20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

Date: PRACTICAL-1 Introduction to Bioassay AIM: Introduction to bioassay of drugs. DEFINITION: It is an estimation of the potency of an active principle in a unit quantity of preparation and measurement of the concentration of the substance in a preparation using biological method (i.e. observation of pharmacological effect on living tissues, microorganisms or immune cells or animal) is known as biological assay or bioassay. IMPORTANCE OF BIOASSAYS: Bioassays are essential in the development of new drugs. In the preclinical assessment of a new compound, the biological activity is compared with that of known compounds using appropriate test systems. The precision, reliability and reproducibility of bioassay depend on the proper selection of the tissue or method with highest selectivity and sensitivity for the drug. In spite of the tremendous advancement in the analytical chemistry and modern instrumentations, bioassay procedures continue to be used as successful tools not only in the estimation of bioactive substances but also for the discovery of biologically active substances. Bioassays are generally employed:  When active principle of drug is unknown or cannot be isolated.  When a chemical assay for the substance is not available or interacting with chemicals as the case with hormones inactivates the substance. Chemical method is too complex, insensitive or requires higher dose.  When the quantity of the sample is too small. In such situation a matching type of bioassay is conveniently done to compare the biological response with the standard drug.  To estimate the concentrations of active principles present in the tissue extracts, the endogenous mediators like acetyl choline, 5-HT, prostaglandins  To measure the pharmacological activity of new or chemically unidentified substances  To measure drug toxicity and  When bioassay is more sensitive than chemical assay. The purpose of bioassay is to ascertain the potency of a drug and hence serves as the quantitative part of any screening procedure. Other purpose of bioassay is to standardize the preparation so that each contains the uniform specified pharmacological activity, serve as pointer for the commercial production of drugs, and help diagnosis of various conditions. PRINCIPLE OF BIOASSAY: The basic principle of bioassay is to compare the substance with the international preparation of the same and to find out how much test substance is required to produce the same biological effect, as produced by standard. The problem of biological variation must
M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE -1-

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

be minimized as far as possible. For that one should keep uniform experimental conditions and assure the reproducibility of the responses. USE OF STANDARDS: Bioassays are designed to measure relative potency of two preparations, usually a standard and an unknown. It is unsatisfactory to designate a unit of particular drug at that amount and causes a particular effect because biological effects vary from animal to animal, time to time & from lab to lab. Use of standard substance for comparison also helps in solving problems arising from biological variations. The observed response/effect of the unknown would be always relative to the effect that produced by a standard substance. The standard substance is a pure substance and in official bioassays it refers to pharmacopoeial standards. In case of hormones, biological products and vaccines it is often necessary to establish the standard response of the standard substances against which unknown samples can be calibrated. DISADVANTAGES OF BIOASSAY:  Less accurate  Less elaborate  More laborious  More troublesome  More expensive PRECAUTIONS IN BIOASSAYS TO MINIMIZE BIOLOGICAL VARIATIONS:  All the experimental conditions should be constant.  The response studied should be reproducible.  The biological response being studied should be sensitive to the drug.  The animals should be of same species, strain, approximate of same age and weight and sex. Also should be kept on a similar diet and housed under similar conditions.

METHODS OF BIOASSAY FOR AGONISTS: An agonist may produce two types of response. [1] Quantal Response: Quantal means that the response is in the form of “all or none” i.e. either no response or maximum response. The drugs producing quantal effect can be bioassayed by end point method. End Point Method Here the threshold dose producing a positive effect is measured on each animal and the comparison between the average results of two groups of animals is done. For ex. Bioassay of digitalis in cats. Here the cat is anaesthetized with chloralose and its blood pressure is recorded. The drug is taken 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
M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE -2-

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

series of such experiments-one using standard digitalis and other using test preparation of digitalis is done and then potency is calculated as follows: Conc. of Unknown = (Threshold dose of Std. /Threshold dose of test) X Conc. of Std. In case, if it is not possible to measure individual effective dose or if animals are not available, fixed doses are injected into groups of animals and the percentage of mortality at each dose level is determined. The percentage of mortality is taken as the response and then the comparison is done in the same way as done for graded response. [2] Graded Response: Graded response means that the response is proportional to the dose and response may lie between no response and the maximum response. Graded response assays are based on the proportionate increase in the response in the response observed with an increase in the concentration or the dose of the drug. The parameter employed in such bioassays are based on the nature of the effect, the drug or substance is expected to produce. For ex. Contraction of smooth muscle of rat ileum for bioassay of acetylcholine, Relaxation of smooth muscle of rabbit ileum for bioassay of Adrenaline. The drugs producing graded responses can be bioassayed by: (A) Graphical method or interpolation method (B) Matching or bracketing method (C) Multiple point method The choice of the procedure or method depends upon precision or accuracy of assay, the quantity of test sample available, the availability of experimental animals. (A) Graphical method: This method is based on the assumption of the dose-response relationship. Log-doseresponse curve is plotted and the dose of standard producing the same response as produced by the test sample is directly read from the graph. In simpler design, 5-6 responses of the graded doses of the standard are taken and then two equiactive responses of the test sample are taken. The height of concentration is measured and plotted against the log-dose. The dose of standard producing the same response as produced by the test is read directly from the graph and the concentration of test sample is determined by the following formula. Conc. of Unknown = (Threshold dose of Std. /Threshold dose of test) X Conc. of Std. The characteristic of log-dose response curve is that it is linear in the middle (20-80%). Thus, the comparison should be done within this range only. In other words, the response of test sample must lie within this range. Advantages:  It is a simple method.  Chances of errors are less if the sensitivity of the preparation is not changed.
M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE -3-

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

(B)Matching Method: In this method a constant dose of the test is bracketed by varying doses of standard till the exact match is obtained between test dose and the standard dose. Initially, two responses of the standard are taken. The does are adjusted such that one is giving response of approximately 20% and other 70% of the maximum. The response of unknown that lies between two responses of standard dose is taken. The panel is repeated by increasing or decreasing the doses of standard till all three equal responses are obtained. The dose of test sample is kept constant. In the end, a response of the double dose of the standard and test that match each other are taken. These should give equal responses. Concentration of the test sample can be determined as follows: Conc. of Unknown = (Dose of Std. / Dose of Test) X Conc. of Std. Advantage: Useful when sensitivity is not stable.

Limitations: It occupies a larger area of the drum as far as tracings are concerned. The match is purely subjective, so chances of error are there and one cannot determine them. It does not give any idea of dose-response relationship. Method is not accurate and not reliable. Ex. Bioassay of histamine on guinea pig ileum is preferably carried out by this method. (C) Multiple point Bioassay: These methods include 3 point, 4 point, 5 point and 6-point methods. In these methods, the responses are repeated several times and the mean of each is taken. Thus, chances of error are minimized in these methods. In 3-point assay method, 2 doses of the standard and one dose of the test are used. Initially a graded dose response curve for the standard drug is taken. From this response two doses of the standard drug S1 & S2 are selected. The two doses should preferably be in the ratio of 1:2. The test dose is fixed in such a way that it gives the response between the responses produced by S1 & S2. These three selected doses are repeated by the Latin Square design method i.e. S1, S2, T – S2, T, S1 – T, S1, S2. In order to avoid bias. The mean responses are calculated and plotted against log-dose and amount of standard producing the same response as produced by the test is determined mathematically:
M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE -4-

