BP503T Unit 4-6

Study Material
B.Pharm 5th Semester
Sub: PHARMACOLOGY-II (Theory)
Sub. Code: BP503T
UNIT-IV
Topic: 1. Pharmacology of drugs acting on endocrine system
Chapters:
a. Basic concepts in endocrine pharmacology.
b. Anterior Pituitary hormones- analogues and their inhibitors.
c. Thyroid hormones- analogues and their inhibitors.
d. Hormones regulating plasma calcium level- Parathormone, Calcitonin and Vitamin-D.
d. Insulin, Oral Hypoglycemic agents and glucagon.
e. ACTH and corticosteroids.
Prepared by:
Dr. Bimalendu Chowdhury, M.Ph, PhD.
Professor, Roland Institute of Pharmaceutical Sciences, Berahampur
Teacher Regd. No:T080326711

a. Basic concepts in endocrine pharmacology
The word endocrine implies internal secretions poured directly into the blood; hence the
endocrine glands are also called the ductless glands. The chemicals secreted by the endocrine
glands are called hormones. Chemically hormones may be peptides, steroids, amines or
derivatives of amino acids.
Characteristics of hormones:
1. Act in very low concentration
2. Synthesized and stored in glands (except posterior pituitary hormones)
3. Produce their effect at the target organ far away from the gland
4. Have a regulatory role on the physiological functions
5. Rapidly destroyed after their functions are over
Table.1. Endocrine glands, their hormone and their chemical nature
Endocrine gland Hormone secretions

1. Pituitary (Anterior) Growth hormone (GH), Prolactin
Thyroid Stimulating Hormone (TSH)
Follicle Stimulating Hormone (FSH)
Leutenizing Hormone (LH)
Adrenocorticotropic Hormone (ACTH)
Pituitary (Posterior) Oxytocin, Vasopressin
2. Thyroid Tri-iodo-thyronine (T3), Thyroxin (T4), Calcitonin
3. Parathyroid Parathormone
4. Adrenal gland Glucocorticoids, Mineralocorticoids, Sex-steroids (estrogen,
progesterone and androgens), Adrenaline
5. Pancreas Gluocagon, Insulin, Somatostatin
6. Testis Testosterone
7. Ovary Estrogen, Progesterone
1
Mechanism of action of hormones:
Many hormones bind to their specific receptors that may be nuclear or on the cell surface of the
target cells and produce two effects:
1. Activation of genome and increase their protein synthesis

2. Activation of second messenger like cyclic AMP
b. Anterior Pituitary hormones- analogues and their inhibitors
1. Growth hormone:
Growth hormone (GH) or somatotropic hormone (STH) is secreted by acidophil cell of anterior
pituitary gland. It is a polypeptide with 191 aminoacids residue. The secretion of GH from the
anterior pituitary is controlled by hypothalamus through GH releasing factor.
Mechanism of action:
GH bind to growth hormone receptor located on cell surface and induces dimerization of a
protein janus kinase 2 (JAK2). This JAK2 undergo autophosphorylation and activates signaling
pathways involving various kinase, finally affects gene expression in the nucleus and increase
synthesis of various proteins.
Functions of GH:
1. Increase body growth

2. Protein metabolism
3. Fat metabolism
4. Carbohydrate metabolism
Hypersecretion of GH in the young causes “Gigantasim” and in adult it causes “acromegali”.

Hyposecretion of GH in young causes “Dwarfism”.
GH analogues:
There are three types of GH analogues:

1. Somatropin: DNA recombinant GH
2. Somatrem: Derivative of GH
3. Sermorelin acetate: Synthetic form of GH releasing factor
Use: These are used for replacement therapy in GH deficient children and for patients with
Turner’s Syndrome to improve adult height.
Adverse effect: Intracranial hypertension, headache, nausea, vomiting and type-2 diabetes
2
GH inhibitors:
These are somatostatin analogues like Octeretide, Lanreoptide and Valpreotide, these analogues
decrease GH secretion. GH antagonist like Pegvisomant have been developed for the treatment
of acromegali.
Adverse effect: GI adverse effect like diarrhea, nausea, abdominal pain and gall stone
2. Prolactin:
It is a peptide hormone secreted by acidophilic cells of anterior pituitary. It is responsible for

lactation in the post partum state of women. The secretion of prolactin is also controlled by
hypothalamus through prolactin releasing factor.
Prolactin inhibitor:
1. Pergolide and Cabergoline: These are ergot alkaloids.

2. Bromocriptine: A semisynthetic ergot alkaloid
3. Quinagolide: It is a non ergot
These are dopamine D2 receptor stimulant and used in the treatment of hyperprolactinemia,
galactorrhoea and purperal lactation. It also induce ovulation and permits a women to become
pregnant.
c. Thyroid stimulating hormones- analogues and their inhibitors
It is a glycoprotein released from the anterior pituitary and its secretion is controlled by
hypothalamus through TSH releasing factor. TSH controls the activity of thyroid gland by
regulating the uptake of iodide by the gland. It also affects the enzymes involved in the various
stages of synthesis and release of the thyroid hormones. The blood level of TSH can be assessed
by radioimmunoassay. The main use of TSH is as a diagnostic agent to differentiate primary
hypothyroidism and secondary hypothyroidism.
Synthesis and release of thyroid hormones:
The synthesis and release of thyroid hormones from the thyroid gland takes place by following
steps:
1. Uptake of iodide
2. Oxidation of iodide to free iodine and iodination of tyrosine
3. Formation of thyroxine (T4) and triodothyronine (T3) by coupling of iodotyrosines
4. Proteolysis of thyroglobulin and release of T4 and T3
5. Peripheral conversion of T4 to T3 by deiodination
3
Fig.1. Biosynthesis and release of T3 and T4
1. Iodide trapping 2. Oxidation of iodide 3. Coupling of iodotyrosines

4. Proteolysis of thyroglobulin (Mit= Monoiodotyrosine; Dit= Diodotyrosine)
Function of Thyroid hormones:
1. Increase calorigenesis and raise basal metabolic rate (BMR)

2. Increase carbohydrate and protein metabolism
3. Hypolipidemic effect
4. Increase heart rate, contractility and cardiac output
5. Increase calcium mobilization from bone
6. Normal development of nervous system
7. Essential for normal body growth and development
Deficiency of thyroid hormones called hypothyroidism, the disease associated with

hypothyroidism is called myxoedema. Excess secretion of thyroid hormones is called
hyperthyroidism, it occurs in two major forms; (i) Diffuse toxic goiter (Graves’ disease) and (ii)
Toxic nodular goiter (Pulmmer’s disease)
Thyroid hormones analogues:
1. Levothyroxine: This is a synthetic thyroxine (T4), readily absorbed from the gut and is
the drug of choice for management of hypothyroid disorder. Average dose is 150 mcg
daily.
4
2. Liothyronine: This is a synthetic triodothyronine (T3), it has rapid onset and short
duration of action. It is the drug of choice in an emergency situation like myxoedema
coma. Dose 5 to 10 mcg daily orally.
3. Liothrix: It is 4:1 mixture of synthetic T4 and T3. It is orally active.
Anti-thyroid drug or thyroid hormone inhibitor:
1. Inhibitors of thyroxine synthesis: Propylthiouracil, Methylthiouracil, Methimazole,

Carbimazole
2. Drug that destroy thyroid tissue: Radioactive iodine (I131)
3. Drugs with uncertain mode of action: Potassium iodide, Sodium iodide, Lugol’s solution
1. Propylthiouracil, Methylthiouracil, Methimazole, Carbimazole: These are thioamide, inhibits

the synthesis of thyroid hormones. The antithyroid effect may range from several days to
weeks. When thyroxine synthesis is diminished, the secretion of TSH is increased, this leads
to thyroid hyperplasia.
MOA: The thioamides inhibit the formation of thyroxin mainly by interfering with: (i) the
iodination of tyrosine and (ii) the coupling and condensation of iodotyrosine.
Adverse effect: Goitrogenic action, allergic reactions, leucopenia, and agranulocytosis
Therapeutic uses: Hypothyroidism (Graves’ disease)
Contraindication: Use should be minimized in pregnancy and lactating mother
2. Radioactive iodine (I131): I131 is mostly as antithyroid drug and accumulates in the thyroid
gland, emits beta and gama radiation.
MOA: It emits beta and gama radiation, the biological activity is attributed to ionizing beta
radiation, which destroy the thyroid cells within few weeks.
Adverse effect: Hypothyroidism, bone marrow depression, thyroid carcinoma and chromosomal
abnormalities.
Therapeutic uses: Selected cases of hyperthyroidism, thyroid carcinoma
Contraindication: Pregnancy and lactation
3. Iodide: It is used in the treatment of iodine deficiency goiter but paradoxically, if iodide is
administered to hyperthyroid patients, there is a reduction in the vascularity and swelling of
glands. The gland shrinks and this response reaches its maximum in 10 to 15 days.
Therapeutic uses: Used to prepare hyperthyroid patients for surgery
5
d. Hormones regulating plasma calcium - Parathormone, Calcitonin and Vitamin-D
Calcium metabolism in the body is mainly controlled by two hormone, parathormone from the
parathyroids and calcitonin from the parafolicular cells of the thyroid gland. In addition, vitamin-
D also regulate plasma calcium level.
1. Parathormone (PTH): It is a large polypeptide hormone containing 84 amino acids

secreted from the parathyroid gland present posterior to the thyroid gland. Release of PTH is
stimulated by a fall and inhibited by a rise in the ionized Ca+2 level in plasma.
Action: Produce hypercalcemia by:
(i) Bone: PTH increase resorption of bone by promoting osteoclastic activity and decreasing
osteoblastic activity.
(ii) Kidney: PTH decrease the calcium and phosphate clearance
(iii) Intestine: Promote intestinal calcium absorption in presence of Vit-D
Therapeutic use: Parathyroid injection 20 to 40 USP units twice daily subcutaneously or

intramuscularly used for the early control of tetany due to hypoparathyroidism.
Adverse effect: Over dose with PTH causes hypercalcaemia, vomiting, diarrhea. Prolonged use
of PTH can leads to demineralization of bone and metastatic calcification in the kidney.
2. Calcitonin: Calcitonin is a small polypeptide hormone with 32 amino acids, synthesized

and secreted by the parafollicular cells (C cells) of the thyroid gland. Calcitonin release is
regulated by the ionized calcium concentration in the blood. Hypercalcaemia promotes its
release and vice versa.
Action: Produce hypocalcemia by:
(i) Bone: Calcitonin inhibit bone resorption by decreasing osteoclastic activity and
increasing osteoblastic activity
(ii) Kidney: Promoting urinary excretion of calcium and phosphate
Calcitonin Preparation: Synthetic compounds that resemble the polypeptide hormone is salmon
calcitonin and human calcitonin.
Therapeutic Use: Salmon calcitonin 40 to 160 units daily, sc or im or iv and human calcitonin in
a dose of 0.5 mg daily is used in hypercalcemic state, Paget’s disease.
Adverse effect: Mild hypocalcemia
3. Vitamin-D: Vitamin-D designated as a group of related sterols (contain cyclopentano-

perhydrophenanthrene ring). It has more than 6 components, of them Vit-D2 and D3 are most
potent. Vit D2 (ergocalciferol) an isomer of ergosterol and Vit-D3 (cholecalciferol) are
sources for Vit-D activity and are referred as provitamins. Both cholecalciferol and
ergocalciferol are the substrate for both 25-hydroxylase enzyme of liver and α-hydroxylase
enzyme of kidney to form 25(OH)D3 (Calcifediol) followed by 1,25(OH)2 D3 (calcitriol).
This conversion step is modulated by PTH.
6
Source: The source of Vit-D2 (ergocalciferol) is vegetable where as Vit-D3 (cholecalciferol) is
produced in the skin from 7-dehydrocholesterol by the action of UV light from the sun and also
from egg, milk, fish.
Action of calcitriol:
(i) Bone: Maintains the store of calcium in the mitochondria, thus is essential for PTH-
induced bone resorption
(ii) Kidney: Increase calcium reabsorption
(iii) Intestine: Promote the absorption of calcium
Deficiency: Vit-D deficiency gives rise to rickets in children and osteomalacia in adults.
Therapeutic use: Treatment of rickets and osteomalaia
Adverse effect: Hypervitaminosis-D, hypercalcaemia, hyperphosphataemia and metastatic

calcification.
e. Insulin, Oral Hypoglycemic agents and glucagon
The pancreas has both endocrine and exocrine functions. The exocrine secretions are mostly
the digestive enzymes such as amylase, trypsine, chemotrypsine and lipase.The pancreas
contains islets of Langerhans. The islets of Langerhans contain four types of secretory cells:
(i) Αlpha cells-secrets glucagon
(ii) Beta cells-secrets insulin
(iii) Delta cells-secrets somatostatin and
(iv) PP (F) cells-secrets digestive enzymes
1. Glucagon: It is derived by the proteolytic cleavage of proglucagon (a large peptide). Its

half life is 3-5 min. Decrease blood level of glucose, exercise, high protein diet stimulate
glucagon release. Glucagon raise blood glucose by accelerating breakdown of glycogen into
glucose in liver (glycogenolysis) and conversion of lactates and amino acid into glucose in the
liver (gluconeogenesis), releasing glucose into blood.
Therapeutic uses: To treat severe hypoglycaemia or hypoglycemic coma due to insulin in

patients of type-1 diabetes.
2. Insulin: Insulin is a polypeptide, composed of an A-chain (acidic) made up of 21 amino

acids and a B-chain (basic) of 30 amino acids, linked by two disulphide (-S-S-) bridges and
both A and B-chain is joined by a connecting peptide (C-peptide) composed in man of 31
amino acids.The immediate precursor of insulin in beta cell is proinsulin.
The most important factor controlling insulin secretion is glucose. An increase in blood
glucose promotes insulin secretion from beta cell. The total insulin content of pancreas is about
200 units, normal man secrets about 50 units of insulin per day. Insulin produces its action
through specific insulin receptors which consist of α and β sub unit.
7
Fig.2. Mechanism of insulin secretion
Function of insulin:
1. Carbohydrate metabolism: It stimulates transport of glucose into the cell, increase glycogen
synthesis in muscle and liver, reduce glucogenolysis in liver. The net result is decrease
blood glucose.
2. Protein metabolism: It stimulates protein synthesis, increase amino acid uptake in muscle
and decrease gluconeogenesis in liver.
3. Lipid metabolism: In adipose tissues insulin increases fatty acid synthesis, glycerol
phosphate synthesis and triglyceride deposition. Also decrease lypolysis.
4. Miscellaneous: Insulin prevents ketone body formation.
Fig.2. Action of insulin, It stimulates the formation of glycogen, protein, fatty acid
and triglycerides from their precursors
8
Deficiency of insulin leads to type-1 diabetes mellitus, known as insulin dependent diabetes
mellitus. The treatment for type-1 diabetes mellitus is administration of insulin.
Pharmacokinetics:
Insulin cannot be given orally because it is destroyed in the gastrointestinal tract. Injection of
soluble crystalline insulin is given by subcutaneous injection and is quickly absorbed. Peak effect
achieved quickly and excreted quickly within a few hours. Whereas zinc suspensions, protein
and globulin insulin preparations like semilente, lente and ultralente are absorb slowly. The peak
is reached slowly and sustained, their excretion is also slow.
Table.2. Insulin preparations

Type/Names Onset Peak Duration
(hours) (hours) (hours)
I. Short acting insulin
Crystalline zinc insuline 0.5-1 2 5-8
Prompt zinc insulin suspension 0.5-1 2 5-8
II. Intermediate acting insulin
Insulin zinc suspension (Lente) 1-3 6-8 16-20
Neutral protamine hagedorn 1-2 10-12 16-20
III. Long acting insulin
Insulin glargine 1.5-2 14-16 15-24
Extended insulin zinc crystalline (Ultralente) 4-6 14-16 20-36
Resistance:
Insulin resistance develops in course of time and hence doses have to be increased. This occurs
because of development of insulin antibodies.
Therapeutic use:
1. It is used as the specific replacement therapy in diabetes mellitus.

