Pharmacology-II Unit-V Past Paper PDF

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 Ana...

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 5th Semester Pharmacology-II (Theory) Unit-V 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 5th Semester Pharmacology-II (Theory) Unit-V 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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V 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 5th Semester Pharmacology-II (Theory) Unit-V 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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V  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. Mechanism of action:  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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V 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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V  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. Mechanism of 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 5th Semester Pharmacology-II (Theory) Unit-V 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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V 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 5th Semester Pharmacology-II (Theory) Unit-V  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. Mechanism of action:  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 5th Semester Pharmacology-II (Theory) Unit-V 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 5th Semester Pharmacology-II (Theory) Unit-V 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 5th Semester Pharmacology-II (Theory) Unit-V 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 5th Semester Pharmacology-II (Theory) Unit-V 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 5th Semester Pharmacology-II (Theory) Unit-V 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 5th Semester Pharmacology-II (Theory) Unit-V 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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V 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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V 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 5th Semester Pharmacology-II (Theory) Unit-V 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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V  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 5th Semester Pharmacology-II (Theory) Unit-V produces effects by acting on the histamine receptors (H1, H2, H3 and H4) present