BIOASSAY OF ANTAGONISTS: Commonly used method for the bioassay of antagonist is simple graphical method. The response of agonist is repeated every ten min. The minimum dose of standard. plotted against logdose and the concentration of unknown is determined as usual. of Std. in 5 point method three doses of standard and two doses of test. in 6 point method three doses of standard and three doses of test are used. DRUGS PREPARATION ACTIVITY ASSAYED -5- M. dose = Higher std. till recovery is obtained. In this method. n1 n2 t S1 S2 T Cs = Lower std. three to four doses of the standard. two responses of the same dose of agonist (sub maximal giving approximately 80% of the maximum response) are taken. Following chart gives different preparations and the pharmacological activity for which a particular drug is assayed: Sr. antagonist is added and responses are taken as before. different. BIOASSAY ON SOME IMPORTANT DRUGS: Depending upon pharmacological action of various drugs. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . of Unknown= Cs X ---. preparations may be used. Similarly one can design 8-point method also. Antagonist is used and than one to two doses of test sample of the antagonist is used similarly. dose = Test dose = Response of n1 = Response of n2 = Response of t = Conc.X antilog -----------. The higher dose of standard. antagonist is added in the bath and then the response of the same dose of agonist is taken in presence of antagonist. The responses are determined in the form of the percentage inhibition of the fixed dose of agonist. These are then plotted against the log dose of the antagonist and the concentration of unknown is determined by finding out the amount of standard producing the same effect as produced by the test.X log ---X Dilution Factor t S2 – S1 n1 Where. No. In 4 point method two doses of standard and two doses of test. The percentage inhibition is calculated.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs n1 T – S1 n2 Conc.

Rat ileum and leech dorsal muscle Isolated mouse heart Rat / Cat blood pressure Isolated. Increase in width of epiphyseal cartilage Increase in testicular weight. Enlargement of prostate gland -6- 09 10 11 Antibiotics Vitamin D. Vasodepressor activity. Insulin 12 Oxytocin Adult cockerel. Hypophysectomized male rats Hypophysectomized Female rats Immature male rats. Isolated atropinized rat uterus. Isolated heart of cat Perfused rabbit ear Rabbit Rat diaphragm with phrenic nerve. Suitable micro-organism grown on suitable nutrient agar medium Rats maintained on richetogenic diet. Increased metabolism of glucose. Isolated rat uterus Blood pressure of the pitched cat. Isolated rat uterus. Fall in blood pressure. d-tubocurarine Heparin Constriction of blood vessels Dropping of head Inhibition of the contractile effect Prolongation of blood clotting time.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs 01 02 Digitalis Adrenaline 03 04 Nor adrenaline Acetylcholine Cat blood pressure Guinea pig Blood pressure Blood pressure of the spinal cat Isolated rabbit duodenum. Fall in blood pressure. Contractile effect 06 5 HydroxyTryptamine 07 08 Curariform drugs e. Inhibition of growth of microorganism. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . atropinized terminal ileum of guinea – pig. Rabbits (female) Rat blood-pressure Hypophysectomized rats. Increase in weight of ovaries. Isolated Terminal colon of rat. indicated by increase CO2 production. Isolated rectus abdominus of frog. Cat Gastrocnemius muscle with sciatic nerve Sulfated whole blood of ox with thrombokinase extract and acetone dried ox brain. Alleviation of rachitic stage Lowering of blood-sugar level Convulsions and/or death due to hypoglycemia Increase in glycogen content. 13 14 15 16 Vasopressin Growth hormone Gonadotrophin (FSH) Gonadotrophin M. Contractile effect Ejection of milk from mammary duct Vasopressor activity. Rabbits Mice Isolated rat diaphragm Rat’s epididymal fat Fall in blood pressure or stoppage of heart and death Rise in blood pressure Inhibition of tone Rise of B. Isolated Caucus of fowl. P 05 Histamine Inhibition of cardiac contractions. Isolated fundus Strip of rat stomach. Gain in weight.g. Contractile effect. Anaesthetized and atropinized cat.

Castrated male rats. availability of advanced sophisticated and more reliable analytical method the scenario for bioassay has changed dramatically. Increase in weight of uterus. Secretory changes in mammary gland. which were assayed by biological methods.  Newer drugs have been included for which bioassay recommended. 22 23 Estrogen Progesterone Rat or mouse (Female) Sexual immature rabbits *Radioimmunoassay or radio receptor assay methods are also available CURRENT STATUS OF BIOASSAY: Above-mentioned discussion is an overview of bioassay. Mice or rats. Increase in size of comb Increase in weight of prostate gland and seminal vesicles. Cloves of pigeons.  Most of drugs. 19 20 *Corticotrophin *Thyrotropin Hypophosectomized rats. which is prevailing. If one reviews the emphasis of bioassay in pharmacopoeia published before 1980 as compared to those published recently. in various academic institutions. with advent of technology. 21 *Androgen Castrated capon Castrated male rat. However. Proliferative changes in endometrium of uterus or Increase In Carbonic anhydrase-activity in uterus. M. are now being recommended to be assayed by chemical methods.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs 17 18 (LH) Gonadotrophin (FSH & LH) *Prolactin Immature female rate. Increase in weight of levatorani muscles. Inhibition of estrogen upon vaginal smear Depletion of ascorbic acid from adrenal gland Release of previously administered 131I (Iodine) from thyroid gland. Hypophysectomized rat. Lengthening of estrous cycle and function of corpus luteum. Female guinea-pig or rabbit Female rats. It will be clear that:  There are very few drugs which are now recommended to be assayed by biological method. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE -7- . Increase in weight of crop sac. Increase in weight of uterus.

Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE -8- .20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Teacher’s sign M.

the terminal ileum was cut after discarding 10 cm nearest to the ileocaecal junction because of the presence of excitatory α . 5-6 responses of the graded doses of the standard are taken and then two equi-active response of the test sample are taken. the ileocaecal junction exposed. ADVANTAGES:  Most simple method.) Drugs : Acetylcholine (10 µg/ml. Mammalian isolated organ bath. 1 mg/ml) Apparatus : Student’s physiograph. Acetylcholine produces a dose dependent contraction of rat ileum smooth muscle. organ tube.  A rat fasted over night was anaesthetized by chloroform and sacrificed by cutting neck blood vessels and bleeding the animal to death.  The abdominal cavity was quickly opened through a midline incision.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Date: PRACTICAL-2 Bioassay of agonist-acetylcholine by Graphical Method using rat ileum preparation AIM: To find out the concentration of unknown sample of acetylcholine by graphical method using rat ileum. aeration tube cum Tissue holder. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE -9- . The dose of standard producing the same response as produced by the test sample is directly read from the graph and the concentration of unknown is determined by the formula. 100 µg/ml. Thermostat.adrenoreceptor near the M. ink etc. EXPERIMENTAL CONDITIONS: Physiological salt solution : Temperature : Basal Tension on lever : Sensitivity : Aeration : Contact Time : PROCEDURE:  The assembly was set up and arrangements were made for experimental conditions mentioned above. recording drum. The height of the contraction is measured and Log-dose-response curve is plotted. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g. haemostatic forceps. Graded responses of acetylcholine are taken and two equipotent responses to unknown sample are taken.  Chances of error are less if the sensitivity of the preparation is not changed. In simpler design. sketch pen tip. PRINCIPLE: This method is based on the assumption of the dose-response relationship. isotonic frontal writing lever.