2. For emergency treatment of diabetic ketoacidosis (diabetic coma)
3. For emergency treatment of hyperkalaemia
Adverse effect: Hypoglycemia, lipodystrophy, allergic manifestations, insulin resistance
3. Oral hypoglycemic agent:
Oral hypoglycemic agents are agents used to treat type-2 diabetes mellitus, they are of two type:
(i) Agent which promote insulin secretion: Sulfonylureas and meglitinides
(ii) Antihyperglycemic agent: Biguanides, Alpha glucosidase inhibitor and Thiazolidinediones
1. Sulfonylureas: These are chemically related to sulfonamides but are deprived of

antibacterial activity. The examples of sulfonylureas are Tolbutamide, Tolzamide,
Chlorpropamide, Glibenclamide, Glipizide, Glyburide. These are readily absorbed from the
9
gastrointestinal tract, appear in the blood within 1-2 hrs and peak levels are attained within 4-6
hrs. They are partially protein bound and metabolized in liver.
MOA: 1. It blocks the ATP dependant K+ channel in the beta cell of the pancreas and cause
degranulation of beta cell to release insulin.
2. Inhibits hepatic glycogenolysis.
Therapeutic uses: (i) Maturity onset diabetes mellitus

(iii) Insulin resistant diabetes mellitus
Adverse effect: Hypoglycemia, allergic skin reaction, bone marrow depression, cholestatic
jaundice, Chlorpropamide may produce disulfiram like reaction.
2. Meglitinides: These are quick and short acting insulin secretion enhancer, examples are
Repaglinide, Nateglinide. Their mode of action is similar to sulfonylureas. Because of their
rapid onset of action, these drugs are administered shortly before a meal to reduce the post-
prandial glucose rise in type-2 diabetic patient.
Adverse effect: Hypoglycemia is a great risk if the meal is delayed or skipped.
3. Biguanides: The examples are: Phenformin and Metformin, it do not lower blood
glucose level in normal non-diabetic person.
MOA: The action of biguanides is extrapancreatic, and does not depend on presence of
endogenous insulin in the body. They stimulate the peripheral utilization of glucose and reduce
the intestinal absorption of glucose. Also lower LDL, VLDL and elevates HDL.
Therapeutic uses: Phenformin is used for the treatment of maturity onset diabetes mellitus and it
may be combined with sulfonylureas.
Adverse effect: Phenformin induce a metallic taste, nausea, anorexia, and diarrhea. Phenformin
may cause ketonuria and lactic acidosis.
4. Αlpha-glucosidase inhibitors: Example of α-glucosidase inhibitor is acarbose and

miglitol. It inhibits intestinal α-glucosidase and inhibits the digestion and absorption of starch
and sucrose from the gut, therefore reduces post-prandial digestion and absorption of
carbohydrate and lower post meal hyperglycemia. Regular use also tends to lower HbA1c,
body weight and serum triglycerides.
Adverse effect: Flatulence, diarrhea and abdominal pain.
5. Thiazolidinediones (Glitazones): Examples are Rosiglitazone, Pioglitazone and

Troglitazone are a newer class of antihyperglycemic agents.
MOA: These are agonist of nuclear receptor called Peroxisome Proliferator-Activated Receptor-
gamma (PPAR-γ). The PPAR-γ receptor is expressed mainly in adipose tissue but also in muscle
10
and liver. Activation of PPAR-γ receptor by glitazones increases lipogenesis and promote uptake
of fatty acid and glucose. It also reduce hepatic glucose output by inhibiting hepatic
gluconeogenesis, promote glucose uptake into muscle by decreasing insulin resistance and
decrease HbA1c level.
Adverse effect: Weight gain, fluid retention, oedema and hepatotoxicity.
f. ACTH and corticosteroids
Corticosteroids are the hormone secreted by the adrenal cortex under the control of ACTH
released from the anterior pituitary into the circulation in response to corticotrophin releasing
hormone (CRH) secreted from hypothalamus.
Table.3. Hormones of adrenal cortex
Sl Layer of adrenal cortex Hormones

1 Zona glomerulosa Aldosterone (Mineralocorticoids)
2 Zona fasciculate Cortisone, cortocosterone ( Glucocorticoids)
3 Zona reticularis Androgen, estrogen and progesterone (Sex hormones)
All the hormones of adrenal cortex have a common basic structure cyclopentanoperhydro-
phenanthrene or steroid ring. All steroid hormones are derived from cholesterol. Cholesterol
under the influence of ACTH is converted into pregnenolone, the precursor of all steroid
hormones. The steroid hormones can be divided into three major groups on the basis of no of
carbon atoms in their structure. These are:
1. The C-21 steroids: Progesterone, glucocorticoids, and mineralocorticoids

2. The C-19 steroids: Androgen
3. The C-18 steroids: estrogen
1. Glucocorticoids: They are classified into:

(i) Natural: Cortisol,
(ii) Synthetic: (a) Short acting e.g. Cortisone, Hydrocortisone
(b) Intermediate acting e.g. Prednisone, Prednisolone
(c) Long acting e.g. Betamethasone, Dexamethasone
Physiological action:
1. Carbohydrate metabolism: Stimulate the formation of glycogen in liver and muscles and
increase gluconeogenesis in liver.
2. Protein metabolism: It increases the rate of deamination and breakdown of tissue proteins
into amino acids. Thus body proteins are lost, increasing nitrogen excretion.
3. Fat metabolism: They act directly to breakdown triglycerides to fatty acid (lipolysis).
However, they also indirectly increase the formation and storage of fat (lipogenesis).
4. Other metabolism: Glucocorticoids have minimal action on mineral metabolism causing
sodium and water retention and increased excretion of K+ and PO4.
11
Pharmacological action:
1. Anti-inflammatory: Glucocorticoids inhibits the inflammatory responses of body tissue to

all kind of noxious stimuli.
2. Immunosupressant: The corticosteroids inhibit antibody formation and antibody reaction.
3. Anti-allergic activity: They suppress immediate hypersensitivity reaction by interfearing
the release of histamine and bradykinins.
Effect on organ system:
1. Cardiovascular system: Corticosteroids exerts positive inotropic effect.

2. Central nervous system: Increases CNS excitability, euphoria, psychosis
3. Gastrointestinal system: Increase secretion of gastric hydrochloric acid, pepsinogen and
pancreatic trypsinogen
4. Skeletal muscle: Excess corticosteroid leads to skeletal muscles weakness and fatigue
rapidly
5. Body growth: Large dose of glucocorticoids retard growth of children
Mode of action: The natural and synthetic glucocorticoids bind to specific intracellular receptors
located on the cytoplasm of the target cells to form steroid receptor complex. The complex is
then entering to the nucleus where it binds to the nuclear acceptor site. In the nucleus the
complex interacts with the DNA and alters transcription and translation of proteins that produce
various pharmacological effects.
Fig.3. Cellular mechanism of glucocorticoids
Therapeutic uses: Primary adrenocortical insuffiency, Rheumatoid arthritis, Allergic disorders,

Bronchial asthma.
12
Adverse effects: Prolong use for more than 2 weeks produce iatrogenic Cushing’s syndrome,
steroid induced glaucoma, adrenal suppression, superinfection.
Contraindications: Peptic ulcer, infection, hypertension, psychosis, diabetes mellitus,

osteoporosis, glaucoma, pregnancy.
2. Mineralocorticoids: Aldosterone is the natural mineralocorticoid in man, but unsuitable

for medical use as it is inactivated when given orally. Fludrocortisone, a synthetic
corticosteroid, is the most often used compound when mineralocorticoid treatment is required.
It is the only compound available for oral use, and is employed for the management of chronic
primary adrenocortical insufficiency and salt losing forms of congenital adrenal hyperplasia.
MOA: It produces its effect by binding to the cytosolic aldosteron receptor, a nuclear receptor.
The drug receptor complex then enters into the nucleus and binds to the specific site of the
DNA and alters transcription and translation of the protein. The protein produces desired
pharmacological activity.
Adverse effect: Oedema, hypertension and hypokalaemia.
Aldosterone antagonist: e.g. Spironolactone is used clinically as a potassium sparing diuretics.
3. Adrenocortical antagonist: e.g. Metyrapone, Aminoglutethamide and Trilostane.
Metyrapone blocks the biosynthesis of corticosteroids by inhibiting the enzyme 11-β-

hydroxylase. It decrease the synthesis of hydrocortisone, corticosterone, and aldosterone. It is
used as a diuretic in case of resistant oedema associated with excess aldosterone.
Aminoglutethamide blocks the conversion of cholesterol to pregnenolone, thus reduce

synthesis of adrenocorticosteroids. It is used in case of adrenocortical malignancy.
Trilostane is a synthetic steroid, a competitive inhibitor of the 3-β-hydroxysteroid

dehydrogenase. It is used for the treatment of Cushing’s disease.
References:
1. Barar FSK. Textbook of Pharmacology. 1st ed. New Delhi (India):S.Chand

Publisher;2013.
2. Goyal RK, Mehta AA, Balaraman R, Burande MD. Elements of Pharmacology. 17th ed.
Ahmedabad (India):B.S. Shah Publisher;2007.
3. K.D.Tripathi. Essentials of Medical Pharmacology. 6th ed. New Delhi (India) Jaypee
Brothers Medical Publishers; 2008.
4. Rang HP, Dale M M, Ritter J M, Flower R J. Rang and Dale’s Pharmacology, Churchill
Livingstone Elsevier; 2008.
13
5th Semester Pharmacology-II (Theory) Unit-V
BP503.T. PHARMACOLOGY-II (Theory)
UNIT-V 07hours
5. Pharmacology of drugs acting on endocrine system
a. Androgens and Anabolic steroids.
b. Estrogens, progesterone and oral contraceptives.
c. Drugs acting on the uterus.
6. Bioassay
a. Principles and applications of bioassay.
b.Types of bioassay.
c. Bioassay of insulin, oxytocin, vasopressin, ACTH,d-tubocurarine,digitalis, histamine
and 5-HT.
Subject note prepared by: Dr. Ghanshyam Panigrahi,

Associate Professor,
Royal College of Pharmacy and Health Sciences,
Berhampur, Odisha
1
5. Pharmacology of drugs acting on endocrine system

A. ANDROGENS AND ANABOLIC STEROIDS
Androgens (Male Sex Hormones):
 Testosterone is the main natural androgen. It is synthesised mainly by the interstitial
cells of the testis, and in smaller amounts by the ovaries and adrenal cortex.
 These are substances which cause development of secondary sex characters in the
castrated male. That testes are responsible for the male characters is known since
prehistoric times.
 Its endocrine function was established by Berthold in 1849. Testosterone was isolated,
its structure worked out and synthesized by the year 1935.
 Androgens are may be natural and synthetic.
Natural androgens:
 Testes of adult male produce 5-12 mg of testosterone daily, a part of which is
converted in extraglandular tissues to the more active dihydrotestosterone; by the
enzyme steroid 5 α-reductase; cholesterol is the starting material.
 Adrenal cortex produces small quantities of dehydroepiandrosterone and
androstenedione which are called 'weak androgens' (potency 1/20 to 1 /30), but are
infact inactive as such and derive their weak activity from partial conversion to
testosterone by peripheral tissues. Adrenals themselves do not produce significant
quantity of testosterone.
 In women ovary produces small quantity of testosterone; this together with that
derived indirectly from adrenals amounts to 0.25-0.5 mg/ day.
 Androsterone is a metabolite of testosterone which is excreted in urine. It has 1 /10
the activity of testosterone.
Synthetic androgens:
 Methyltestosterone and fluoxymesterone are 17-alkyl substituted derivatives of
testosterone which are orally active because of resistance to first pass metabolism, but
have submaximalandrogenic efficacy and potential to cause cholestatic jaundice.
 Other orally active compounds are testosterone undecanoatewhich administered as
oily solution is absorbed through lymphatics bypassing the liver and mesterolone.
 A number of lipidsoluble esters of testosterone have been produced, suitable for
injection in oily vehicle, from which they are absorbed slowly and exert prolonged
action afterdeesterification in the body.
2
Regulation of secretion
 Testosterone is secreted by the interstitial (Leydig) cells of the testes under the
influence of pulsatile secretion of LH from pituitary.
 FSH is mainly responsible for promotion of spermatogenesis intubular (Sertoli) cells.
 While relatively high concentration of testosterone inhibits LH secretion and in time
causes atrophy of interstitial cells, it has only weak inhibitory action on FSH
secretion.
 Estrogens are more potent inhibitors of Gn secretion even in males and it is believed
that the small amount of estradiol produced by testes and that resulting from
conversion of testosterone to estradiol plays a role in feedback inhibition.
 Inhibin, (a protein) produced by Sertoli cells, has strong FSH inhibitory action and
may be mediating the feedback inhibition.
 Testosterone and estradiol act on hypothalamus to reduce GnRH as well as act
directly on pituitary.
 The plasma level of testosterone in adult males ranges from 0.3 to 1 pg/ dl and in
females from 20 to 60 ng/ dl.
Fig.1: Regulation and production of sex steroids in the male

