Then the mixture is incubated at 37°C for 30 minutes. A drop of the
mixture is placed immediately on a slide and at least five fields were
microscopically observed under high power (x400) for assessment of sperm
morphological changes and motility.
Effective agents can immobilize and kill the sperms.
PHARMACOLOGY
43

Immobilization Assay
Procedure:
The cauda portion of epididymes of ram is isolated and minced in 0.9 % saline
solution (pH 7.5) and filtered through a piece of cheese cloth to get sperm
suspension.
For human sample, ejaculates (n=10) from normal subjects after 72-96 h of sexual
abstinence are subjected to routine semen analysis following liquefaction at 37°C.
Sperm count above 100 million/ml and viability above 60 % with normal
morphology, rapid and progressive motility is employed for the test.
Ram epididymal sperm suspension (100 million/ml to 200 million/ ml) or human
ejaculate (100 million/ml to 150 million/ml) is mixed thoroughly in 1:1 ratio with
different concentration of drugs.
A drop of the mixture is placed immediately on a slide and at least five fields
were microscopically observed under high power (x400) for assessment of sperm
motility. The mixture is then incubated at 37°C for 30 minutes and the above
process is repeated.
PHARMACOLOGY
44

Androgenic activity
In vivo methods
Chicken comb method
PURPOSE AND RATIONALE
This classical bioassay based on growth of the capon comb has been used
by many authors for androgenic activity and found to be extremely useful for
the isolation and structural elucidation of natural androgens.
PROCEDURE
Prior to assay, the surface area (sum of the length plus height of each
individual comb) is determined by a millimeter rule placed directly on the
comb.
The capons are injected daily intramuscularly for 5 consecutive days with a
solution or suspension of the test compound or the standard in 1 ml olive oil.
PHARMACOLOGY
45

Cont..
Twenty-four hours after the last injection, the combs are re-measured and the
growth of the comb is expressed as the sum of length and height in
millimeters.
Groups of 8 animals are used for at least 2 doses of the test compound and the
standard.
EVALUATION
The mean values of each group are calculated and plotted as dose-response
curve for the test compound and the standard in order to calculate potency
PHARMACOLOGY
46

Chick comb method
PURPOSE AND RATIONALE
This description is purely of historical interest. Several modifications of
the chick comb method were described for the androgen as well as the anti-
androgen applied either systemically or locally.
PROCEDURE
One- to 3-day old male or female White Leghorn chicks are housed at constant
temperature in a heated incubator. Testosterone is incorporated into the finely ground
chick starting mash at a concentration of 80 mg per kilogram of food.
The chicks are placed on this diet on day one. The test compound is dissolved in
sesame oil. Each day for 4 days 0.1 ml of the oil solution is injected subcutaneously.
Control chicks receive onlythe vehicle. Groups of 10 chicks are treated with various
doses of the test compound or the standard.
SCREENING METHODS IN
REPRODUCTIVE PHARMACOLOGY
47

• Twenty four hours after the last injection, the animals are sacrificed, the
combs removed and, after blotting of the cut edge, weighed rapidly to the
nearest 0.5 mg. Body weights are also determined.
EVALUATION
The results are either expressed as absolute com weights or as mg of comb
per g of body weight. Results of groups treated with various doses of the
inhibitor are statistically compared with controls receiving the vehicle only
PHARMACOLOGY
48

References
 Drug discovery and evaluation pharmacological assays second edition 2nd
edition /Wolfgang H.Vogel Bernward A. Schölkens, Jürgen Sandow, Günter
Müller ,Wolfgang F. Vogel.
 Drug screening and methods / SK Gupta / Jaypee publishers/antifertility
AGENTS.
 MEDICAL PHARMACOLOGY by PADMAJA UDAYKUMAR.
49
PHARMACOLOGY

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