10 - . It was placed in a petridish containing tyrode solution maintained at 370C.  Starting with the equipotent responses. Undue stretching. x dilution factor M. and the lumen of the isolated piece thoroughly cleaned by running warm salt solution repeatedly through the proximal opening with the help of 50 ml volumetric (bulb) pipette (held at an angle of about 20 – 30 degrees). and the friction at the writing surface reduced to a minimum by smooth point. Dose of STD. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . The tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min.  The height of contraction was measured and plotted against the log dose. depending on the size of the organ tube) was cut: a thread was passed through the lumen and the wall near the mesenteric attachment at each end with the help of a fine curved sewing needle and tied securely but without occluding the lumen.  For maximum sensitivity the lever was nearly balanced.  Two equi-active responses of test sample were taken in such a manner that the height of response lay in between 20% to 80% response of the acetylcholine (It is better to start with the diluted unknown solution). they should be better placed directly in ice-cold aerated salt solution and kept in refrigerator overnight: about two hour before use they should be transferred to salt solution at room temperature and actively aerated.  The clean strip of the intestine was then placed in fresh warm salt solution for a short period for acclimatization before being put up. of STD. The intestine was then cut across.  The tissue was mounted in mammalian organ bath in the up-right position and connected to isotonic frontal writing lever under a tension of 500 mg. The dose of standard producing the same response as produced by the test was read directly from the graph and the concentration of test sample was determined by the formula as mentioned below. ----------------Dose of TEST Concentration of unknown = X Conc. the responses of acetylcholine were taken till maximum effect was obtained. (If strips were to be kept for further use.  The mesenteric attachment was cut as close to the gut as possible without injury for a distance of about 20-25 cm.)  A small segment (2-3 cm.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs ileocaecal junction. ballooning or handling of the gut was avoided.

However. Among whole animal experiments cat or rat blood pressure experiments have also been suggested. M. It is ligated on both sides and suspended in mammalian at 37O C and oxygenated continuously. overall experience dose not recommend these method as reliable.11 - .20302: Pharmacometrics and Methods of Biological Evaluation of Drugs STANDARD PATTERN: OTHER METHODS FOR BIOASSAY OF ACETYLCHOLINE: Apart from using rectus abdominis muscle of frog or rat-ileum one can perform bioassay of acetylcholine on guinea-pig ileum and leech dorsal muscle. intestine and tracheal preparations. these are tiresome and not commonly used. The extent of contraction produced by the test sample is compared with the standard preparation of acetylcholine. Some laboratories have reported use of isolated heart. Acetylcholine contracts the ileum. However. The abdominal wall is dissected out so as to isolate the ileum. the faecal matter. reproducible or accurate. This principle is utilized for its bioassay. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . Guinea-pig’s Ileum: Guinea pig is killed by a below on the head and bled to death. mesentery and blood vessels are removed from the piece of ileum.

 GRAPH: M. This muscle is highly sensitive (picograms) to acetylcholine.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Leech Dorsal Muscle: Compare the contractions produced by the standard and test samples on eserinised dorsal muscle of the leech. Anaesthetized Rat’s Blood Pressure: Compare the extent of fall in blood pressure of the test sample with that produced by the standard preparation. Cat’s Blood Pressure: A cat is anaesthetized with suitable anesthetic. CALCULATION & OBSERVATIONS: Drug: Stock Concentration: Bath capacity: OBSERVATION TABLE: Concentration of the drug (µg/ml) Dose of Drug (ml) Height of Response of the drug (mm) Sr. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . No. Trachea is cannulated for giving artificial respiration. The extent to which blood pressure falls due to the test sample is compared with the fall by the standard preparation. The carotid artery is cannulated for recording blood pressure femoral vein is cannulated for injecting acetylcholine. Acetylcholine produces a fall in blood pressure by dilating peripheral blood vessels. rabbit’s heart or venous mercenerials’ heart is used. Rabbit’s Intestine and Tracheal chain: Ach contracts these tissues.12 - . Isolated Heart Preparations: Rabbit’s auricle. Ach decreases the force of contraction and rate of the heart. This principle is utilized for its bioassay. frog’s heart.

----------------Dose of TEST X Conc.13 - . of STD.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Plot Graph: Height of contraction (Y-axis) vs the Log dose of drug (X-axis) Height of contraction of test drug on y-axis = Dose on x-axis (taken as dose of standard in the formula)  CALCULATION: Concentration of unknown = Dose of STD. x Dilution Factor RESULT: M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE .

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs The concentration of given unknown sample of the drug ._______________ is _____________ µg/ml. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . Teacher’s sign M.14 - .

Mammalian isolated organ bath. The dose of test sample is kept constant. PRINCIPLE: Acetylcholine produces a dose dependent contraction of rat ileum smooth muscle. ink etc. The does are adjusted such that one is giving response of approximately 20% and other 70% of the maximum. and the lumen of the isolated piece thoroughly cleaned by running warm salt solution repeatedly through the M. the terminal ileum was cut after discarding 10 cm nearest to the ileocaecal junction because of the presence of excitatory α . The intestine was then cut across.  A rat fasted over night was anaesthetized by chloroform and sacrificed by cutting neck blood vessels and bleeding the animal to death. a response of the double dose of the standard and test that match each other are taken. In the end.  The mesenteric attachment was cut as close to the gut as possible without injury for a distance of about 20-25 cm.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Date: PRACTICAL-3 Bioassay of agonist-Acetylcholine by Matching Method using rat ileum preparation AIM: To find out the concentration of unknown sample of acetylcholine by matching method using rat ileum. and the concentration of unknown is determined by the formula.adrenoreceptor near the ileocaecal junction. EXPERIMENTAL CONDITIONS: Physiological salt solution : Temperature : Basal Tension on lever : Sensitivity : Aeration : Contact Time : PROCEDURE:  The assembly was set up and arrangements were made for experimental conditions mentioned above. 100 µg/ml. isotonic transducer & Coupler. It was placed in a petridish containing tyrode solution maintained at 370C.15 - .) Drugs : Acetylcholine (10 µg/ml. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . the ileocaecal junction exposed. organ tube. 1 mg/ml) Apparatus : Student’s physiograph. The abdominal cavity was quickly opened through a midline incision. The panel is repeated by increasing or decreasing the doses of standard till all three equal responses are obtained. haemostatic forceps. Two responses of the standard acetylcholine are taken. aeration tube cum Tissue holder. The response of unknown that lies between two responses of standard acetylcholine dose is taken. Thermostat. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g.

and the friction at the writing surface reduced to a minimum by smooth point.  Starting with the equipotent responses. they should be better placed directly in ice-cold aerated salt solution and kept in refrigerator overnight: about two hour before use they should be transferred to salt solution at room temperature and actively aerated. till all the equal responses are obtained. are taken. Dose of STD.  Initially. depending on the size of the organ tube) was cut: a thread was passed through the lumen and the wall near the mesenteric attachment at each end with the help of a fine curved sewing needle and tied securely but without occluding the lumen. (If strips were to be kept for further use.  The response of unknown which lies between two responses of std dose is taken. Undue stretching. The panel is repeated by increasing or decreasing the dose of std.16 - . The dose of test sample is kept constant. Concentration of unknown = ----------------Dose of TEST STANDARD PATTERN: X Conc. of test is obtained.  At the end a response of the double dose of the std & test which match each other are taken.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs proximal opening with the help of 50 ml volumetric (bulb) pipette (held at an angle of about 20 – 30 degrees). There should give equal responses. x dilution factor CALCULATION & OBSERVATIONS M.20% and other 70% of the maximum. two responses of the std.  For maximum sensitivity the lever was nearly balanced.)  A small segment (2-3 cm. The tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . The doses are adjusted such that one is giving responses of aprrox.  Then by using the following formula corresponding conc. of STD. the responses of acetylcholine were taken till maximum effect was obtained. ballooning or handling of the gut was avoided. The clean strip of the intestine was then placed in fresh warm salt solution for a short period for acclimatization before being put up. The tissue was mounted in mammalian organ bath in the up-right position and connected to isotonic frontal writing lever under a tension of 500 mg.