Actions:
1. Sex organs and secondary sex characters(Androgenic)
Testosterone is responsible for all the changes that occur in a boy at puberty:
 Growth of genitals-penis, scrotum, seminalvesicles, prostate.
 Growth of hair-pubic, axillary, beard, moustache,bodyhairandmalepattern
ofitsdistribution.
3
 Thickening of skin which becomes greasy due toproliferation and increased activity
of sebaceousglands.
 Subcutaneous fat is lost and veins lookprominent.
 Larynx grows and voice deepens.
 Behavioral effects are-increased physical vigour,aggressiveness, penile erections.
Male libidoappears to be activated by testosterone directly aswell as by estradiol
produced from testosterone.
 Testosterone is also important for the intrauterinedevelopment of the male
phenotype;relativelylarge amounts are produced by the foetal testesduring the first
half of intrauterine life.
2. Testes:
 Moderately large doses cause testicular atrophy by inhibiting Gn secretion
frompituitary. Still larger doses have a direct sustaining effect and atrophy is less
marked. Testosterone is needed for normal spermatogenesis andmaturation of
spermatozoa. High concentrationof testosterone attained locally in the spermatogenic
tubules by diffusion from the neighbouringLeydig cells stimulates spermatogenesis.
3. Skeleton and skeletal muscles (Anabolic):
 Testosterone is responsible for the pubertal spurt of growth in boys and to a smaller
extent in girls.
 There is rapid bone growth, both in thickness as well as in length. After puberty, the
epiphyses fuse and linear growth comes to a halt. There is evidence now that estradiol
produced from testosterone, and not testosterone itself, is responsible for fusion of
epiphyses in boys as well as girls. Moreover, estradiol appears to supplement the
effect oftestosterone on bone mineralization.
 Testosterone also promotes muscle building, especially if aided by exercise. There is
accretion of nitrogen, minerals (Na,K,Ca,P,S) and water-body weight
increasesrapidly, more protoplasm is built.
 Appetite is improved and a sense of well-being prevails.
4. Erythropoiesis
 Testosterone also accelerates erythropoiesis by increasing erythropoietin production
and probably direct action on haemesynthesis.
Mechanism of action
 In most target cells, testosterone works through an active metabolite,
dihydrotestosterone, to which it is converted locally by a 5α-reductase enzyme.
4
 In contrast, testosterone itself causes virilisation of the genital tract in the male
embryo and regulates LH/ICSH production in anterior pituitary cells.
 Testosterone and dihydrotestosterone modify gene transcription by interacting with
cytoplasmic receptors. The effects are expressed through modification of protein
synthesis.
Preparations:
 Testosterone itself can be given by subcutaneous implantation or by transdermal
patches.
 Various esters (e.g. enanthate and proprionate) are given by intramuscular depot
injection.
 Testosterone undecanoate and mesterolone can be given orally.
Pharmacokinetic:
 If given orally, testosterone is rapidly metabolised in the liver. It is therefore usually
injected.
 Virtually all testosterone in the circulation is bound to plasma protein-mainly to the
sex steroid-binding globulin.
 The elimination half-life of free testosterone is short (10-20 minutes). It is inactivated
in the liver by conversion to androstenedione.
 This has weak androgenic activity in its own right and can be reconverted to
testosterone, although approximately 90% of testosteroneis eliminated as metabolites
rather than the parent compound.
 Synthetic androgens are less rapidly metabolised, and some are excreted in the urine
unchanged.
Unwanted effects:
 Unwanted effects of androgens include eventual decrease of gonadotrophin release,
with resultant infertility, and salt and water retention leading to oedema.
 Adenocarcinoma of the liver has been reported. Androgens impair growth in children
(via premature fusion of epiphyses), cause acne, and lead to masculinisation in girls.
 Adverse effects of testosterone replacement and monitoring for these are reviewed by
Rhoden & Morgentaler (2004).
 Gynaecomastia: may occur, especially in children and in patients with liver disease.
This is due to peripheral conversion of testosterone toestrogens.
Contraindications:
5
 Androgens are contraindicated in carcinoma of prostate and male breast, liver and
kidney disease and during pregnancy (masculinization of female foetus).
 They should be used cautiously in patients who may be adversely affected by fluid
retention-such asCHF, epilepsy, migraine.
Clinical use:
Androgens (testosterone preparations) as hormone replacement in:
 male hypogonadism due to pituitary or testicular disease (e.g. 2.5 mg/day patches)
 hyposexuality following ovariectomy (e.g. 300μg/day patches).
 Testosterone therapy has been shown to improve weakness and muscle wasting in
AIDS patients with lowtestosterone levels.
 Because testosterone levels decline in old age, it has been administered to elderly
males and found to improve bone mineralizationand muscle mass.
Anabolic Steroids:
 These are synthetic androgens with supposedlyhigher anabolic and lower androgenic
activity. Androgens can be modified chemically to alter the balance of anabolic and
other effects.
 The anabolic : androgenic ratio of testosterone is considered as 1; The anabolic
selectivity of thesesteroids is modest with ratios between 1 to 3 inthe rat model, and
probably still lower in man.
 Drugs as anabolic steroids are Nandrolone, Oxyrnetholone, Stanozololand
Methandienone.
 The anabolic effects are similar to that of testosterone and are mediated through the
same receptor as the androgenic effects.
 Such 'anabolic steroids' (e.g. nandrolone) increase protein synthesis and muscle
development, but clinical use (e.g. in debilitating disease) has been disappointing.
 They may be used in catabolic states like: acute illness, sever trauma, major surgery,
etc. are associated with negative N balance. They are used in the therapy of aplastic
anaemia, Osteoporosis, Suboptimal growth in boys,Renal insufficiency and
(notoriously) abused by some athletes.
 Unwanted effects are described above, under Androgens. In addition, cholestatic
jaundice, liver tumours and increased risk of coronary heart disease are recognised
adverse effects of high-dose anabolic steroids.
6
Antiandrogens:
 Both oestrogens and progestogens have antiandrogen activity, oestrogens mainly by
inhibiting gonadotrophin secretion and progestogens by competing with androgens in
target organs.
 Cyproterone is a derivative of progesterone and has weak progestational activity. It is
a partial agonist at androgen receptors, competing with dihydrotestosterone for
receptors in androgen-sensitive target tissues. Through its effect in the hypothalamus,
it depresses the synthesis of gonadotrophins. It is used as an adjunct in the treatment
of prostatic cancer during initiation of GnRH treatment. It is also used in the therapy
of precocious puberty in males, and of masculinisation and acne in women. It also has
a central nervous system effect, decreasing libido, and has been used to treat
hypersexuality in male sexual offenders.
 Danazol is an orally active ethisterone derivative having weak androgenic, anabolic
and progestational activities. Though labelled as an impeded/ attenuated androgen,
because it binds to the androgen receptor and induces some androgen-specific mRNA
production, the most prominent action is suppression of Gn secretion from pituitary in
both men and women leads to inhibition of testicular / ovarian function.It suppresses
gonadal function directly as well byinhibiting steroidogenic enzymes.
 Flutamide is a non-steroidal antiandrogen used with GnRH in the treatment of
prostate cancer.It is having specific antiandrogenic, but no other hormonal activity. Its
active metabolite 2-hydroxyflutamide competitively blocks androgen action on
accessory sex organs as well as on pituitary-increases LH secretion by blocking
feedback inhibition. Bicalutamide is more potent and longer acting congener of
flutamide and is suitable for once daily administration in metastatic carcinoma of
prostate.
 Drugs can have antiandrogen action by inhibiting synthetic enzymes. Finasteride
inhibits the enzyme (5α-reductase) that converts testosterone to dihydrotestosterone,
which has greater affinity than testosterone for androgen receptors in the prostate
gland. Finasteride is well absorbed after oral administration, has a half-life of about 7
hours, and is excreted in the urine and faeces. It is used to treat benign prostatic
hyperplasia.
 Dutasteride is the newer congener of Finasteride inhibits both type 1 and type 2 5α-
reductase and reduces dihydrotestosterone level.
7
Clinical uses of antiandrogens

 Antiandrogens (e.g. flutamide, cyproterone) are used as part of the treatment of
prostatic cancer.5α-Reductase inhibitors (e.g. finasteride) are used in benign prostatic
hypertrophy.
B. ESTROGENS, PROGESTERONE AND ORAL CONTRACEPTIVES:

At puberty, an increased output of the hormones of the hypothalamus and anterior
pituitary stimulates secretion of oestrogenic sex steroids. These are responsible for the
maturation of the reproductive organs and the development of the secondary sexual
characteristics, and also for a phase of accelerated growth followed by closure of the
epiphyses of the long bones. Sex steroids are thereafter involved in the regulation of the
cyclic changes expressed in the menstrual cycle, and are important in pregnancy. A
simplified outline of the inter-relationship of these substances in the physiological control of
the menstrual cycle is given in the following figures.
Fig. 2: Hormonal control of the female reproductive system.

The Graafian follicle (GF) is shown developing on the left, then involuting to form the corpus luteum (CL)
on the right, after the ovum (•) has been released. FSH, follicle-stimulating hormone; GnRH,
gonadotrophin-releasing hormone; LH, luteinising hormone.
Hormonal control of the female reproductive system
 The menstrual cycle starts with menstruation.
8
 Gonadotrophin-releasing hormone, released from the hypothalamus, acts on the

anterior pituitary to release follicle-stimulating hormone (FSH) and luteinising
hormone (LH).
 FSH and LH stimulate follicle development in the ovary. FSH is the main hormone
stimulating oestrogen release. LH stimulates ovulation at mid-cycle and is the main
hormone controlling subsequent progesterone secretion from the corpus luteum.
 Oestrogen controls the proliferative phase of the endometrium and has negative
feedback effects on the anterior pituitary. Progesterone controls the later secretory
phase, and has negative feedback effects on both hypothalamus and anterior pituitary.
 If a fertilised ovum is implanted, the corpus luteum continues to secrete progesterone.
 After implantation, human chorionic gonadotrophin from the chorion becomes
important, and later in pregnancy progesterone and other hormones are secreted by
the placenta.
Estrogens (Oestrogens):
 Estrogensare synthesised by the ovary and placenta, and in small amounts by the testis
and adrenal cortex.
 As for other steroids, the starting substance for oestrogen synthesis is cholesterol.
 The immediate precursors to the estrogens are androgenic substances-androstenedione
or testosterone.
 These are substances which can induce estrus inspayed animals.
Fig. 3: The biosynthetic pathway for the oestrogens, with sites of drug action
Natural estrogens:
 There are three main endogenous estrogens in humans: estradiol, estrone and estriol.
Estradiol is the most potent and is the principal estrogen secreted by the ovary.
 It is synthesized in the graafian follicle, corpus luteum and placenta from cholesterol.
9
 Estradiol is rapidly oxidized in liver to estronewhich is hydroxylated to form estriol.

 All three are found in blood, but estradiol is the most potent estrogen.
 At the beginning of the menstrual cycle, the plasma concentration is 0.2 nmol/l, rising
to ∼2.2 nmol/l in mid-cycle.
 Small quantity (2-20 pg/ day) of estradiol is derived in human males also
fromaromatization of testosterone in the testes andextraglandular tissues.
 In mare, large quantity ofequilin is produced which has 1 /5 estrogenicpotency of
estradiol.
Fig. 4: Structure of natural and synthetic estrogens

