Mbe Part 1

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-
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
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.
(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:
n1 T S1 n2 Conc. of Unknown= Cs X ---- X antilog ------------ X log ---X Dilution Factor t S2 S1 n1
Where,
n1 n2 t S1 S2 T Cs
= Lower std. dose = Higher std. dose = Test dose = Response of n1 = Response of n2 = Response of t = Conc. of Std.
In 4 point method two doses of standard and two doses of test, 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. Similarly one can design 8-point method also. BIOASSAY OF ANTAGONISTS: Commonly used method for the bioassay of antagonist is simple graphical method. 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. In this method, two responses of the same dose of agonist (sub maximal giving approximately 80% of the maximum response) are taken. The minimum 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 response of agonist is repeated every ten min. till recovery is obtained. The higher dose of standard, antagonist is added and responses are taken as before, three to four doses of the standard. Antagonist is used and than one to two doses of test sample of the antagonist is used similarly. The percentage inhibition is calculated, plotted against logdose and the concentration of unknown is determined as usual. BIOASSAY ON SOME IMPORTANT DRUGS: Depending upon pharmacological action of various drugs, different, preparations may be used. Following chart gives different preparations and the pharmacological activity for which a particular drug is assayed:
Sr. No.
DRUGS
PREPARATION
ACTIVITY ASSAYED
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M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE
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, Isolated Caucus of fowl, Isolated rat uterus Blood pressure of the pitched cat. Isolated rectus abdominus of frog, Rat ileum and leech dorsal muscle Isolated mouse heart Rat / Cat blood pressure Isolated, atropinized terminal ileum of guinea pig. Anaesthetized and atropinized cat. Isolated atropinized rat uterus, Isolated Terminal colon of rat, Isolated fundus Strip of rat stomach, Isolated heart of cat Perfused rabbit ear Rabbit Rat diaphragm with phrenic nerve, Cat Gastrocnemius muscle with sciatic nerve Sulfated whole blood of ox with thrombokinase extract and acetone dried ox brain. Suitable micro-organism grown on suitable nutrient agar medium Rats maintained on richetogenic diet. Rabbits Mice Isolated rat diaphragm Rats epididymal fat
Fall in blood pressure or stoppage of heart and death Rise in blood pressure Inhibition of tone Rise of B. P
05
Histamine
Inhibition of cardiac contractions. Fall in blood pressure. Contractile effect. Fall in blood pressure. Contractile effect
06
5 HydroxyTryptamine
07
08
Curariform drugs e.g. d-tubocurarine Heparin
Constriction of blood vessels Dropping of head Inhibition of the contractile effect Prolongation of blood clotting time. Inhibition of growth of microorganism. Alleviation of rachitic stage Lowering of blood-sugar level Convulsions and/or death due to hypoglycemia Increase in glycogen content. Increased metabolism of glucose, indicated by increase CO2 production. Vasodepressor activity. Contractile effect Ejection of milk from mammary duct Vasopressor activity. Gain in weight, Increase in width of epiphyseal cartilage Increase in testicular weight. Increase in weight of ovaries. Enlargement of prostate gland
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09 10 11
Antibiotics Vitamin D. Insulin
12
Oxytocin
Adult cockerel. Isolated rat uterus. Rabbits (female) Rat blood-pressure Hypophysectomized rats. Hypophysectomized male rats Hypophysectomized Female rats Immature male rats.
13 14 15 16
Vasopressin Growth hormone Gonadotrophin (FSH) Gonadotrophin
17 18
(LH) Gonadotrophin (FSH & LH) *Prolactin
Immature female rate. Cloves of pigeons. Female guinea-pig or rabbit Female rats.
Increase in weight of uterus. Increase in weight of crop sac. Secretory changes in mammary gland. Lengthening of estrous cycle and function of corpus luteum. Inhibition of estrogen upon vaginal smear Depletion of ascorbic acid from adrenal gland Release of previously administered 131I (Iodine) from thyroid gland. Increase in size of comb Increase in weight of prostate gland and seminal vesicles. Increase in weight of levatorani muscles. Increase in weight of uterus. Proliferative changes in endometrium of uterus or Increase In Carbonic anhydrase-activity in uterus.
Hypophysectomized rat. 19 20 *Corticotrophin *Thyrotropin Hypophosectomized rats. Mice or rats.