Teacher’s sign Date: PRACTICAL-4 Bioassay of agonist-Acetylcholine by Three Point Method using rat ileum preparation M.17 - . of STD. No._______________ is _____________ µg/ml. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE .  CALCULATION: Dose of STD. Concentration of unknown = ----------------X Conc. x Dilution Factor Dose of TEST RESULT: The concentration of given unknown sample of the drug .20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Drug: Stock Concentration: Bath capacity: OBSERVATION TABLE: Concentration of the drug (µg/ml) Dose of Drug (ml) Height of Response of the drug (mm) Sr.

18 - . isotonic transducer & Coupler. It was placed in a petridish containing tyrode solution maintained at 370C.  A rat fasted over night was anaesthetized by chloroform and sacrificed by cutting neck blood vessels and bleeding the animal to death. The mean responses are calculated and plotted against log-dose and amount of standard producing the same response as produced by the test is determined mathematically.S2. The abdominal cavity was quickly opened through a midline incision.e. S1. The clean strip of the intestine was then placed in fresh warm salt solution for a short period for acclimatization before being put up. PRINCIPLE: Acetylcholine produces contractions of smooth muscle of rat ileum.) Drugs : Acetylcholine (10 µg/ml. S1 & S2 are the doses of standard that are 20% & 70% of the maximum response.S2. M.  The mesenteric attachment was cut as close to the gut as possible without injury for a distance of about 20-25 cm. Undue stretching. organ tube. 1 mg/ml) Apparatus : Student’s physiograph. haemostatic forceps. The test dose is fixed in such a way that it gives the response between the responses produced by S1 & S2.T. S2) and one dose of the test acetylcholine (T) are used. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE .S1. Thermostat. and the lumen of the isolated piece thoroughly cleaned by running warm salt solution repeatedly through the proximal opening with the help of 50 ml volumetric (bulb) pipette (held at an angle of about 20 – 30 degrees). These three selected doses are repeated by the Latin Square design method i. ballooning or handling of the gut was avoided.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs AIM: To perform the bioassay of acetylcholine by three point method using rat ileum. In 3-point assay method. The intestine was then cut across.T – S2. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g. 2 doses of the standard acetylcholine (S1. the ileocaecal junction exposed. Mammalian isolated organ bath.adrenoreceptor near the ileocaecal junction. EXPERIMENTAL CONDITIONS: Physiological salt solution : Temperature : Basal Tension on lever : Sensitivity : Aeration : Contact Time : PROCEDURE:  The assembly was set up and arrangements were made for experimental conditions mentioned above. ink etc. the terminal ileum was cut after discarding 10 cm nearest to the ileocaecal junction because of the presence of excitatory α . (If strips were to be kept for further use. aeration tube cum Tissue holder. 100 µg/ml.S1 – T.

of Std. n1 n2 t S1 S2 T Cs T – S1 n2 ---------.  Responses of the test solution were taken in such a way that the height of response produced lie between the responses produced by n1 and n2 dose.X antilog t Where. Consider the dose as t.19 - .X log ---S2 – S1 n1 = Lower std. dose = Test dose = Response of n1 = Response of n2 = Response of t = Conc.  Response of higher dose of acetylcholine approximately producing 80% of the response (S2) was taken.  The mean responses were calculated and plotted against log dose and amount of standard producing the same response as produced by the test was determined mathematically.  Response of smaller dose of acetylcholine approximately producing 20% of the response (S1) was taken. and the friction at the writing surface reduced to a minimum by smooth point. depending on the size of the organ tube) was cut: a thread was passed through the lumen and the wall near the mesenteric attachment at each end with the help of a fine curved sewing needle and tied securely but without occluding the lumen. Consider the dose as n1. The tissue was mounted in mammalian organ bath in the up-right position and connected to isotonic frontal writing lever under a tension of 500 mg. of Unknown= Cs X ---.  n1 Conc.)  A small segment (2-3 cm. dose = Higher std. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . Consider the dose as n2. Then responses were taken in pattern of S2-T-S1 and then T-S1-S2. X dilution factor STANDARD PATTERN: M. The tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min.  The sequence of responses was followed as per the Latin square method of randomization in order to avoid bias.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs they should be better placed directly in ice-cold aerated salt solution and kept in refrigerator overnight: about two hour before use they should be transferred to salt solution at room temperature and actively aerated.  For maximum sensitivity the lever was nearly balanced.

20 - . of Unknown= Cs X ---.X log ---S2 – S1 n1 X Dilution Factor .20302: Pharmacometrics and Methods of Biological Evaluation of Drugs S1 S2 T S2 T S1 T S1 S2 CALCULATION & OBSERVATIONS: Drug: Stock Concentration: Bath capacity: OBSERVATION TABLE: Concentration of the drug (µg/ml) S1 S2 T Dose of Drug (ml) Height of Response of the drug (mm) I II III Mean  CALCULATION: n1 Conc.X antilog t M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE T – S1 n2 ---------.

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Where. n1 n2 t S1 S2 T Cs = Lower std. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . of Std. RESULT: The concentration of given unknown sample of the drug . dose = Higher std. dose = Test dose = Response of n1 = Response of n2 = Response of t = Conc.21 - ._______________ is _____________ µg/ml. Teacher’s sign M.

100 µg/ml. In 3-point assay method. The test dose is fixed in such a way that it gives the response between the responses produced by S1 & S2. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . the terminal ileum was cut after discarding 10 cm nearest to the ileocaecal junction because of the presence of M. All the four responses (S1. The higher volume of the test solution selected would be D such that the dose ratio B/A=D/C. aeration tube cum Tissue holder. 2 doses of the standard acetylcholine (S1. 1 mg/ml) Apparatus : Student’s physiograph. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g.S2) and bearing a dose ration 1:2 preferentially. organ tube. Thermostat. S1 & S2 are the doses of standard that are 20% & 70% of the maximum response. Select two concentrations (A. Select two suitable volumes of the test solution by trial and error method in such a way that the response (T1) due to the lower dose of the test (C) lies preferentially between S1 & S2. eliciting sub maximal responses (S1. The abdominal cavity was quickly opened through a midline incision.C. isotonic transducer & Coupler.  A rat fasted over night was anaesthetized by chloroform and sacrificed by cutting neck blood vessels and bleeding the animal to death. EXPERIMENTAL CONDITIONS: Physiological salt solution : Temperature : Basal Tension on lever : Sensitivity : Aeration : Contact Time : PROCEDURE:  The assembly was set up and arrangements were made for experimental conditions mentioned above.22 - .T1.S2) and one dose of the test acetylcholine(T) are used. These four selected doses are repeated by the Latin Square design method The mean responses are calculated and concentration of the test is determined mathematically. Mammalian isolated organ bath.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Date: PRACTICAL-5 Bioassay of agonist-Acetylcholine by Four point method using rat ileum preparation AIM: To perform the bioassay of acetylcholine by four point method using rat ileum.D) must lie on the linear part of the standard (sigmoid) curve. the ileocaecal junction exposed.B. haemostatic forceps. ink etc.B) of the standard drug. PRINCIPLE: Acetylcholine produces contractions of smooth muscle of rat ileum.S2.T2) due to the doses thus selected (A.) Drugs : Acetylcholine (10 µg/ml.