Synthetic estrogens:
 Natural estrogens are inactive orally and have a short duration of actiondue to rapid
metabolism in liver.
 To overcomethis, synthetic compounds have been produced:
o Steroidal:Ethinylestradiol, Mestranol, Tibolone.
o Nonsteroidal:Diethylstilbestrol (stilbestrol) Hexestrol, Dienestrol
Regulation of secretion:
 The daily secretion of estrogens in menstruating women varies from 10-100 g
depending on the phase of the cycle.
 Secretion starts from the graafian follicle under the influence of FSH and the blood
level risesgradually during the follicular phase.
 Due to themodest preovulatory FSH surge, estrogens furtherrise transiently.
 After ovulation, corpus luteumcontinues to secrete estrogens till about two daysbefore
menstruation.
 Estrogens exercise feedbackinhibition of FSH (also LH at higher concentrations) by
direct action on pituitary as well asthrough hypothalamus.
 During pregnancy, placenta secretes largequantities of estrogens, reaching a peak of
upto30 mg/ day at term.
 Their level declines sharplyafter delivery.
10
 In the postmenopausal women,daily production of estrogen has been estimatedas 2-10

g-derived primarily by extra glandulararomatization of adrenal androgens.
Actions:
1) Sex organs:
 The estrogens bring about pubertal changes in the female including growthof uterus,
fallopian tubes and vagina. Vaginalepithelium gets thickened, stratified and cornified.
 They are responsible for the proliferation ofendometrium in the preovulatory phase
and it isonly in concert with estrogens that progesteronebrings about secretory
changes.
 Oestrogen acts in concert with progesterone, and induces synthesis of
progesteronereceptors in uterus, vagina, anterior pituitary and hypothalamus.
 Conversely, progesterone decreases oestrogen receptor expression in the reproductive
tract.
 Prolactin also influences oestrogen action by increasing the numbers of oestrogen
receptors in the mammary gland, but has no effect on oestrogen receptor expression in
the uterus.
2) Secondary sex characters
 Estrogensproduced at puberty cause growth ofbreasts-proliferation of ducts and
stroma, accumulation of fat.
 The pubic and axillary hair appear, feminine bodycontours and behaviour are
influenced.
3) Metabolic effects:
 Estrogens are anabolic similar to but weaker than testosterone. Therefore, small
amount of androgen may be contributing to the pubertal growth spurt even in
females.
 Oestrogens have several metabolic actions, including mineralocorticoid (retention of
salt and water) and mild anabolic actions.
 They increase plasma concentrations of high-density lipoproteins, a potentially
beneficial effect that may contribute to the relatively low risk of atheromatous disease
in premenopausal women compared with men of the same age.
 Oestrogens increase the coagulability of blood, and increase the risk of
thromboembolism. This effect is dose-related.
Effects of exogenous oestrogen
11
 depend on the state of sexual maturity when the oestrogen is administered:

o in primary hypogonadism: oestrogen stimulates development of secondary
sexual characteristics and accelerates growth
o in adults with primary amenorrhoea: oestrogen, given cyclically with a
progestogen, induces an artificial cycle
o in sexually mature women: oestrogen (with a progestogen) is contraceptive
o at or after the menopause: oestrogen replacement prevents menopausal
symptoms and bone loss.
 As with other steroids, oestrogen binds 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, blood vessels, heart, CNS and in certain hormone responsive
breastcarcinoma cells.
 There are two types of ER, termed ERα; and ERβ, have been identified, cloned and
structurally characterized. Most tissues express both subtypes but ERα predominates
in uterus, vagina, breast, hypothalamus and blood vessels, while ERβ predominates in
prostate gland of males andovaries in females.
 Binding is followed by interaction of the resultant complexes with nuclear sites and
subsequent genomic effects-either gene transcription (i.e. DNA-directed RNA and
protein synthesis) or gene repression (inhibition of transcription).
 Estradiol binds to both ERαand ERβ with equal affinity, but certain ligandsmay have
differing affinities.
Preparations
 Many preparations (oral, transdermal, intramuscular, implantable and topical) of
oestrogens are available for a wide range of indications.
 These preparations include natural (e.g. estradiol, estriol) and synthetic (e.g.
mestranol, ethinylestradiol, stilbestrol) estrogens.
 Estrogensare presented either as single agents or combined with progestogen.
Pharmacokinetic
 Natural as well as synthetic oestrogens are well absorbed in the gastrointestinal tract,
but after absorption the natural oestrogens are rapidly metabolised in the liver,
whereas synthetic oestrogens are degraded less rapidly.
 Estradiol esters injected i.m. are slowly absorbedand exert prolonged action.
12
 There is a variable amount of enterohepatic cycling, which forms the basis for drug
interaction, because broad-spectrum antibiotic use alters bowel flora and can thereby
render oral contraception ineffective.
 Most estrogens are readily absorbed from skin and mucous membranes. They may be
given topically in the vagina as creams or pessaries for local effect.
 In the plasma, natural oestrogens are bound to albumin and to a sex steroid-binding
globulin.
 Natural oestrogens are excreted in the urine as glucuronides and sulfates.
Clinical use:
 Hormone replacement therapy:
o primary ovarian failure (e.g. Turner's syndrome)
o secondary ovarian failure (menopause) for flushing, vaginal dryness and to
preserve bone mass.
 Contraception
 Prostate and breast cancer
Unwanted effects
 Unwanted effects of oestrogens include tenderness in the breasts, nausea, vomiting,
anorexia, retention of salt and water with resultant oedema, and increased risk of
thromboembolism.
 Used intermittently for postmenopausal replacement therapy, oestrogens cause
menstruation-like bleeding. Oestrogen causes endometrial hyperplasia unless given
cyclically with a progestogen. When administered to males, oestrogens result in
feminisation.
 Oestrogen administration to pregnant women can cause genital abnormalities in their
offspring. Carcinoma of the vagina was more common in young women whose
mothers were given stilbestrol in early pregnancy in a misguided attempt to prevent
miscarriage
Antiestrogens and Selective Estrogen Receptor Modulators (SERMs)
Antiestrogens:
 Antioestrogens compete with natural oestrogens for receptors in target organs.
 Two nonsteroidal compounds clomiphenecitrate and tamoxifencitrate previously
grouped asestrogen antagonists have been in use since 1970s.
 Clomiphene inhibits oestrogen binding in the anterior pituitary, so preventing the
normal modulation by negative feedback and causing increased secretion of GnRH
13
and gonadotrophins. This results in a marked stimulation and enlargement of the

ovaries and increased oestrogen secretion. The main effect of their antioestrogen
action in the pituitary is that they induce ovulation. It is used in treating infertility
caused by lack of ovulation. Twins are common, but multiple pregnancy is unusual.
SERMs:
 The recent discovery of two estrogen receptors (ERs) and that ligand binding could
change their configuration in multiple ways allowing interaction with different
coactivators and corepressors in a tissue specific manner has paved the way for
development of compounds with unique profile of agonistic and antagonistic actions
in different tissues.
 These drugs have been designated 'selective estrogen receptor modulators' and two
new compounds Raloxifene and Ormeloxifene have been marketed. It has been
demonstrated that the conformation of ER after binding tamoxifen or raloxifene is
different fromthat after binding estradiol.
 Tamoxifen has antioestrogenic action on mammary tissue but oestrogenic actions on
plasma lipids, endometrium and bone. It produces mild oestrogen-like adverse effects
consistent with partial agonist activity. The tamoxifen-oestrogen receptor complex
does not readily dissociate, so there is interference with the recycling of receptors.
Tamoxifen up-regulates transforming growth factor-β, decreased function of which is
associated with the progression of malignancy, and which has a role in controlling the
balance between bone-producing osteoblasts and bone-resorbing osteoclasts.Side
effects are hot flushes. vomiting, vaginal bleeding, vaginal discharge, menstrual
irregularities, increased risk of venous thromboembolism,dermatitis, anorexia,
depression and mild leucopenia. It is much less toxic than other anticancer drugs.
 Raloxifene, a 'selective oestrogen receptor modulator', has antiestrogenic effects on
breast and uterus but estrogenic effects on bone, lipid metabolism and blood
coagulation. It is used for prevention and treatment of postmenopausal osteoporosis
and reduces the incidence of estrogen receptor-positive breast cancer, although its role
in therapy of breast cancer is undefined. Unlike estrogen, it does not prevent
menopausal flushes.Side effects are hot flushes, leg cramps are generally mild;
vaginal bleeding is occasional. The only serious concern is 3-fold increase in risk of
deep vein thrombosis and pulmonary embolism.However, similar risk attends
estrogen HRT.
14
 Ormeloxifene a distinct new SERM which acts as estrogen antagonist in breast and
uterus. lt suppresses endometrial proliferation by regulating their ER. Excessive
uterine bleeding with anovular cycles occurring near menopause is normalized.
However, vaginal epithelium and cervical mucus are not altered. It may have
contraceptive property. Ormeloxifene is approved for treatment of dysfunctional
uterine bleeding. Side effects are nausea, headache, fluid retention, weight gain,rise in
BP and prolongation of menstrual cycles.
Clinical use of antiestrogens
 To treat oestrogen-sensitive breast cancer (tamoxifen).
 To induce ovulation (clomiphene) in treating infertility.
Progesterone:
 The natural progestational hormone (progestogen) is progesterone. This is secreted by
the corpus luteum in the second part of the menstrual cycle, and by the placenta
during pregnancy. Small amounts are also secreted by testis and adrenal cortex.
 These are substances which convert the estrogen primed endometrium to secretory
and maintain pregnancy in animals spayed after conception(Progestin = favouring
pregnancy).
Natural progestin:
 Progesterone, a 21 carbon steroid, is the natural progestin and is derived from
cholesterol.
 It is secreted by the corpus luteum (10-20 mg; day) in the later half of menstrual cycle
under the influence of LH.
 Its production declines a few days before the next menstrual flow.
 If the ovum gets fertilized and implants the blastocytes immediately starts producing
chorionic gonadotropin which is absorbed and sustains the corpusluteum in early
pregnancy.
 Placenta starts secreting lots of estrogens and progesterone from 2ndtrimester till term.
Men produce 1-5 mg progesterone per day from adrenals and testis, its role if any, in
males is not known.
Synthetic progestins:
 A number of synthetic progestins with high oral activity have been produced.
 These are either progesterone derivatives (21 C) or 1 9-nortestosterone derivatives
also called estranes(18 C).
15
 The progesterone derivatives are almost pure progestins, have weaker anti-ovulatory
action and are used primarily as adjuvants to estrogens for HRT.
Progesterone derivatives:
 Medroxyprogesterone acetate, Megestrol acetate, Dydrogesterone,
Hydroxyprogesterone caproate and Newer compound- Nomegestrol acetate.
 19- Nortestosterone derivatives:
o Older compounds: Norethindrone (Norethisterone), Lynestrenol
(Ethinylestrenol), Allylestrenol, Levonorgestrel (Gonane).
o Newer compounds: (Gonanes) Desogestrel, Norgestimate, Gestodene
Actions:
 The main function of progesterone is preparationof the uterus for nidation and
maintenance ofpregnancy. The latter is due to prevention of endometrial shedding,
decreased uterine motility and inhibition of immunological rejection of the foetus:
progesterone depresses T -cell function and cell-mediated immunity (CMI).
1. Uterus:
 Progesterone brings about secretory changes in the estrogen primed endometrium:
hyperemia, tortuocity of glands and increased secretion occurs while epithelial
proliferation issuppressed.
 It is lack of progestational supportwhich causes mucosal shedding during
menstruation.
2. Cervix:
 Progesterone converts the watery cervical secretion induced by estrogens to viscid,
scanty and cellular secretion which is hostile tosperm penetration.
3. Vagina:
 Progesterone induces pregnancy like changes in the vaginal mucosa-
leukocyteinfiltration of cornified epithelium.
4. Breast:
 Progesterone causes proliferation ofacini in the mammary glands.
 Cyclic epithelialproliferation occurs during luteal phase, butcontinuous exposure to
progesterone duringpregnancy halts mitotic activity and stabilizesmammary cells.
 Acting in concert with estrogens, it prepares breast for lactation. Withdrawal of these
hormones after delivery causes release ofprolactin from pituitary and milk secretion
starts.
5. CNS:
16
 High circulating concentration of progesterone (during pregnancy) appears to have

asedative effect.
6. Body temperature:
 It causes a slight (0.5 0C) rise in body temperature by resetting the hypothalamic
thermostat and increasing heat production. This is responsible for the higherbody
temperature seen during the luteal phase.
7. Respiration:
 Progestins in relatively higher doses stimulate respiration, as occurs duringpregnancy.
8. Metabolism:
 Prolonged use of oral contraceptives impairs glucose tolerance in some women. This
has been ascribed to the progestational component.
 Progestins, especially those with androgenic activity (19-nortestosterone derivatives)
tend to raise LDL and lower HDLlevels.
 This may reduce the beneficial effect of estrogen used concurrently in HRT or
incontraceptives.
 Micronized oral progesterone formulation (referred to as 'natural progesterone'
introduced recently has been shown not to counteract the beneficial effect of estrogen
on LDLand HDL.
9. Pituitary:
 Progesterone is a weak inhibitor of Gn secretion from pituitary. It decreases the
frequency of LH pulses by action on hypothalamic pulse generator but increases the
amount ofLH secreted per pulse.
 Administration of progestin during follicular phase suppresses the preovulatory LH
surge and prevents ovulation;synergises with estrogen for this action.
 Unlike other steroid receptors, the progesterone receptor (PR) has a limited
distribution in the body: confined mostly to the female genital tract, breast, CNS and
pituitary. The PR is normallypresent in the nucleus of target cells.
 Analogous to ER, upon binding the hormone PR undergoes dimerization, attaches to
progesterone response element (PRE) of target genes and regulates transcription
through coactivators. TheTheantiprogestins also bind to PR, but the conformation
assumed is different from agonist bound receptor and opposite effects are produced by
interaction with corepressors.
17
Pharmacokinetic:
 Injected progesterone is bound to albumin, not to the sex steroid-binding globulin.
Some is stored in adipose tissue. It is metabolised in the liver, and the products,
pregnanolone and pregnanediol, are conjugated with glucuronic acid and excreted in
the urine.
Unwanted effects:
 Unwanted effects of progestogens include weak androgenic actions. Other unwanted
effects include acne, fluid retention, weight change, depression, change in libido,
breast discomfort, premenstrual symptoms, irregular menstrual cycles and
breakthrough bleeding. There is an increased incidence of thromboembolism.
Clinical use of progestogens:
 Contraception:
o with oestrogen in combined oral contraceptive pill
o as progesterone-only contraceptive pill
o as injectable or implantable progesterone-only contraception
o as part of an intrauterine contraceptive system.
 Combined with oestrogen for oestrogen replacement therapy in women with an intact
uterus, to prevent endometrial hyperplasia and carcinoma.
 For endometriosis.
 In endometrial carcinoma; use in breast and renal cancer has declined.
 Poorly validated uses have included various menstrual disorders.
Antiprogestogens:
Mifepristone:
 Mifepristone is a 19-norsteroid with potent competitive antiprogestational and
significant antiglucocorticoid as well as antiandrogenic activity.
 It is a partial agonist and competitive antagonist.It sensitises the uterus to the action of
prostaglandins.
 Given during the follicular phase, its anti progestin action results in attenuation of
midcycle Gn surge from pituitary and slowing of folliculardevelopment and delay/
failure of ovulation.
18
 During the luteal phase, it prevents secretory changes normally brought about by
progesterone.Later in the cycle, it blocks progesterone support to the endometrium,
unrestrains PG release fromit and this stimulates uterine contractions.
 It is given orally and has a plasma half-life of 21 hours.
Clinical use of antiprogestogens
 Mifepristone is used, in combination with a prostaglandin, as a medical alternative to
surgical termination of pregnancy.
 Surgical ripening: 24-30 hours before attemping surgical abortion or induction of
labour, Mifepristone600 mg results in softening of cervix.
 Postcoital contraceptive:Mifepristone 600 mggiveen within 72 hr of intercourse.
 Once-a-month contraceptive: A single 200 mg of mifepristone given 2 days after mid-
cycle each month prevents conception on mostof the occasion.
Oral Contraceptives:
Female Contraception:
 Over 100 million women worldwide are currentlyusing hormonal contraceptives.
 With these drugs, fertility can be suppressed at will, for as long as desired, with
almost 100% confidence and complete return of fertility on discontinuation. The
efficacy, convenience, low cost and overall safety of oral contraceptives (OCs) has
allowed women to decide if and when they will become pregnantand to plan their
activities.
 A variety of oral andparenteral preparations are now available offering individual
choices.
OralContraceptives:
 There are two main types of oral contraceptives:
o combinations of an oestrogen with a progestogen (the combined pill)
o progestogen alone (the progestogen-only pill).
1. Combined pill:
 It contains an estrogen and a progestin. With accumulated experience, it hasbeen
possible to reduce the amount of estrogenand progestin in the 'second generation' OC
pillswithout compromising efficacy, but reducing sideeffects and complications.
'Third generation' pillscontaining newer progestins like desogestrel withimproved
profile of action have been introducedin the 1990s.
19
 Ethinylestradiol 30 g daily isconsidered threshold but can be reduced to 20g/ day if

a progestin with potent antiovulatoryaction is included.
 The progestin is a 19- nortestosterone because these have potent antiovulatory action.
 Used alone the ovulation inhibitory dose (per day) of the currently used progestins is
estimated to be- levonorgestrel 60 g, desogestrel 60 g, norgestirnate 200 g,
gestodene 40 g, but the amount in the pill is 2-3 times higher to attain100%
certainty.
 While both estrogens and progestins synergise to inhibit ovulation, the progestin
ensures prompt bleeding at the end of a cycle and blocks the risk of developing
endometrial carcinoma due to the estrogen.
 One tablet is taken daily for 21 days, starting on the 5th day of menstruation. The next
course is started after a gap of 7 days in which bleeding occurs.Thus, a cycle of 28
days is maintained. Calendarpacks of pills are available.
 This is the mostpopular and most efficacious method.
 The mode of action of combined oral contraceptive pill is as follows:
o oestrogen inhibits secretion of FSH via negative feedback on the anterior
pituitary, and thus suppresses development of the ovarian follicle.
o progestogen inhibits secretion of LH and thus prevents ovulation; it also
makes the cervical mucus less suitable for the passage of sperm.
o oestrogen and progestogen act in concert to alter the endometrium in such a
way as to discourage implantation.
2. Phased regimens:
 These have been introduced to permit reduction in total steroid dose without
compromising efficacy. These are biphasic ortriphasic.
 The estrogen dose is kept constant (orvaried slightly between 30-40 g), while
theamount of progestin is low in the first phase andprogressively higher in the
second and thirdphases.
 Phasic pills are particularly recommended forwomen over 35 years of age or
when other riskfactors are present.
3. Minipill (progestin only pill):
 It has been devised to eliminate the estrogen, because many of the long-term
risks have been ascribed to thiscomponent.
20
 A low-dose progestin only pill istaken daily continuously without any gap.
Themenstrual cycle tends to become irregular andovulation occurs in 20-30%
women, but other mechanisms contribute to the contraceptiveaction.
 The efficacy is lower (96-98%) compared to 98-99.9% with combined pill-
look for pregnancy if amenorrhoea of more than 2 months occurs. This
method is less popular.
4. Postcoital (emergency) contraception:
Currently 3 regimens are used in a woman not taking any contraceptive who had a sexual
intercourse risking unwanted pregnancy.
 Levonorgestrel 0.75 mg two doses 12 hours apart or 1.5 mg single dose taken as soon
as possible, but before 72 hours of unprotected intercourse.Levonorgestrel 0.75 mg +
ethinylestradiol 0.1 mg(Yuzpe method) 2 doses at 12 hour interval within 72 hours of
exposure was used earlier.
 Ulipristal 30 mg single dose as soon as possible, but within 120 hoursof unprotected
intercourse.
 Mifepristone 600 mg single dose taken within 72 hours of intercourse has been used.
Common adverse effects:
 The common effects are: weight gain, owing to fluid retention or an anabolic effect,
or both mild nausea, flushing, dizziness, depression or irritability skin changes (e.g.
acne and/or an increase in pigmentation) amenorrhoea of variable duration on
cessation of taking the pill.
C. DRUGS ACTING ON THE UTERUS:
Motility of the uterus:
Uterine muscle contracts rhythmically both in vitro and in vivo, the contractions
originating in the muscle itself. Myometrial cells in the fundus act as pacemakers and give
rise to conducted action potentials. The electrophysiological activity of these pacemaker cells
is regulated by the sex hormones.
The non-pregnant human uterus contracts spontaneously but weakly during the first
part of the cycle, and more strongly during the luteal phase and during menstruation. Uterine
movements are depressed in early pregnancy because oestrogen, potentiated by progesterone,
hyperpolarises myometrial cells. This suppresses spontaneous contractions. Towards the end
of gestation, however, contractions recommence; these increase in force and frequency, and
become fully coordinated during parturition. The nerve supply to the uterus includes both
excitatory and inhibitory sympathetic components: adrenaline, acting on β2-adrenoceptors,
21
inhibits uterine contraction, whereas noradrenaline, acting on α- adrenoceptors, stimulates

contraction.
Uterine Stimulants (Oxytocics, Abortifacients):
These drugs increase uterine motility, especiallyat term.
1. Posterior pituitary hormone: Oxytocin,Desamino oxytocin
2. Ergot alkaloids: Ergometrine (Ergonovine),Methylergometrine
3. Prostaglandins: PGE2, PGF2α, 15-methylPGF2α, Misoprostol
Oxytocin:
 Oxytocin is a nonapeptide secreted by the posterior pituitary along with vasopressin
(ADH).Both oxytocin and ADH are synthesizedwithin the nerve cell bodies in
supraoptic andparaventricular nuclei of hypothalamus; aretransported down the axon
and stored in thenerve endings within the neurohypophysis. Theyare stored in separate
neurones as complexes withtheir specific binding proteins (neurophysins).
 Oxytocin released by stimuli by -coitus, parturition, suckling.
Actions:
 Uterus: Oxytocin contracts the uterus. Oestrogen induces oxytocin receptor synthesis
and, consequently, the uterus at term is highly sensitive to this hormone. Given by
slow intravenous infusion to induce labour, oxytocin causes regular coordinated
contractions that travel from fundus to cervix. Both amplitude and frequency of these
contractions are related to dose, the uterus relaxing completely between contractions
during low-dose infusion. Larger doses further increase the frequency of the
contractions, and there is incomplete relaxation between them. Still higher doses
cause sustained contractions that interfere with blood flow through the placenta and
cause fetal distress or death.
 Breast:Oxytocin contracts myoepithelium of mammary alveoli and forces milk into
the bigger milk sinusoids-'milk ejection reflex' (milk letdown in cattle) is initiated by
suckling so that it may be easily sucked by the infant. It has been used in milch cattle
to facilitate milking.
 CVS: Conventional doses used in obstetrics have no effect on BP but higher doses
cause vasodilatation leads to brief fall in BP, reflex tachycardia and flushing. This
action is most marked in chicken-used for bioassay. The umbilical vessels are
markedly constricted; oxytocin may help intheir closure at birth.
22
 Kidney: Oxytocin in high doses exerts anADH-like action-urine output is

decreased:pulmonary edema can occur if large amounts of i.v. fluids and oxytocin are
infused together.Conventional doses are without any effect.
 Action of oxytocin on myometrium is independent of innervation. There are specific
G-protein coupled oxytocin receptors which mediate the response mainly by
depolarization of muscle fibres and influx of Ca2+ ions as well as through
phosphoinositide hydrolysis and IP3 mediated intracellular release of Ca2+ ions. The
number of oxytocin receptors increases markedly during later part of pregnancy.
Oxytocin increases PG synthesis and release by the endometrium which may
contribute to the contractile response. Distinct subtypes of oxytocin receptors have
been shown on the myometrium and the endometrium.
Clinical use:
 Oxytocin is used to induce or augment labour when the uterine muscle is not
functioning adequately. It can also be used to treat postpartum haemorrhage.
Pharmacokinetic aspects
 Oxytocin can be given by intravenous injection or intramuscularly, but is most often
given by intravenous infusion. It is inactivated in the liver and kidneys, and by
circulating placental oxytocinase.
Unwanted effects of oxytocin
 Unwanted effects of oxytocin include dose-related hypotension (arising from its
vasodilator action), with associated reflex tachycardia. Its antidiuretic hormone-like
effect on water excretion by the kidney causes water retention and, unless water
intake is curtailed, consequent hyponatraemia.
Ergometrine
 Ergot (Clavicepspurpurea) is a fungus that grows on rye and contains a surprising
variety of pharmacologically active substances. Ergot poisoning, which was once
common, was often associated with abortion. In 1935, ergometrine was isolated and
was recognised as the oxytocic principle in ergot.
Actions
 Ergometrine contracts the human uterus. This action depends partly on the contractile
state of the organ. On a contracted uterus (the normal state following delivery),
23
ergometrine has relatively little effect. However, if the uterus is inappropriately

relaxed, ergometrine initiates strong contraction, thus reducing bleeding from the
placental bed (the raw surface from which the placenta has detached). Ergometrine
also has a moderate degree of vasoconstrictor action per se.
 The mechanism of action of ergometrine on smooth muscle is not understood. It is
possible that it acts partly on α adrenoceptors, like the related alkaloid ergotamine,
and partly on 5-hydroxytryptamine receptors.
Pharmacokinetic aspects
 Ergometrine can be given orally, intramuscularly or intravenously. It has a very rapid
onset of action and its effect lasts for 3-6 hours.
Clinical use:
 Ergometrine can be used to treat postpartum haemorrhage.
 A preparation containing both oxytocin and ergometrine is used for the management
of the third stage of labour; the two agents together can also be used, prior to surgery,
to control bleeding due to incomplete abortion.
Unwanted effects
 Ergometrine can produce vomiting, probably by an effect on dopamine D2 receptors
in the chemoreceptor trigger zone. Vasoconstriction with an increase in blood
pressure associated with nausea, blurred vision and headache can occur, as can
vasospasm of the coronary arteries, resulting in angina.
Prostaglandins:
Endogenous prostaglandins:
 Prostaglandins like PGE2, PGF2a and 15-methyl PGF2u are potent endogenous
uterine stimulants. The endometrium and myometrium have substantial prostaglandin-
synthesising capacity, particularly in the second, proliferative phase of the menstrual
cycle.
 Prostaglandin (PG) F2α is generated in large amounts, and has been implicated in the
ischaemic necrosis of the endometrium that precedes menstruation (although it has
relatively little vasoconstrictor action on many human blood vessels, in contrast to
some other mammalian species). Vasodilator prostaglandins, PGE2 and PGI2
(prostacyclin), are also generated by the uterus.
 In addition to their vasoactive properties, the E and F prostaglandins contract the non-
pregnant as well as the pregnant uterus. The sensitivity of uterine muscle to
24
prostaglandins increases during gestation. Their role in parturition is not fully

understood, but as cyclo-oxygenase inhibitors can delay labour.
 Prostaglandins also play a part in two of the main disorders of menstruation:
dysmenorrhoea (painful menstruation) and menorrhagia (excessive blood loss).
Prostaglandin preparations
 Prostaglandins of the E and F series promote coordinated contractions of the body of
the pregnant uterus, while relaxing the cervix. E and F prostaglandins reliably cause
abortion in early and middle pregnancy, unlike oxytocin, which generally does not
cause expulsion of the uterine contents at this stage.
 The prostaglandins used in obstetrics are dinoprostone (PGE2), carboprost (15-methyl
PGF2α) and gemeprost or misoprostol (PGE1 analogues). Dinoprostone can be given
intravaginally as a gel or as tablets, or by the extra-amniotic route as a solution.
Carboprost is given by deep intramuscular injection. Gemeprost or misoprostolare
given intravaginally.
Clinical use
 Dinoprostone given by the extra-amniotic route is used for late (second trimester)
therapeutic abortion; given as vaginal gel, it is used for cervical ripening and
induction of labour.
 Gemeprost, given as vaginal pessary following mifepristone, is used as a medical
alternative to surgical termination of pregnancy (up to 63 days of gestation).
Unwanted effects
 Unwanted effects include uterine pain, nausea and vomiting, which occur in about
50% of patients when the drugs are used as abortifacients.
 Dinoprost may cause cardiovascular collapse if it escapes into the circulation after
intra-amniotic injection.Phlebitis can occur at the site of intravenous infusion.
 When combined with mifepristone, a progestogen antagonist that sensitises the uterus
to prostaglandins, lower doses of the prostaglandins (e.g. misoprostol) can be used to
terminate pregnancy and side effects are reduced.
Drugs that inhibit uterine contraction
 Selective β2-adrenoceptor agonists, such as ritodrine or salbutamol, inhibit
spontaneous or oxytocin-induced contractions of the pregnant uterus. These uterine
relaxants are used in selected patients to prevent premature labour occurring between
22 and 33 weeks of gestation in otherwise uncomplicated pregnancies. They can delay
delivery by 48 hours, time that can be used to administer glucocorticoid therapy to the
25
mother so as to mature the lungs of the baby and reduce neonatal respiratory distress,
and to optimise logistics such as making sure the baby is born in a facility with
neonatal intensive care.
 Cyclo-oxygenase inhibitors (e.g. indometacin) inhibit labour, but their use could
cause problems in the baby, including renal dysfunction and delayed closure of the
ductus arteriosus, both of which are influenced by endogenous prostaglandins.
 An oxytocin receptor antagonist, atosiban, provides an alternative to a β2-
adrenoceptor agonist. It is given as an intravenous bolus followed by an intravenous
infusion for not more than 48 hours. Adverse effects include symptoms of
vasodilation, nausea, vomiting, and hyperglycaemia.
6. Bioassay
Bioassay may be defined as the determination of the concentration of a biologically
active substance, of physical, chemical or biological origin, by using a biological indicator
with reference to standard. Bioassay is comparative in nature.
Bioassay is the determination or estimation of the amount of biological activity in a
unit quantity of the preparation. It is a process of determining relative potency of a substance
or active principle of a substance by comparing its biological activity with that of a reference
standard. Bioassay includes both quantitative assays of drugs as well as the application of
qualitative biological tests. The purpose of bioassay is to determine the potency of a drug and
hence it serves as the quantitative part of any screening procedure. Other purpose of bioassay
is to standardize the preparation so that each contains uniform specified pharmacological
activity.
Biological indicator:
The biological activity of a substance can be studied by using different experimental
animals, isolated tissues of the experimental animals, immune cells or microorganisms are
termed as biological indicator.
Biological standardization:
Comparison and adjustment of the strength of the unknown sample with that of the
standard under rigidity controlled conditions is termed as bio-standardization. It is a process
of increasing or decreasing the potency of a biologically active substance to match it with the
biological activity of a reference standard. Matching of material of unknown potency with an
26
international/national standard objective of providing a reliable preparation for the use in