B. and the lumen of the isolated piece thoroughly cleaned by running warm salt solution repeatedly through the proximal opening with the help of 50 ml volumetric (bulb) pipette (held at an angle of about 20 – 30 degrees). It was placed in a petridish containing tyrode solution maintained at 370C. and T2). STANDARDIZATION AND FOUR CYCLES USING LATIN SQUARE DESIGN ABCD ABCD ABCD ABCD BCDA BADC BDAC BADC CDAB CDBA CADB CDAB DABC DCAB DCBA DCBA  Measure various responses were measure to calculate the mean of each response (S1. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . ballooning or handling of the gut was avoided. The tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min. Any of the following latin squares may be used to ensure good randomization and to account for the fluctuating sensitivity of the tissue. T2) due to the doses thus selected (A.adrenoreceptor near the ileocaecal junction. The higher volume of the test solution selected would be D such that the dose ratio B/A=D/C.)  A small segment (2-3 cm.  For maximum sensitivity the lever was nearly balanced. S2.  Select two suitable volumes of the test solution by trial and error method in such a way that the response (T1) due to the lower dose of the test (C) lies preferentially between S1 & S2. Record four sets of responses due to A. T1.  Record graded response with the standard solution of acetylcholine until peak effect is obtained. and the friction at the writing surface reduced to a minimum by smooth point. S2. All the four responses (S1. T1. Undue stretching. The intestine was then cut across.  Select two concentrations (A.S2) and bearing a dose ration 1:2 preferentially.  The mesenteric attachment was cut as close to the gut as possible without injury for a distance of about 20-25 cm.23 - . The tissue was mounted in mammalian organ bath in the up-right position and connected to isotonic frontal writing lever under a tension of 500 mg. they should be better placed directly in ice-cold aerated salt solution and kept in refrigerator overnight: about two hour before use they should be transferred to salt solution at room temperature and actively aerated.  The sequence of responses was followed as per the Latin square method of randomization in order to avoid bias. (If strips were to be kept for further use. when repeated). C. D) must lie oh the linear part of the standard (sigmoid) curve. depending on the size of the organ tube) was cut: a thread was passed through the lumen and the wall near the mesenteric attachment at each end with the help of a fine curved sewing needle and tied securely but without occluding the lumen. eliciting sub maximal responses (S1.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs excitatory α .B) of the standard drug. The clean strip of the intestine was then placed in fresh warm salt solution for a short period for acclimatization before being put up.  Standardize the tissue with concentration A.  Calculate the potency ratio (M) using formula: M. (Tissue is said to be standardized when it responds identically to the same concentration.D adding them to the organ bath in a randomized fashion.B.C.

X log (x2/x1) (T2-T1) + (S2-S1) Where. STANDARD PATTERN: Fu pi tmhd or o n eo t T 2 S 2 T 1 S 1 S 2 T 1 S 1 T 2 A B T 2 C D B T 2 S 2 C D S 2 A T 1 S 1 S 1 T 1 C D A B D A B C CALCULATION & OBSERVATIONS: Drug: Stock Concentration: Bath capacity: OBSERVATION TABLE: M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE .24 - .20302: Pharmacometrics and Methods of Biological Evaluation of Drugs (T2-S2) + (T1-S1) Potency ratio = M = (x1/y1) X antilog -------------------. Drug(B) y1 = Lower volume of test drug(C) S & T = Represent mean response  Determine the strength of unknown solution of acetylcholine using the concentration of the standard (1 mg/ml). Drug(A) x2 = Higher volume of std. dilution factor for the test solution and the potency ratio (M). x1 = Lower volume of std.

_______________ is _____________ µg/ml.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Concentration of the drug (µg/ml) S1 S2 T1 T2 Dose of Drug (ml) Height of Response of the drug (mm) I II III IIII Mean  CALCULATION: (T2-S2) + (T1-S1) Potency Ratio= M =(x1/y1) X antilog ------------------. Drug(B) = Lower volume of test drug(C) = Represent mean response RESULT: The concentration of given unknown sample of the drug . Teacher’s sign M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE .25 - . x1 x2 y1 S&T = Lower volume of std. Drug(A) = Higher volume of std.X log(x2/x1) (T2-T1) + (S2-S1) Where.

Atropine (1 µg/ml. 100 µg/ml. Thermostat. the ileocaecal junction exposed. Thus recovery is slow.  A rat fasted over night was anaesthetized by chloroform and sacrificed by cutting neck blood vessels and bleeding the animal to death. the terminal ileum was cut after discarding 10 cm nearest to the ileocaecal junction because of the presence of excitatory α . 10 µg/ml. Also large dose of Ach would not speed up the removal of atropine. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . EXPERIMENTAL CONDITIONS: Physiological salt solution : Temperature : Basal Tension on lever : Sensitivity : Aeration : Contact Time : PROCEDURE:  The assembly was set up and arrangements were made for experimental conditions mentioned above. 1 mg/ml). organ tube. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g. PRINCIPLE: Acetylcholine produces contractile responses on rat ileum through muscarinic receptors. Mammalian isolated organ bath. It was placed in a petridish containing tyrode solution maintained at 370C.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Date: PRACTICAL-6 Bioassay of antagonist-Atropine by Graphical Method using rat ileum preparation AIM: To find out the concentration of unknown sample of atropine by graphical method using rat ileum. ink etc. M.26 - . 100 µg/ml) Apparatus : Student’s physiograph. The percent inhibition is plotted against log dose of atropine and the concentration of unknown is determined by finding out the amount of standard producing same response (inhibition) as produced by unknown. Atropine is a muscarinic receptor blocker. haemostatic forceps. One has to wait for a longer period of 10-15 min for the recovery to come. The abdominal cavity was quickly opened through a midline incision. Atropine has a very slow dissociation rate. Hence graded responses of atropine in the form of inhibition of the fixed dose of acetylcholine can be determined.) Drugs : Acetylcholine (10 µg/ml. A blocking agent produces dose dependent competitive and reversible antagonist of acetylcholine.adrenoreceptor near the ileocaecal junction. aeration tube cum Tissue holder. isotonic transducer & Coupler.

At least 4 such dose dependent inhibitions were recorded.  The responses to acetylcholine were taken after every 5 minutes till the recovery to the control height was achieved. ----------------Dose of TEST CALCULATION & OBSERVATIONS: Concentration of unknown = M. The intestine was then cut across. Dose of STD. x Dilution Factor . and the friction at the writing surface reduced to a minimum by smooth point.)  A small segment (2-3 cm. and the lumen of the isolated piece thoroughly cleaned by running warm salt solution repeatedly through the proximal opening with the help of 50 ml volumetric (bulb) pipette (held at an angle of about 20 – 30 degrees). In case the recovery was not achieved even after repeated doses then either a large dose of acetylcholine was given for recovery or the preceding height was taken as control for the next dose.  Two equipotent responses to sub maximal doses of acetylcholine were recorded.X 100 control  The percent inhibition was plotted on log graph and the concentration of unknown was then calculated out. The response to acetylcholine in presence of higher dose of atropine was taken. The drum was moved for 30 seconds and the lowest dose of Atropine was added in bath. of STD. responses to the same dose of acetylcholine were taken in the presence of atropine. depending on the size of the organ tube) was cut: a thread was passed through the lumen and the wall near the mesenteric attachment at each end with the help of a fine curved sewing needle and tied securely but without occluding the lumen.  In the same fashion the responses to acetylcholine were produced in presence of unknown solution of atropine at least twice.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs  The mesenteric attachment was cut as close to the gut as possible without injury for a distance of about 20-25 cm. The clean strip of the intestine was then placed in fresh warm salt solution for a short period for acclimatization before being put up. ballooning or handling of the gut was avoided.  Heights of control (s = in absence of atropine) and test (t = in presence of atropine) were measured and the % inhibition was calculated as follows: control – test % Inhibition = -----------------. The 5-minute cycle was followed as usual. After 2 minutes. (If strips were to be kept for further use. The tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min. they should be better placed directly in ice-cold aerated salt solution and kept in refrigerator overnight: about two hour before use they should be transferred to salt solution at room temperature and actively aerated. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE X Conc. The tissue was mounted in mammalian organ bath in the up-right position and connected to isotonic frontal writing lever under a tension of 500 mg.  For maximum sensitivity the lever was nearly balanced. Undue stretching.27 - .