therapeutics research. For example, matching of LD50 of digitalis tincture (which contain
variable quantities of the active principle) to the standard preparation.
Principles of Bioassay:
The basic principle of bioassay is to compare the test substance with the international
standard preparation of the same and to find out how much test substance is required to
produce the same biological effect, as produced by the standard. The followings are the
principles of bioassay:
1. All bioassays (lab studies, toxicity studies, clinical studies) must be comparative &
compared against a standard drug or preparation. The potency of biologically assayed
compounds is compared with the activity of internationally accepted standards.
2. The standard and new drugs should be as far as possible identical to each other, then their
dose response curve (DRC) will have the same stop and would be constant all along the
response level.
3. The biological response should be closely related to the therapeutic use of the drug as
possible. For example, in estimating the potency of insulin, blood glucose levels would be of
the ideal index, but the method commonly used is the mouse convulsion method, in which the
% of animals developing convulsions are recorded. Similarly for estimating digitaloid drug,
the arrest of heart in pigeon or guinea pig is used as end point which is as the toxic effect of
the drug and not the therapeutic effect.
4. An estimate of relative potency is made by comparing the log dose of the test and the log
dose of the standard that produces an equal magnitude of effect.
5. The problem of biological variation must be minimized as far as possible. For that one
should keep uniform experimental conditions and assure the reproducibility of the responses.
The results of the test should be subjected to statistical analysis to minimize errors due to
biological variations.
Applications of bioassay:
1. To standardize drugs of natural origin, especially when the chemical identity of the active
has not been elucidated fully. e.g. parathyroid hormones, antitoxins etc.
2. To standardize those drugs from which no adequate chemical assay has been devised even
though the chemical structure of the active principle has been established. e.g. hormones like
insulin, oxytocin etc.
3. To standardize the drugs which are composed of a complex mixture of substances varying
structure and activity. e.g. digitalis, posterior pituitary extract etc.
27
4. If the chemical assay is not a valid indication of the biological activity, e.g. due to lack of
differentiation between active and inactive isomers.
5. To standardize when the drugs have same pharmacological actions but different chemical
structures existing together. e.g. glycosides in digitalis preparation.
6. To estimate the dose of a drug required to produce a therapeutic or toxic response. e.g.
calculation of ED50, LD50 etc.
7. When the physical property of rotation (dextro or levo) shows difference in action. e.g. d-
Chloramphenicol is active. Levo adrenaline is 80 times more active than dextro.
b. Types of bioassay:
Bioassay may be broadly classified into two type depending upon the type of response
recorded i.e. i) quantal bioassay and ii) graded bioassay.
(i) Quantal bioassay:

Quantal means the response is in the form of "all or none", i.e. either maximum
response or no response. In this type of bioassay, there is no intermediate response. For
example animal receiving a convulsant drug either show convulsions or does not. Examples
of drugs assayed by quantal bioassay method are:
Digitalis: by cardiac arrest in guinea pig or pigeon.
d-Tubocurarine: by rabbit head drop method.
Insulin: by hypoglycaemic convulsions produced in mice.
Though the method is not very accurate, it is employed for comparison of threshold
response and comparison of ED50 or LD50 values. The quantal bioassay is an end point
method.
End point method: Here the threshold dose showing a positive effect is measured on each
animal and the comparisons between two groups of animals are done i.e. one receiving
standard and other the test compounds. e.g. bioassay of digitalis in cats. Here the cat is
anaesthetized with chloralose and its blood pressure is recorded. The drug is slowly infused
into the animal and the moment the heart stops beating and blood pressure falls to zero, the
volume of fluid infused is noted down. Two groups of animals, one using standard digitalis
and the other using test preparation of digitalis and then potency is calculated as follows:
Concentration of unknown = Threshold dose of standard Threshold dose of test ×
Concentration of standard
(ii)Graded bioassay:
28
Graded response means the response is proportional to the dose and response may lie
between no response and the maximum response. These responses are based on the
observations that there is a proportionate increase in the observed response with a subsequent
increase in the concentration or dose. This procedure depends on the precision of assay
demand, quality of the sample available, availability of experimental animals. Types of
graded assay are:(a)Matching, (b)Bracketing, (c)Interpolation, (d)Multiple point bioassay
(three point, four point, six point, eight point).
BIOASSAY OF INSULIN:
 Insulin is a hormone made by pancreatic β cell.Insulin is synthesised as pro-insulin in
the endoplasmic reticulum and is processed to the biologically active form inside the
secretory granules.
 Bioassay of insulin is generally carried out by rabbit method and mice method.
 Some of the other methods are: Rat diaphragm method,Epididymal fat pad of rats,
 Standard preparation and unit: It is pure, dry and crystalline insulin. One
unitcontains 0.04082 mg. This unit is specified by Ministry of Health, Government
ofIndia and is equivalent to international unit.
 Preparation of standard solution: Accurately weigh 20 units of insulin and dissolve
it in normal saline. Acidify it with HCl to pH 2.5. Add 0.5% phenol as preservative.
Add 1.4% to 1.8% glycerin. Final volume should contain 20units/ml. Store the
solution in a cool place and use it within six months.
 Preparation of test sample solution: The solution of the test sample isprepared in
the same way as the standard solution.
1) Rabbit method:
 Selection of rabbits: They should be healthy, weighing about 1800-3000 gms. They
should then be maintained on uniform diet but are fasted for 18 hrs.before assay.
Water is withdrawn during the experiment.
 Standard and Sample Dilutions: These are freshly prepared by diluting withnormal
NaCl solution so as to contain 1 unit/ml and 2 units/ml.
29
 Doses: The dose which can produce suitable fall in blood sugar level iscalculated for
the standard.
 Principle: The potency of a test sample is estimated by comparing the hypoglycemic
effect of the sample with that of the std. preparation of insulin.Any other suitable
method can also be used.
 Experimental Procedure: Animals are divided into 4 groups of 3 rabbits each. The
rabbits are then put into an animal holder. They should behandled with care to avoid
excitement.
 First part of the Test: A sample of blood is taken from the marginal ear vein of each
rabbit. Presence of reducing sugar is estimated per 100 ml of blood by a suitable
chemical method. This concentration is called‘Initial Blood Sugar Level’.
 The four groups of rabbits are then given sc. injections of insulin as follows:
12 Rabbits
3Rabbits 3Rabbits 3Rabbits 3Rabbits

Standard dilution Standard dilution Test dilution Test dilution
Group-I Group-II Group-III Group-IV
 From each rabbit, a sample of blood is withdrawn up to 5 hrs. at the interval of 1 hr.
each. Blood sugar is determined again. This is known as‘Final Blood Sugar Level’.
 Second part of the test (Cross over test) : The same animals are used for the second
part. The experiment can be carried out after one week. Again they are fasted and
initial blood sugar is determined. The grouping is reversed, that is to say, those
animals which received the standard are given the test and those which received the
test are now given the standard. Those animals which received the less dose of the
standard are given the higher dose of the test sample and vice-versa. This test isknown
as ‘Twin Cross Over Test’.
 Mean percentage of decrease in blood sugar of first part and second part is calculated.
2) Mouse Method:
 Mice show characteristic convulsions after s.c. inj. of insulin at elevated temperatures.
The percentage convulsions produced by the test and standard preparations are
compared.
30
 Experimental procedure: Minimum 100 mice weighing between 18-22gms. of the

same strain are used. They should be maintained on constantdiet. They should be
fasted 18 hrs. prior to the experiment.
 Standard and sample dilutions: Dilutions are prepared with sterile salinesolution, so
as to contain 0.064 units/ml. (std dilution I) and 0.096untis/ml. (std. dilution II).
Similarly, test sample solutions are alsoprepared.
 Mice are divided into 4 groups each containing 25 mice and insulin is injected s.c. as
follows:
100 Mice
25 Mice 25 Mice 25 Mice 25 Mice

Standard dilution Standard dilution Test dilution Test dilution
(0.064 units/ml) (0.096 units/ml)
Group-I Group-II Group-III Group-IV
 Mice are put in an air incubator at 33oC and observed for one and a halfhour.
 The mice which convulse or die are taken out of the incubator and observed. These
mice usually convulse severely but failure of the animal to upright itself when placed
on its back, should as well be considered asconvulsion.
 Convulsive mice may be saved by an inj. Of 0.5 ml of 5% dextrose solution.
 Percentage convulsions produced by test samples are compared with those of the
standard sample. Those animals which survive may be used again for another
experiment after an interval of one week.
BIOASSAY OF OXYTOCIN:
 Oxytocin is a peptide hormones and Neuropeptide. Oxytocin is normally produced by
the paraventricular nucleus of the hypothalamus and released by the
posteriorpituitary.
 Oxytocin is a natural hormone that causes the uterus to contract. Oxytocin is used to
induce labor or strengthen labor contractions during childbirth, and to control
bleeding after childbirth.
 Oxytocin is also used to stimulate uterine contractions in a woman with an incomplete
or threatenedmiscarriage.
 Different bioassay methods used for oxytocin are
o By contraction of the rat uterus
31
o By depression of the blood pressure in chicken