x dilution factor RESULT: The concentration of given unknown sample of the drug . Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . Concentration of the antagonist (µg/ml) Dose of antagonist (ml) Height of response of agonist in absence of antagonist (control response “C” mm) Height of response of agonist in presence of antagonist (Test response “T” mm) Percentage inhibition of control response C-T x 100 C (%)  GRAPH: Plot Graph: Percentage inhibition of the control response (Y-axis) vs.28 - . No.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Drug: Agonist Antagonist Stock Concentration: Agonist Antagonist Bath capacity: OBSERVATION TABLE: Sr. The Log dose of antagonist (X-axis) Height of contraction of test drug on y-axis = dose on x-axis (taken as dose of standard in the formula)  CALCULATION: Concentration of unknown = Dose of STD._______________ is _____________ µg/ml. of STD. Teacher’s sign Date: PRACTICAL-7 M. ----------------Dose of TEST X Conc.

ink etc. 100 µg/ml. The dose of standard producing the same response as produced by the test sample is directly read from the graph and the concentration of unknown is determined by the formula. The translucent fundus (rumen) was cut along the pylorus (thick and red ) leaving a thin band of the pyloric tissue attached to the fundus and its contents were washed clean. 5-6 response of the graded doses of the standard are taken and then two equi-active response of the test sample are taken. Chances of error are less if the sensitivity of the preparation is not changed. and the stomach dissected out and placed in warm salt solution. 1 mg/ml) Apparatus : Student’s physiograph.  A rat fasted over night was anaesthetized by chloroform and sacrificed by cutting neck blood vessels and bleeding the animal to death. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g. Alternative transverse cuts were then made to preserve the longitudinal muscle. isotonic transducer & Coupler. The height of the contraction is measured and Log-dose-response curve is plotted. Mammalian isolated organ bath. Thermostat. - EXPERIMENTAL CONDITIONS: Physiological salt solution : Temperature : Basal Tension on lever : Sensitivity : Aeration : Contact Time : PROCEDURE:  The assembly was set up and arrangements were made for experimental conditions mentioned above. Advantages: Most simple method. Graded responses of acetylcholine are taken and two equipotent responses to unknown sample are taken. organ tube. In simpler design.  The fundus was then cut open along the lesser curvature and spread on a cork mat soaked in salt solution.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Bioassay of agonist-Histamine by Graphical Method using rat fundus preparation AIM: To find out the concentration of unknown sample of histamine by graphical method using rat fundus strip PRINCIPLE: This method is based on the assumption of the dose-response relationship. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . Histamine produces a dose dependent contraction of rat fundus smooth muscle. aeration tube cum Tissue holder.) Drugs : Histamine (10 µg/ml. The abdominal cavity was quickly opened through a midline incision. The strip was then pulled out by cotton thread tied on each end and M. haemostatic forceps.29 - .

It is also used for the assay of PGE2.30 - . Teacher’s sign M. DISSCUSSION : It is suitable for assay of 5-Hydroxytryptamine.  Two equi-active responses of test sample were taken in such a manner that the height of response lay in between 20% to 80% response of the histamine (It is better to start with the diluted unknown solution). Dose of STD. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . It is 10 times less sensitive to acetylcholine. the effect of which can be blocked by hyoscine. the responses of histamine were taken till maximum effect was obtained. The tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min._______________ is _____________ µg/ml. The dose of standard producing the same response as produced by the test was read directly from the graph and the concentration of test sample was determined by the formula as mentioned below. of STD.  The height of contraction was measured and plotted against the log dose. x dilution factor RESULT: The concentration of given unknown sample of the drug.  For maximum sensitivity the lever was nearly balanced. and the friction at the writing surface reduced to a minimum by smooth point.  Starting with the equipotent responses. being very sensitive to its action.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs protrusion and fringes of the pyloric tissue trimmed away to give a long clean thin strip for suspension in the bath. while it is over 1000 times less sensitive to histamine. ----------------Dose of TEST Concentration of unknown = X Conc.  The tissue was mounted in mammalian organ bath in the up-right position and connected to isotonic frontal writing lever under a tension of 500 mg.

31 - . No.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Date: PRACTICAL-7 Bioassay of agonist-histamine by Graphical Method using rat fundus preparation CALCULATION & OBSERVATIONS: Drug: Stock Concentration: Bath capacity: OBSERVATION TABLE: Sr. of STD. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . the Log dose of drug (X-axis) Height of contraction of test drug on y-axis = dose on x-axis (taken as dose of standard in the formula)  CALCULATION: Dose of STD. ----------------Dose of TEST Concentration of unknown = X Conc. Concentration of the drug (µg/ml) Dose of Drug (ml) Log dose of drug Height of Response of the drug (mm) Percentage height of contraction (%)  GRAPH: Plot Graph: Height of contraction (Y-axis) vs. x Dilution Factor M.

heating coil. aeration tube cum Tissue holder. 5-6 response of the graded doses of the standard are taken and then two equi-active response of the test sample are taken. 1 mg/ml) Apparatus : Reservoir. sketch pen tip. organ tube. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g. Chances of error are less if the sensitivity of the preparation is not changed. EXPERIMENTAL CONDITIONS: Physiological salt solution : Temperature : Basal Tension on lever : Sensitivity : Aeration : Contact Time : PROCEDURE:  The assembly was set up and arrangements were made for experimental conditions mentioned above. tubing. Advantages: Most simple method. 100 µg/ml.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Date: PRACTICAL-8 Bioassay of agonist-Serotonin by Graphical Method using rat fundus preparation AIM: To find out the concentration of unknown sample of Serotonin by graphical method using rat fundus strip PRINCIPLE: This method is based on the assumption of the dose-response relationship. and the stomach dissected out and placed in warm salt solution. Mammalian isolated organ bath. The dose of standard producing the same response as produced by the test sample is directly read from the graph and the concentration of unknown is determined by the formula. Thermostat. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . The abdominal cavity was quickly opened through a midline incision. Graded responses of acetylcholine are taken and two equipotent responses to unknown sample are taken. M. In simpler design. Serotonin produces a dose dependent contraction of rat fundus smooth muscle. recording drum.  A rat fasted over night was anaesthetized by chloroform and sacrificed by cutting neck blood vessels and bleeding the animal to death. isotonic frontal writing lever. haemostatic forceps.32 - - .) Drugs : Serotonin (10 µg/ml. ink etc. The translucent fundus (rumen) was cut along the pylorus (thick and red ) leaving a thin band of the pyloric tissue attached to the fundus and its contents were washed clean. The height of the contraction is measured and Log-dose-response curve is plotted.