o By measurement of milk ejection pressure in a lactating rat etc.
Principle:
 The potency of oxytocin injection is determined by comparing its activity with that of
the standard preparation of oxytocin under the conditions of the followingmethod of
assay.
Standard preparation:
 The standard preparation is consisting of a freeze dried preparation of oxytocin with
human albumin and citric acid (supplied in ampoules containing 12.5 units) or any
other suitable preparation, the potency of which had been determined in relation to the
International standard.
 The Unit is the specific oxytocin activity corresponding to that yielded by 0.0005 g of
the Standard preparation.
 The standard preparation is as per the 4th international standard for Oxytocin,
established in 1978.
Experimental Methods:
1) By contraction of the rat uterus:
 Use female rats weighingbetween 120 and 200 g. Immediately before the assay
confirm the rat is in oestrous or preoestrus by vaginal smear.
 Inject 100 microgram of oestradiol benzoate intramuscularly 18-24 hours before the
assay.Kill the rat and suspend one horn of the uterus in a bath containing a solution of
the following composition:
Composition (% w/v)
Sodium chloride 0.662
Potassium chloride 0.045
Calcium chloride 0.007
Sodium bicarbonate 0.256
Disodium hydrogen phosphate 0.029
Sodium dihydrogen phosphate 0.003
Magnesium chloride 0.010
Dextrose 0.050
 Maintain the bath at a temperature of 32º C so that spontaneous contraction of the
uterus are abolished and preparation maintain its sensitivity.
 Oxygenate the solution with a mixture of 95% of oxygen and 5% of carbon dioxide.
32
 Record the contractions of the muscles produced by the addition to the bath of two
doses of the Standard Preparation suitably diluted with the above solution. The doses
should be such as to produce clearly discriminated, submaximal contractions.The
required doses normally lie between 10 and 50 micro Unitsper ml of bath liquid.
 When maximal contraction has reached replace the bath liquidby a fresh solution.
 The doses should be added at regular intervals of 3 to 5minutes depending upon the
rate of recovery of the muscle.
 Dilute the preparation being examined so as to obtain the responses on the addition of
two doses similar to thoseobtained with the Standardpreparation.
 The ratio between the two doses of the preparation being examined should be the
same as that between the twodoses of the Standard Preparation and this ratio should
keptconstant throughout the assay.
 The two doses of Standard preparation and the two doses of the test preparation
should be given according to a randomized order or Latin square design and at least
six responses toeach should be recorded.
 Measure all the responses and calculate the result of theassay by statistical methods.
2) By depression of the blood pressure in chicken:
 Anaesthetize a young healthy adult cockerel weighing 1.2 to 2.3 kg with an anesthetic
that will maintain aprolonged and constant high blood pressure.
 Expose the gluteus primus muscle in one thigh and cut and retract it to reveal the
popliteal artery and cruralvein.
 Cannulate the popliteal artery and record the bloodpressure.Cannulate the crural or
brachial vein.
 Prepare standard solution with saline so that the volume to be injected is between 0.1-
0.5 ml.
 Inject 2 doses of standard solution into cannulated vein and record blood pressure.
The doses should be such that as to produce clearly discriminated, precipitious,
submaximal decreases in B.P.
 The required doses normally lie between 20 and 100 milli units.
 The interval between injections should be constant and lie between 3-10 minutes
depending on the rate at which B.P. returns tonormal.
 Dilute test sample before use with saline solution so as to obtain responses similar to
those obtained with the standard preparation.
33
 The ratio between the two doses of the test preparation being examined should be
same as that between the two doses of the standard preparation and the ratio should be
kept constant throughout the assay.
 The two doses of Standard preparation and the two doses of the test preparation
should be given according to a randomized order or Latin square design and at least
six responses toeach should be recorded.
 If animal rapidly become insensitive due to repeated injections of the solutions
another animal must be used. Measure all responses and calculate the result by
standard statistical methods.
3) By measurement of milk-ejection pressure in a lactating rat:
 Select alactating rat, in the third to twenty-first day after parturition and weighing
about 300 gseparate it from the litter and 30 to 60 minutes later anaesthetise (for
example, by theintraperitoneal injection of a solution of Pentobarbitone Sodium).
 Tie the rat to anoperating table, maintained at 37, by its hind legs leaving the front
legs free. Cannulatethe trachea with a short polyethylene tube of internal diameter
about 2.5 mm in such amanner so as to ensure a free airway; apply artificial
respiration only if necessary.
 Cannulate an external jugular or femoral vein with a polyethylene tube of
internaldiameter about 0.4 mm which is filled with saline solution and closed with a
pin.
 Shave the skin surrounding the inguinal and abdominal teats and excise the tip of
oneteat, preferably the lower inguinal teat. Insert a polyethylene tube of internal
diameterabout 0.3 mm and external diameter about 0.6 mm, to a depth sufficient to
obtainappropriate measurement of pressure (3 to 10 mm depth), into the primary teat
ductwhich opens onto the cut surface and tie firmly in place with a ligature.
 Connect thiscannula with a suitable strain gauge transducer (such as that used for
recording arterialblood pressure in the rat) and fill the whole system with a 3.8% w/v
solution of sodiumcitrate or saline solution containing 50 Units of heparin sodium per
ml to prevent clottingof milk.
 After cannulation, inject a small volume (0.05 to 0.2 ml) of this solution into theteat
duct through the transducer to clear the milk from the tip of the cannula.
(Thisprocedure may be repeated during the assay should obstruction arise from milk
ejectedinto the cannula).
34
 Clamp the strain gauge so that a slight tension is applied to the teat andits natural
alignment is preserved and connect the gauge to a potentiometric recorderadjusted to
give full-scale deflection for an increase in milk-ejection pressure of about 5.3kPa.
 Inject all solutions through the venous cannula using a 1-ml syringe graduated in0.01
ml and wash them in with 0.2 ml of saline solution.
 Prepare a solution of the Standard Preparation and a solution of the preparation
beingexamined in saline so that the volume to be injected is between 0.1 ml and
0.4ml.
 Choose two doses of the Standard Preparation such that the increase in milk-
ejectionpressure is about 1.35 kPa for the lower dose and about 2.7 kPa for the higher
dose.
 As aninitial approximation, a lower dose of between 0.1 and 0.4 milliUnit and an
upper dose of1.5 to 2 times this amount may be tried.
 Choose two doses of the preparation beingexamined with the same inter-dose ratio,
matching the effects of the doses of theStandard Preparation as closely as possible.
 Inject the four doses (two doses of theeStandard Preparation and two doses of the
preparation being examined) at intervals of 3to 5 minutes.
 The two doses of Standard Preparation and the two doses of the preparationbeing
examined should be given according to a randomised block or a Latin squaredesign
and at least four responses to each should be recorded. Measure all the responsesand
calculate the result of the assay by standard statistical methods.
BIOASSAY OF VASOPRESSIN:
Biological Assay for Vasopressor Activity
Principle:
 The vasopressor activity is estimated by comparing the activity of thepreparation
being examined with that of the Standard Preparation of arginine vasopressinunder
the conditions of a suitable method of assay.
Standard Preparation:
 The Standard Preparation is the Ist International Standard forArginine
vasopressin,established in 1978,consisting of freeze-dried synthetic
argininevasopressin peptide acetate with human albumin and citric acid(supplied in
ampoulescontaining 8.20 Units),or another suitable preparation the potency of which
has beendetermined in relation to that of the International Standard.
Suggested Method:
35
 Inject slowly into the tail vein of a male albino rat weighing about300 g a solution of
a suitable alpha-adrenoceptor blocking agent.for example 10 ml perkg of body weight
of a solution prepared by dissolving 5 mg of phenoxybenzaminehydrochloride in 0.1
ml of ethanol(95%),adding 0.05 ml of 1M hydrochloride acid anddiluting to 5 ml with
saline solution.
 After 18 hours,anaesthetise the rat with ananaesthetic that will maintain a prolonged
and uniform blood pressure.After 45 to 60minutes,tie the rat on its back to the
operating table by its hind legs.
 Cannulate thetrachea with a short polyethylene tube of external diameter about 2.5
mm and dissect acarotid artery ready for cannulation.Then cannulate the femoral vein
close to the inguinalligament.
 Retract the abdominal muscles to expose the inguinal ligament.Retract thesuperficial
pudendal vein to one side and dissect the femoral vein towards the inguinalligament
rom the corresponding artery.
 When dissecting,a deep branch reaching thefemoral vein must be found and tied off to
prevent bleeding during cannulation.Tie ashort polyethylene cannula of external
diameter about 1 mm into the femoral vein by twoligatures and join by a short piece
of flexible tubing to a 1-ml burette with an attachedthistle funnel containing saline
solution at about 37.
 Firmly fix a wet absorbent cottonswab to the thigh so as to cover the incision and
cannula.
 At this stage inject through thevenous cannula 200 Units of heparin,dissolved in
saline solution,per 100 g of bodyweight.
 Then tie in a carotid cannula of external diameter about 1 mm and connect by
acolumn of saline solution containing heparin with a suitable pressure measuring
devicesuch as a mercury manometer of internal diameter about 2 to 3 mm.
 The central and peripheral nervous system including both vagus and
associatedsympathetic nerves is left intact. No artificial respiration is necessary.
 Taking care that noair is injected, inject all solutions through the venous cannula by
means of a 1-ml syringegraduated in 0.01 ml and wash in with 0.2 ml of saline
solution fronm the burette.
 Dilute the extract of the Standard Preparation and the preparation being examined
withsaline solution so that the volume to be injected is between 0.1 ml and 0.5 ml.
Choosetwo doses of the Standard Preparation such that the elevation of the blood
36
pressureisabout 4 kPa for the lower dose and about 7 kPa but always submaximal for
the higherdose, the ratio of low to high dose being determined by the response and
usually being 3to 5.
 As an initial approximation doses of 3 and 5 milliUnits may be tried. Choose
twodoses of the preparation being examined with the same inter-dose ratio, matching
theeffects of the dose of the Standard Preparation as closely as possible.
 Inject doses atintervals of 10 to 15 minutes. The two doses of the Standard
Preparation and the twodoses of the preparation being examined should be given in a
randomised block or aLatin square design and four to five responses to each should be
recorded.
 Measure allthe responses and calculate the result of the assay by standard statistical
methods.
BIOASSAY OF ACTH:
 ACTH (Adrenocorticotropic hormone, corticotropin) is polypeptide tropic hormone
(39 amino acids) secreted by the anterior pituitary gland.
 ACTH stimulates the production of cortisol, a steroid hormone important for
regulating glucose, protein and lipid metabolism, suppressing the immune system
response, and helping to maintainblood pressure.
Official Preparations
 Corticotropin injection: Is a sterile solution , in a suitable diluent, of the polypeptide
from the pituitary glands of mammals. Potency range should be 80.0 – 120.0 % of
USP cartiotropinunits.
 Corticotropin for injection, antimicrobial agent. Repository corticotropin injection is
corticotropin in a sterile solution of partially hydrolyzedgelatin and is intended for
subcutaneous and intramuscular use. This solution has beenadopted as the reference
standard for the bioassay.
o Packing: Preserve in single-dose or multiple-dose containers of Type-1glass.
o Storage:Store in cold place.
o Labeling:Injection recommends intravenous administration
Purpose and rationale
 This is a historical assay method. Administration of pituitary ACTH decrease the
ascorbic acid present in the adrenals. The depletion of adrenal ascorbic acid is a
function of the dose of ACTH administered. This relationship has been used for a
quantitative assay ofACTH.
37
Solutions:
 Solution A: Five units of test or standard dissolved in 0.25 ml of 0.5% phenol solution
and diluted with 8.1 ml of 15% gelatin solution (Now 0.5ml contain 300 mU ACTH).
 solution B: Three ml of solution A diluted with 6 ml gelatin solution.
Nowconcentration reduced to 100 mU ACTH/ 0.5 ml.
 solution C: Again 3 ml of solution B diluted with 6 ml of gelatin solution, theresulting
solution contains 33 mU ACTH/ 0.5 ml.
Procedure
 Male Wistar rat (100-200 g) are hypophysectomized (pituitary gland removed by
surgery) one day prior to the test.
 For one test with 3 dose of test preparation and standard solution used for the study.
 Number of hypophysectomized rats required: at least 36(preferably 60).
 The hypophysectomized rats are randomly distributed in to sixgroups. Each rat
receives subcutaneous 0.5 ml of the variousconcentrations of test or standard.
 Three hours after injection, the animals are anesthetized andboth adrenals removed,
freed from extraneous tissue andweighed. The rats are sacrificed and the scull opened
to verifycompleteness of hypophysectomy.
 The adrenals are homogenized in glass tubes contains 200 mgpure sand and 8.0 ml of
4% trichloroacetic acid and the ascorbicacid determined. (Roe and Kuether 1943).
 The potency ratio including confidence limits is calculated withthe 3 + 3 point assay.
Ascorbic acid determination:
Reagents
 0.02% ascorbic acid solution
 85 % sulfuric acid (9N H2SO4)
 0.02 g/ml of dinitrophenolhydrazine in 9N H2SO4
 0.06 g/ml of thiourea are dissolved in distilled water
 Charcoal
Preparation of 0.02% ascorbic acid solution
 100 mg L-ascorbic acid are dissolved in 100 ml of 4% trichloroacetic acid (1mg/ml
solution) (Solution A= 1 % solution)
 2 ml of Solution A diluted in 10 ml of 4% trichloroacetic acid to achieve a 0.2%
ascorbic acid solution (solution B)
38
 1 ml of solution B diluted in 10 ml of 4% trichloroacetic acid to achieve a 0.02%

ascorbic acid solution (solution C)
Preparation of other solutions
 Sulfuric acid (85%) is obtained by adding 900 ml concentrated sulfuric acid to 100 ml
distilled water.
 Two g dinitrophenolhydrazine are dissolved in 100 ml 9 N H2SO4 (75 ml distilled
water and 25 ml concentrated sulfuric acid).
 Six g thiourea are dissolved in 100 ml distilled water.
Calibration
 Trichloroacetic acid (4%) is added to 0.0, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0 ml of the
0.02% ascorbic acid solution (solution C) and 1.0, 1,5 and 2.0 ml of the 0.2% ascorbic
acid solution to reach a final volumeof 8.0 ml (Solution B).
 100 mg charcoal is added to each sample and thoroughly mixed byshaking for 1 min.
 After 5 min the solutions are filtered.
 An aliquot of 0.1 ml of the 6% thiourea solution is added to 2.0 mlof the filtrate
followed by 0.5 ml dinitrophenylhydrazine solution.
 The mixture is shaken and heated for 45 min at 57°C in a waterbath.
 The solutions are placed in an ice-cold water bath and withfurther cooling 2.5 ml of
the 85% sulfuric acid are added.
 The calibration curve is established at a wave length of 540 mmusing the solutions
without ascorbic acid as blank.
BIOASSAY OF d-TUBOCURARINE:
1. Rabbit Head-drop Method :
Principle:
 d-Tubocararine hydrochloride is injected into the marginal vein of a rabbit’s ear till
the rabbit’s neck muscles are relaxed such that the animal cannot hold its head up.
The total amount of test sample required to produce the endpoint is compared with the
total amount of the standard sample required to producesimilar endpoint.
Selection of Rabbits:
 Rabbits weighing 2 kg are used. Animals should be free from disease, obtained from a
healthy colony and should beaccustomed with the experimental procedure.
Experimental Procedure:
39
 Rabbit is placed in a holder with its head protruding outside.The head should be freely
movable.
 Minimum 8 rabbits are used.They are divided into two groups eachcontaining 4
rabbits.
 First group will receive standard sample andthe second group will receive the
sampleunder test.d-Tubocurarine solution is injected at aconstant speed by infusion
apparatus throughthe marginal vein.
 Injection should be given at a rate of 0.4ml/min and should take about 10 min.
Dose0.012% w/v in saline.
 Infusion is continued till the rabbit will not bein a position to hold its head erect or
therewill be no response by focusing light on theeyes.Rabbits recover immediately
from the effectof curarization.
 During the experiment there is a possibility ofrespiratory embarrassment which is
treatedby injecting neostigmine methyl sulphate(0.05 mg.) and atropine sulphate
immediatelythrough the marginal ear vein.
 Cross-over test is carried out to minimizebiological error due to animal variation.
 Those rabbits which received the standardsample on the first day will be given
testsample on the second day of experiment andvice versa.
 Mean dose which produces head drop of thetest sample is compared with the mean
doseof standard preparation.
2. Frogs Rectus Abdominis muscle Preparation:

 A frog is pithed and laid on its back on a cork covered board to which it is pinned.
The skin covering the abdomen is cut away and the rectus abdominis muscle of one
side is dissected from the pelvic girdle to itsinsertion in the cartilage of the pectoral
girdle.
 The muscle is then pinned to the cork byfour pins to keep its normal length while a
thread is sewn through each end.
 It is then mounted in the organ bath containing frog's Ringer solution which contains:
NaCl, 6.5gm.; KCl, 0.29 gm.; CaCl2, 0.24 gm.; NaHCO3, 0.4 gm.; glucose, 1.5 gm.
and distilledwater 2000 ml.
40
 Oxygenation is carried out to keep the tissue alive. The muscle isstabilized for 30-45
min. in order to get critical quantitative response.
 The responses arerecorded using isotonic frontal writing lever with 1 G. tension.
 Two similar contractions with the same concentration of acetylcholine are obtained.
 Three doses of the standard sample and one intermediate dose of the test sample are
selected and the reduction in height of contraction induced by acetylcholine is
noteddown.
 Acetylcholine contraction is recorded on slow moving drum for 90 second. d-
Tubocurarine is allowed to act for 30 sec.
 The percentage reduction at each dose levels is calculated and log dose response
curve of the standard drug is plotted. A linear response will beobtained. The potency
of test sample is calculated from the standard curve.
BIOASSAY OF DIGITALIS:
Principle:
 Potency of the test sample is compared with that of the standard preparation by
determining the action on the cardiac muscle.
Standard Preparation and Units:
 The standardpreparation is a mixture of dried and powdereddigitalis leaves (1 unit =
76 mg.)
Preparation of Extracts:
 Exact amount of thepowder is extracted with dehydrated alcohol in acontinuous
extraction apparatus for six hours. Thefinal extract should contain 10 ml(5 ml. alcohol
+5 ml. water) per 10 g. of digitalis powder. It shouldbe stored in between 5oC and –
5oC.
1. Guinea-pig Method (Endpoint method):