x dilution factor RESULT: The concentration of given unknown sample of the drug . The dose of standard producing the same response as produced by the test was read directly from the graph and the concentration of test sample was determined by the formula as mentioned below.  The tissue was mounted in mammalian organ bath in the up-right position and connected to isotonic frontal writing lever under a tension of 500 mg. Teacher’s sign M. of STD._______________ is _____________ µg/ml. the responses of serotonin were taken till maximum effect was obtained. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . and the friction at the writing surface reduced to a minimum by smooth point.  Starting with the equipotent responses. The tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min.  Two equi-active responses of test sample were taken in such a manner that the height of response lay in between 20% to 80% response of the serotonin (It is better to start with the diluted unknown solution). Alternative transverse cuts were then made to preserve the longitudinal muscle.33 - . DISSCUSSION : It is suitable for assay of 5-Hydroxytryptamine.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs  The fundus was then cut open along the lesser curvature and spread on a cork mat soaked in salt solution. while it is over 1000 times less sensitive to histamine. being very sensitive to its action. Dose of STD.  For maximum sensitivity the lever was nearly balanced. the effect of which can be blocked by hyoscine. It is 10 times less sensitive to acetylcholine.  The height of contraction was measured and plotted against the log dose. It is also used for the assay of PGE2. The strip was then pulled out by cotton thread tied on each end and protrusion and fringes of the pyloric tissue trimmed away to give a long clean thin strip for suspension in the bath. ----------------Dose of TEST Concentration of unknown = X Conc.

34 - . of STD. No. Concentration of the drug (µg/ml) Dose of Drug (ml) Log dose of drug Height of Response of the drug (mm) Percentage height of contraction (%)  GRAPH: Plot Graph: Height of contraction (Y-axis) vs. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE .20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Date: PRACTICAL-8 Bioassay of agonist-serotonin by Graphical Method using rat fundus preparation CALCULATION & OBSERVATIONS Drug: Stock Concentration: Bath capacity: OBSERVATION TABLE: Sr. x dilution factor Date: PRACTICAL-9 M. ----------------Dose of TEST Concentration of unknown = X Conc. the Log dose of drug (X-axis) Height of contraction of test drug on y-axis = dose on x-axis (taken as dose of standard in the formula)  CALCULATION: Dose of STD.

EXPERIMENTAL CONDITIONS: Physiological salt solution : Temperature : Basal Tension on lever : Sensitivity : Aeration : Contact Time : PROCEDURE:  The assembly was set up and arrangements were made for experimental conditions mentioned above.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Bioassay of agonist. The height of the contraction is measured and Log-dose-response curve is plotted. 1 mg/ml) Apparatus : Reservoir. Chances of error are less if the sensitivity of the preparation is not changed. Dopamine produces a dose dependent contraction of rat vas deferens smooth muscle. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . recording drum.) Drugs : Dopamine (10 µg/ml. sketch pen tip.Dopamine by Graphical Method using rat vas deferens preparation AIM: To find out the concentration of unknown sample of Dopamine by graphical method using rat vas deference. organ tube.  A rat fasted over night was anaesthetized by chloroform and sacrificed by cutting neck blood vessels and bleeding the animal to death. ADVANTAGES: Most simple method. 5-6 response of the graded doses of the standard are taken and then two equi-active response of the test sample are taken. tubing. The dose of standard producing the same response as produced by the test sample is directly read from the graph and the concentration of unknown is determined by the formula. isotonic frontal writing lever. Graded responses of acetylcholine are taken and two equipotent responses to unknown sample are taken. 100 µg/ml. Mammalian isolated organ bath. PRINCIPLE: This method is based on the assumption of the dose-response relationship. ink etc. and two vas deference dissected out and placed in warm salt solution. Thermostat. heating coil. haemostatic forceps. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g.   M. In simpler design.35 - . The abdominal cavity was quickly opened through a midline incision. aeration tube cum Tissue holder.

The dose of standard producing the same response as produced by the test was read directly from the graph and the concentration of test sample was determined by the formula as mentioned below.  For maximum sensitivity the lever was nearly balanced. x dilution factor Dose of TEST RESULT: The concentration of given unknown sample of the drug . It has been reported that simply stripping away the serous coat after removing the vas-deference from the animal increases the sensitivity of the vas deference to drugs by facilitating access of drugs to the smooth muscle cells. As it is available in pairs the control and the test preparation can be done from the same animal. They are 5 to 7 cm in length covered by a thin layer of connective tissues and surrounded by fat cells. leads back through the inguinal canal and crosses the ureter before joining the urethra. It is good preparation to study sympathetic nerve trunk and its relations with drug. DISSCUSSION : In comparison with other smooth muscle like aorta.36 - . The vas-deference of rat is supplied with hypo gastric nerve.  Starting with the equipotent responses. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . of STD. Dose of STD. vas-deference is more easy to dissect. The tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min. The muscle contains dense plexus of catecholamine neurons as adrenergic nerves extensively innervate it._______________ is _____________ µg/ml. Concentration of unknown = ----------------X Conc.  The height of contraction was measured and plotted against the log dose. The vas-deference arises from the caudal epididymis which is situated at the posterior end of testes. M.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs  The tissue was mounted in mammalian organ bath in the up-right position and connected to isotonic frontal writing lever under a tension of 500 mg. Thus some of the increase in sensitivity to adrenaline and nor adrenaline Teacher’s sign found after denervation can be attributed to this basis. the responses of Dopamine were taken till maximum effect was obtained.  Two equi-active responses of test sample were taken in such a manner that the height of response lay in between 20% to 80% response of the acetylcholine (It is better to start with the diluted unknown solution). trachea. and the friction at the writing surface reduced to a minimum by smooth point.

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Date: PRACTICAL-9 Bioassay of agonist. of STD. x dilution factor Date: PRACTICAL-10 M.37 - . Concentration of the drug (µg/ml) Dose of Drug (ml) Log dose of drug Height of Response of the drug (mm) Percentage height of contraction (%)  GRAPH: Plot Graph: Height of contraction (Y-axis) vs. No.Dopamine by Graphical Method using rat vas deferens CALCULATION & OBSERVATIONS Drug: Stock Concentration: Bath capacity: OBSERVATION TABLE: Sr. the Log dose of drug (X-axis) Height of contraction of test drug on y-axis = dose on x-axis (taken as dose of standard in the formula)  CALCULATION: Dose of STD. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . ----------------Dose of TEST Concentration of unknown = X Conc.

tubing. EXPERIMENTAL CONDITIONS: Physiological salt solution : Temperature : Basal Tension on lever : Sensitivity : Aeration : Contact Time : PROCEDURE:  The assembly was set up and arrangements were made for experimental conditions mentioned above. The dose of standard producing the same response as produced by the test sample is directly read from the graph and the concentration of unknown is determined by the formula. Graded responses of acetylcholine are taken and two equipotent responses to unknown sample are taken. 5-6 response of the graded doses of the standard are taken and then two equi-active response of the test sample are taken. 24 hours before it was sacrificed. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g. Oxytocin produces a dose dependent contraction of rat uterine horns smooth muscle. haemostatic forceps. Thermostat.  A virgin female rat was injected 100 µg/100 gms body weight of Diethylstilbestrol. ADVANTAGES:  Most simple method. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . recording drum. heating coil. In simpler design.38 - .) Drugs : Oxytocin (1-100 IU) Apparatus : Reservoir. aeration tube cum Tissue holder.  A rat fasted over night was anaesthetized by chloroform and sacrificed by cutting neck blood vessels and bleeding the animal to death The abdominal cavity was quickly M. Mammalian isolated organ bath.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Bioassay of agonist. isotonic frontal writing lever. organ tube. sketch pen tip.Oxytocin by Graphical Method using rat uterine horns AIM: To find out the concentration of unknown sample of Oxytocinn by graphical method using rat uterine horns PRINCIPLE: This method is based on the assumption of the dose-response relationship. The height of the contraction is measured and Log-dose-response curve is plotted. ink etc. The assembly was set up and arrangements were made for experimental conditions mentioned above.  Chances of error are less if the sensitivity of the preparation is not changed.