 Standard and test sample extracts are diluted with normal saline in such a way that
1gof digitalis powder is diluted to 80 ml.
 A guinea pig is anaesthetized with a suitable anaesthetic. It is dissected on the
operation table. The jugular vein is traced out by removing adhering tissues and
cannulated by means of venous cannula. A pin is inserted in the heart, such that it gets
inserted in the apex of the heart. In this way, we can observe the heart beats by up and
down movementsof the pin.
41
 The injection is continued through venous cannula untill the heart is arrested in
systole. The amount of extract required to produce this effect is taken as the lethal
dose ofthe extract.
 Another set of 19 animals of the same species are used for this experiment and the
average lethal dose is determined. It is not necessary to determine the lethal doseof
the standard during each time of the experiment. But it should be occasionally
checked.
 The lethal dose of the test sample is determined in a similar way using minimum
6guinea-pigs of the same strain.
 The potency of the test sample is calculated in relation to that of the std. preparation
by dividing the average lethal dose of the sample to the test and expressed as units per
gram.
2. Pigeon Method
 Minimum 6 pigeons are used for testing eachsample. The weight of the heaviest
pigeon should notexceed twice the weight of the lightest pigeon.Food is withheld 16-
28 hours before theexperiment.
 Pigeons are divided on the basis of their sex,weight and breed, into two groups.They
are anaesthetized with anesthetic ether.
 One side of the wing is dissected and the alarvein is cannulated by means of a venous
 cannula. Dilutions are made with normalsaline.
 The test sample and standard sample isinfused through cannula.In pigeons, stoppage
of heart is associated witha characteristic vomiting response called‘emesis’.
 The milk from the crop sac of pigeons is beingejected out. This may be taken as the
end pointresponse of digitalis.
 The lethal dose per kg. of body weight isdetermined for each pigeon.
 The potency of the test sample is determinedby dividing the mean lethal dose
ofstandard bythe mean lethal dose of the test sample.
BIOASSAY OF HISTAMINE:
 Histamine is present in almost all the animal tissues mostly within mast cell and
basophil granules. Tissues rich in histamine are skin, gastric and intestinal mucosa,
lungs, liver and placenta.
 Non-mast cell histamine is present in brain, epidermis, gastric mucosa and growing
regions. Histamine is also present in blood, most body secretions, venoms and
pathological fluids. It is now known to play important physiological roles. Histamine
42
produces effects by acting on the histamine receptors (H1, H2, H3 and H4) present on
target cells.
 Bioassay of histamine on isolated guinea pig ileum can be determined by graded
bioassay procedure i.e. i) Matching bioassay, ii) Interpolation bioassay, iii) Bracketing
assay, iv) Multiple point assays.
 Bioassay of histamine in biological samples can be studied by using different bioassay
methods. Depending upon pharmacological action, histamine can be assayed by:
o Contractile effect of isolated ileum of guinea pig,
o Contractile effect of uterusof guinea pigand
o Fall in blood pressure of anaesthetized and atropinized cat. Guinea pig's uterus
Bioassay of histamine using guinea pig ileum:
Principle:
 Guinea pig ileum is very useful preparation for the bioassay methods. It is more
sensitive to histamine. Contractile response of histamine to ileum is due to presence of
H1 receptor.
Preparation of Standard and other solution:
 Prepare the stock of Tyrode solution. Also prepare the standard stock solution of
histamine (1 mg/ml) and then different concentrations of histamine by serial dilution
method.
Procedure:
 Sacrifice the 24 hr fasted guinea pig by stunning on the head and carotid bleeding. Fix
the animal on the dissecting board by tying its legs. Open the abdominal cavity by a
small horizontal cut followed by vertical midline incision and expose the abdominal
organs.
 Trace the ileocaecal junction by lifting the caecum, then go upwards up to 8 cm from
the junction and cut the 2-3 cm long segment of ileum muscle (excluding the terminal
5-8 cm, contains excess of excitatory α-adrenergic receptor, which may interfere in
the study).
 Immediately place it in a petri dish containing aerated warm Tyrode solution. Slowly
remove the mesentery attached to the muscle and then gently clean the lumen of ileum
by passing luke warm Tyrode solution through it with a pipette or syringe.
 Mount the preparation in the inner organ bath containing Tyrode solution (20 ml)
maintained at 37ºC. Tie the bottom end of the muscle to the hook of aeration tube and
the upper end to the isotonic frontal lever by a thread without closing the lumen.
43
Adjust the magnification of response to 7-10 fold. Aerate the tissue with O2 or
carbogen slowly (40-60 bubbles per minutes).
 Stabilize the tissue for 30 min by applying a tension of 0.5 g weight attached to the
lever, during which wash the tissue with fresh Tyrode solution once in every 10 min.
 Use 5 min time cycle with contact time of 30s for recording the contraction
dependent responses of tissue due to histamine on the smoked drums.
 Record the responses of standard and test compounds.
 Record the responses of test compound i.e. unknown concentration of histamine with
gradually increasing volume, till obtaining a response (T) which would lie on the
linear portion (30-70%) of the CRC. Fix the response obtained due to volume of T.
 Record the graded responses of standard solution and test solutionof histamine.
gram.
BIOASSAY OF 5-HT:
 5-Hydroxytryptamine (5HT) is the biologically active local hormone, low molecular
weight, and also an important neurotransmitter in the brain and periphery.
 It was first detected in serum (serotonin) and enterochromaffin cells of gut mucosa
(enteramine), and later both were identified to be 5HT. Today the terms 5HT and
serotonin are used interchangeably.
 About 90% of body's content of 5-HT is localized in the intestines; most of the rest is
in platelets and brain. It is also found in wasp and scorpion sting, and is widely
distributed in invertebrates and plants (banana, pear, pineapple, tomato etc.).
 The concentration of 5-HT in biological samples can be assayed by:Isolated fundus
strip of rat stomach,Isolated terminal colon of rat,Isolated rat uterus,Perfused rabbit
ear.
Isolated fundus strip of rat stomach
Principle:
 Rat fundus preparation is a very sensitive tissue for the bioassay methods and is useful
to study the action of several substances like 5-HT, ACh, PGE2 and bradykinin.
Fundus strip preparation is slow contracting muscle. Fundus part of stomach can be
easily identified by its grey colour and situated above the pink coloured thick pyloric
part. A zig-zag preparation of the fundal strip is prepared so as to expose maximum
portion of the tissue to drug.
44
Preparation of Standard and other solution:

 Prepare the stock of Krebs solution. Prepare the standard stock solution of serotonin
(1 mg/ml) and then different concentrations of serotonin by serial dilution method.
Procedure:
 Sacrifice the 24 hr fasted rat by stunning on the head and carotid bleeding. Fix the
animal on the dissecting board by tying its legs. Open the abdominal cavity by a small
horizontal cut followed by vertical midline incision and expose the abdominal organs.
 Identify the stomach and separate it from the abdomen by gently cutting its cardiac
and pyloric end. Place it in a petri dish containing aerated warm Krebs solution.
 Incise the fundus of the stomach (upper grey part) from pyloric part (pink and thick
part). Cut the fundus from the lesser curvature and open it longitudinally. Then cut the
fundus at the midline into two equal parts. Give alternate transverse cuts (zig-zag cut)
on opposite sides of the muscle to make a fundal strip preparation (as shown in fig.
9.1).
 Mount the preparation in the inner organ bath containing Krebs solution (20 ml)
maintained at 37ºC. Tie the bottom end of the muscle to the hook of aeration tube and
the upper end to the isotonic frontal lever by thread. Adjust the magnification of
response to 10-15 fold. Aerate the tissue with O2 or carbogen slowly (40-60 bubbles
per minutes).
 Apply 1 g load and stabilize the tissue for 30 min during which wash the tissue with
fresh Krebs solution once in every 10 min.
 Use a 5 min time cycle with contact time of 90s (since the fundus strip muscle
contracts slowly and relaxes slowly) for recording the contraction due to serotonin.
 Record the graded responses of standard solution and test solutionofserotonin.
gram.
REFERENCES
1. Tripathy KD.Essentials of Medical Pharmacology. 7th ed. Jaypee Brothers Medical
Publishers (P) Ltd, New Delhi, India, 2013.
2. Rang HP, Ritter JM, Flower RJ, Henderson G. Rang and Dale’s Pharmacology. 8th ed.
Churchill Livingstone, Philadelphia,2016.
3. Satoskar RS, Bhandarkar SD, Rege NN. Pharmacology and Pharmacotherapeutics.
21st ed. Popular PrakashanPvt. Ltd., Mumbai, India,2009.
45
4. Katzung BG, Masters SB, Trevor AJ. Basic & Clinical Pharmacology. 12th ed. The
McGraw-Hill Companies, New Delhi, India, 2012.
5. Ghosh MN.Fundamentals of Experimental Pharmacology. 3rd ed.Hliton& Company,
Kolkata, 2011.
6. Goyal RK.. Practical’s in Pharmacology. 8th ed. B.S. Shah Prakashan, Ahmedabad,
2007.
7. Kulkarni SK. Practical pharmacology and clinical pharmacy. 1st ed. Vallabh
Publications, Delhi, 2007.
8. Medhi B, Prakash A. Practical Manual of Experimental and Clinical Pharmacology.
1st ed. Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India, 2010.
9. Panigrahi G, Patra A. Experimental Pharmacology-II (Bridges the Gap Between
Animal Models and Computer Simulation Models). 1st ed.NiraliPrakashan, Pune
India, 2019.
46

BP503T Unit 4-6

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

BP503T Unit 4-6

Uploaded by

Copyright:

Available Formats

Study Material

B.Pharm 5th Semester

Sub: PHARMACOLOGY-II (Theory)

Sub. Code: BP503T

Topic: 1. Pharmacology of drugs acting on endocrine system

Dr. Bimalendu Chowdhury, M.Ph, PhD.

Professor, Roland Institute of Pharmaceutical Sciences, Berahampur

Teacher Regd. No:T080326711

Endocrine gland Hormone secretions

1. Activation of genome and increase their protein synthesis

b. Anterior Pituitary hormones- analogues and their inhibitors

1. Increase body growth

Hypersecretion of GH in the young causes “Gigantasim” and in adult it causes “acromegali”.

There are three types of GH analogues:

It is a peptide hormone secreted by acidophilic cells of anterior pituitary. It is responsible for

1. Pergolide and Cabergoline: These are ergot alkaloids.

c. Thyroid stimulating hormones- analogues and their inhibitors

Synthesis and release of thyroid hormones:

1. Iodide trapping 2. Oxidation of iodide 3. Coupling of iodotyrosines

Function of Thyroid hormones:

1. Increase calorigenesis and raise basal metabolic rate (BMR)

Deficiency of thyroid hormones called hypothyroidism, the disease associated with

Thyroid hormones analogues:

Anti-thyroid drug or thyroid hormone inhibitor:

1. Inhibitors of thyroxine synthesis: Propylthiouracil, Methylthiouracil, Methimazole,

1. Propylthiouracil, Methylthiouracil, Methimazole, Carbimazole: These are thioamide, inhibits

Adverse effect: Goitrogenic action, allergic reactions, leucopenia, and agranulocytosis

Therapeutic uses: Hypothyroidism (Graves’ disease)

Contraindication: Use should be minimized in pregnancy and lactating mother

Therapeutic uses: Selected cases of hyperthyroidism, thyroid carcinoma

Contraindication: Pregnancy and lactation

Therapeutic uses: Used to prepare hyperthyroid patients for surgery

1. Parathormone (PTH): It is a large polypeptide hormone containing 84 amino acids

Therapeutic use: Parathyroid injection 20 to 40 USP units twice daily subcutaneously or

2. Calcitonin: Calcitonin is a small polypeptide hormone with 32 amino acids, synthesized

Adverse effect: Mild hypocalcemia

3. Vitamin-D: Vitamin-D designated as a group of related sterols (contain cyclopentano-

Therapeutic use: Treatment of rickets and osteomalaia

Adverse effect: Hypervitaminosis-D, hypercalcaemia, hyperphosphataemia and metastatic

e. Insulin, Oral Hypoglycemic agents and glucagon

1. Glucagon: It is derived by the proteolytic cleavage of proglucagon (a large peptide). Its

Therapeutic uses: To treat severe hypoglycaemia or hypoglycemic coma due to insulin in

2. Insulin: Insulin is a polypeptide, composed of an A-chain (acidic) made up of 21 amino

Table.2. Insulin preparations

1. It is used as the specific replacement therapy in diabetes mellitus.

Adverse effect: Hypoglycemia, lipodystrophy, allergic manifestations, insulin resistance

3. Oral hypoglycemic agent:

1. Sulfonylureas: These are chemically related to sulfonamides but are deprived of

Therapeutic uses: (i) Maturity onset diabetes mellitus

Adverse effect: Hypoglycemia is a great risk if the meal is delayed or skipped.

4. Αlpha-glucosidase inhibitors: Example of α-glucosidase inhibitor is acarbose and

Adverse effect: Flatulence, diarrhea and abdominal pain.

5. Thiazolidinediones (Glitazones): Examples are Rosiglitazone, Pioglitazone and

Adverse effect: Weight gain, fluid retention, oedema and hepatotoxicity.

f. ACTH and corticosteroids

Table.3. Hormones of adrenal cortex

Sl Layer of adrenal cortex Hormones

1. The C-21 steroids: Progesterone, glucocorticoids, and mineralocorticoids

1. Glucocorticoids: They are classified into:

1. Anti-inflammatory: Glucocorticoids inhibits the inflammatory responses of body tissue to

Effect on organ system:

1. Cardiovascular system: Corticosteroids exerts positive inotropic effect.