While histamine and adrenaline decreases responses of Potassium chloride and also relax the preparation previously contracted with a spsasmogen. and for five to six days after partuation. and the friction at the writing surface reduced to a minimum by smooth point. On this tissue Acetylcholine. Oxytocin .5-1 g. Hence. M.39 - . Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . Barium Chloride and Potassium chloride produces a contractile effect (spasmogenic effect ).20302: Pharmacometrics and Methods of Biological Evaluation of Drugs opened through a midline incision. during natural oestrus. 5-hydroxytryptamine and adrenaline. x dilution factor RESULT: The concentration of given unknown sample of the drug . and the initial excitation phase produced by several sympathomimetic amines is greatly reduced or even abolished by lowering the bath temperature to 25°C. They was placed in a petridish containing de-Jalon solution maintained at 370C.  For maximum sensitivity the lever was nearly balanced. Such drugs are known as Spasmolytics. The α adrenoreceptors are temperature sensitive. The dose of standard producing the same response as produced by the test was read directly from the graph and the concentration of test sample was determined by the formula as mentioned below. ----------------Dose of TEST Concentration of unknown = X Conc. DISSCUSSION : The rat uterus is chiefly used for the assay of Oxytocin. in the late pregnancy. of STD. 5-Hydroxytryptamine.  The entire uterine horn was mounted in mammalian organ bath in the up-right position and connected to isotonic frontal writing lever under a tension of 0. The tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min. Excitatory α-adrenoreceptors have been shown to exist in the rat uterus only under certain conditions such as after estrogen treatment. and the uterine horn’s were dissected out.  The height of contraction was measured and plotted against the log dose. female rats are kept separated from males because pregnant animals are not suitable. For the estimation of oxytocic activity ._______________ is _____________ µg/ml.  Starting with the equipotent responses.  Two equi-active responses of test sample were taken in such a manner that the height of response lay in between 20% to 80% response of the acetylcholine (It is better to start with the diluted unknown solution). such drugs are known as Spasmogenics. the responses of Oxytocin were taken till maximum effect was obtained. Generally blockers act as spasmolytics but it is interesting to note spasmolytic property of histamine and adrenaline. Dose of STD.

Teacher’s sign M. virgin rats are injected with stilbesterol 24 hours before sacrificing. Rats in natural oestrus may also be selected by microscopic examination of the vaginal smear. substance P and adenosine compounds. For 5-hydroy tryptamine assay a relatively large dose of stilbestrol (0. Histamine by acting on H2 receptors releases nor adrenaline which in turn relaxes uterine muscles. The uterus is generally still suitable when kept at 4°C for 24 hours. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . bradykinin.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs The rhythmic contractions normally present are abolished by using de-Jalon’s solution . and to inhibition of adrenaline and noradrenaline.25 mg/ 100 gm) is injected intra-peritoneally for three days before sacrificing. To have relatively quiescent but sensitive uterus for routine assays.40 - . It is very sensitive to stimulation by posterior pituitary extract .

----------------Dose of TEST Concentration of unknown = X Conc. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE .20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Date: PRACTICAL-10 Bioassay of agonist. the Log dose of drug (X-axis) Height of contraction of test drug on y-axis = dose on x-axis (taken as dose of standard in the formula)  Calculation: Dose of STD. Concentration of the drug (µg/ml) Dose of Drug (ml) Log dose of drug Height of Response of the drug (mm) Percentage height of contraction (%)  GRAPH: Plot Graph: Height of contraction (Y-axis) vs. of STD.Oxytocin by Graphical Method using rat uterine horns CALCULATION & OBSERVATIONS: Drug: Stock Concentration: Bath capacity: OBSERVATION TABLE: Sr.41 - . x Dilution Factor Date: M. No.

sketch pen tip.  A transverse cut between the segment of cartilage is given to get a number of rings of tracheal chain. recording drum. aeration tube cum Tissue holder. It was placed in a petridish containing Krebs’s solution maintained at 370C.) Drugs : Acetylcholine (10 µg/ml. isotonic frontal writing lever. is directly read from the graph and the concentration of unknown is determined by the formula. Rings are tied together with cotton thread to form a chain. the trachea dissected out.Acetylcholine by Graphical Method using rat tracheal chains AIM: To find out the concentration of unknown sample of acetylcholine by graphical method using rat tracheal chain. heating coil. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g. organ tube. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . M. Graded responses of acetylcholine are taken and two equipotent responses to unknown sample are taken. Thermostat. The tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min. haemostatic forceps. Preparation can also obtained by cutting tracheal chain into zigzag fashion. ink etc.  The entire tracheal chain was mounted in mammalian organ bath in the up-right position and connected to isotonic frontal writing lever under a tension of 0. EXPERIMENTAL CONDITIONS: Physiological salt solution : Temperature : Basal Tension on lever : Sensitivity : Aeration : Contact Time : PROCEDURE:  The assembly was set up and arrangements were made for experimental conditions mentioned above.  A rat fasted over night was anaesthetized by chloroform and sacrificed by cutting neck blood vessels and bleeding the animal to death. PRINCIPLE: Acetylcholine produces a dose dependent contraction of rat trachea smooth muscle. producing the same response as produced by unknown. Mammalian isolated organ bath.42 - .5-1 g.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs PRACTICAL-11 Bioassay of agonist. The neck portion opened through a midline incision. The responses can be plotted against log dose of standard and the amount of standard. tubing. 1 mg/ml) Apparatus : Reservoir. 100 µg/ml.

 Two equi-active responses of test sample were taken in such a manner that the height of response lay in between 20% to 80% response of the acetylcholine (It is better to start with the diluted unknown solution).43 - .20302: Pharmacometrics and Methods of Biological Evaluation of Drugs  For maximum sensitivity the lever was nearly balanced. the responses of Acetylcholine were taken till maximum effect was obtained. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE . x dilution factor RESULT: The concentration of given unknown sample of the drug _______________is _____________ µg/ml.  The height of contraction was measured and plotted against the log dose. of STD. and the friction at the writing surface reduced to a minimum by smooth point.  Starting with the equipotent responses. The dose of standard producing the same response as produced by the test was read directly from the graph and the concentration of test sample was determined by the formula as mentioned below. Teacher’s sign M. Dose of STD. ----------------Dose of TEST Concentration of unknown = X Conc.

the Log dose of drug (X-axis) Height of contraction of test drug on y-axis = dose on x-axis (taken as dose of standard in the formula)  CALCULATION: Dose of STD. ----------------Dose of TEST Concentration of unknown = X Conc. No. of STD.20302: Pharmacometrics and Methods of Biological Evaluation of Drugs Date: PRACTICAL-11 Bioassay of agonist.44 - . Concentration of the drug (µg/ml) Dose of Drug (ml) Log dose of drug Height of Response of the drug (mm) Percentage height of contraction (%)  GRAPH: Plot Graph: Height of contraction (Y-axis) vs. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE .Acetylcholine by Graphical Method using rat tracheal chains CALCULATION & OBSERVATIONS: Drug: Stock Concentration: Bath capacity: OBSERVATION TABLE: Sr. x dilution factor M.

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