21
*Androgen
Castrated capon Castrated male rat. Castrated male rats.
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, which is prevailing, in various academic institutions. However, with advent of technology, availability of advanced sophisticated and more reliable analytical method the scenario for bioassay has changed dramatically. If one reviews the emphasis of bioassay in pharmacopoeia published before 1980 as compared to those published recently. It will be clear that: There are very few drugs which are now recommended to be assayed by biological method. Most of drugs, which were assayed by biological methods, are now being recommended to be assayed by chemical methods. Newer drugs have been included for which bioassay recommended.
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Teachers sign
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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. PRINCIPLE: This method is based on the assumption of the dose-response relationship. Acetylcholine produces a dose dependent contraction of rat ileum smooth muscle. Graded responses of acetylcholine are taken and two equipotent responses to unknown sample are taken. In simpler design, 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 height of the contraction is measured and Log-dose-response curve is plotted. 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. ADVANTAGES: Most simple method. Chances of error are less if the sensitivity of the preparation is not changed. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g.) Drugs : Acetylcholine (10 g/ml, 100 g/ml, 1 mg/ml) Apparatus : Students physiograph, Mammalian isolated organ bath, organ tube, Thermostat, isotonic frontal writing lever, recording drum, aeration tube cum Tissue holder, haemostatic forceps, sketch pen tip, 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. 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, the ileocaecal junction exposed, the terminal ileum was cut after discarding 10 cm nearest to the ileocaecal junction because of the presence of excitatory - adrenoreceptor near the
ileocaecal junction. It was placed in a petridish containing tyrode solution maintained at 370C. The mesenteric attachment was cut as close to the gut as possible without injury for a distance of about 20-25 cm. The intestine was then cut across, 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). Undue stretching, 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. (If strips were to be kept for further use, 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.) A small segment (2-3 cm, 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. 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 tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min. 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, the responses of acetylcholine 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). The height of contraction was measured and plotted against the log dose. 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 STD. ----------------Dose of TEST
Concentration of unknown =
X Conc. of STD. x dilution factor
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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. Some laboratories have reported use of isolated heart, intestine and tracheal preparations. However, overall experience dose not recommend these method as reliable, reproducible or accurate. Among whole animal experiments cat or rat blood pressure experiments have also been suggested. However, these are tiresome and not commonly used. Guinea-pigs Ileum: Guinea pig is killed by a below on the head and bled to death. The abdominal wall is dissected out so as to isolate the ileum; the faecal matter, mesentery and blood vessels are removed from the piece of ileum. It is ligated on both sides and suspended in mammalian at 37O C and oxygenated continuously. Acetylcholine contracts the ileum. This principle is utilized for its bioassay. The extent of contraction produced by the test sample is compared with the standard preparation of acetylcholine.
M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE - 11 -
Leech Dorsal Muscle: Compare the contractions produced by the standard and test samples on eserinised dorsal muscle of the leech. This muscle is highly sensitive (picograms) to acetylcholine. Isolated Heart Preparations: Rabbits auricle, frogs heart, rabbits heart or venous mercenerials heart is used. Ach decreases the force of contraction and rate of the heart. Rabbits Intestine and Tracheal chain: Ach contracts these tissues. Cats Blood Pressure: A cat is anaesthetized with suitable anesthetic. The carotid artery is cannulated for recording blood pressure femoral vein is cannulated for injecting acetylcholine. Trachea is cannulated for giving artificial respiration. Acetylcholine produces a fall in blood pressure by dilating peripheral blood vessels. This principle is utilized for its bioassay. The extent to which blood pressure falls due to the test sample is compared with the fall by the standard preparation. Anaesthetized Rats Blood Pressure: Compare the extent of fall in blood pressure of the test sample with that produced by the standard preparation. CALCULATION & OBSERVATIONS: Drug: Stock Concentration: Bath capacity:
OBSERVATION TABLE: Concentration of the drug (g/ml) Dose of Drug (ml) Height of Response of the drug (mm)
Sr. No.
GRAPH:
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. ----------------Dose of TEST X Conc. of STD. x Dilution Factor
RESULT:
The concentration of given unknown sample of the drug - _______________ is _____________ g/ml. Teachers sign
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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. PRINCIPLE: Acetylcholine produces a dose dependent contraction of rat ileum smooth muscle. Two responses of the standard acetylcholine 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 acetylcholine 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. and the concentration of unknown is determined by the formula.
REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g.) Drugs : Acetylcholine (10 g/ml, 100 g/ml, 1 mg/ml) Apparatus : Students physiograph, Mammalian isolated organ bath, organ tube, Thermostat, isotonic transducer & Coupler, aeration tube cum Tissue holder, haemostatic forceps, 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. 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, the ileocaecal junction exposed, the terminal ileum was cut after discarding 10 cm nearest to the ileocaecal junction because of the presence of excitatory - adrenoreceptor near the ileocaecal junction. It was placed in a petridish containing tyrode solution maintained at 370C. The mesenteric attachment was cut as close to the gut as possible without injury for a distance of about 20-25 cm. The intestine was then cut across, 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). Undue stretching, 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. (If strips were to be kept for further use, 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.) A small segment (2-3 cm, 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. 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 tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min. 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, the responses of acetylcholine were taken till maximum effect was obtained. Initially, two responses of the std. are taken. The doses are adjusted such that one is giving responses of aprrox.20% and other 70% of the maximum.
The response of unknown which lies between two responses of std dose is taken. The
panel is repeated by increasing or decreasing the dose of std. till all the equal responses are obtained. The dose of test sample is kept constant. At the end a response of the double dose of the std & test which match each other are taken. There should give equal responses. Then by using the following formula corresponding conc. of test is obtained. Dose of STD. Concentration of unknown = ----------------Dose of TEST STANDARD PATTERN:
CALCULATION & OBSERVATIONS

Drug: Stock Concentration: Bath capacity: OBSERVATION TABLE: Concentration of the drug (g/ml) Dose of Drug (ml) Height of Response of the drug (mm)
Sr. No.
CALCULATION: Dose of STD. Concentration of unknown = ----------------X Conc. of STD. x Dilution Factor Dose of TEST
RESULT: The concentration of given unknown sample of the drug - _______________ is _____________ g/ml. Teachers sign
Date: PRACTICAL-4 Bioassay of agonist-Acetylcholine by Three Point Method using rat ileum preparation
AIM: To perform the bioassay of acetylcholine by three point method using rat ileum. PRINCIPLE: Acetylcholine produces contractions of smooth muscle of rat ileum. In 3-point assay method, 2 doses of the standard acetylcholine (S1, S2) and one dose of the test acetylcholine (T) are used. The test dose is fixed in such a way that it gives the response between the responses produced by S1 & S2. S1 & S2 are the doses of standard that are 20% & 70% of the maximum response. These three selected doses are repeated by the Latin Square design method i.e. S1,S2,T S2,T,S1 T,S1,S2. 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. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g.) Drugs : Acetylcholine (10 g/ml, 100 g/ml, 1 mg/ml) Apparatus : Students physiograph, Mammalian isolated organ bath, organ tube, Thermostat, isotonic transducer & Coupler, aeration tube cum Tissue holder, haemostatic forceps, 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. 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, the ileocaecal junction exposed, the terminal ileum was cut after discarding 10 cm nearest to the ileocaecal junction because of the presence of excitatory - adrenoreceptor near the ileocaecal junction. It was placed in a petridish containing tyrode solution maintained at 370C. The mesenteric attachment was cut as close to the gut as possible without injury for a distance of about 20-25 cm. The intestine was then cut across, 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). Undue stretching, 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. (If strips were to be kept for further use,
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.) A small segment (2-3 cm, 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. 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 tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min. For maximum sensitivity the lever was nearly balanced, and the friction at the writing surface reduced to a minimum by smooth point. Response of smaller dose of acetylcholine approximately producing 20% of the response (S1) was taken. Consider the dose as n1. Response of higher dose of acetylcholine approximately producing 80% of the response (S2) was taken. Consider the dose as 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. Consider the dose as t. The sequence of responses was followed as per the Latin square method of randomization in order to avoid bias. Then responses were taken in pattern of S2-T-S1 and then T-S1-S2. 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. n1 Conc. of Unknown= Cs X ---- X antilog t Where, n1 n2 t S1 S2 T Cs T S1 n2 ---------- X log ---S2 S1 n1 = Lower std. dose = Higher std. dose = Test dose = Response of n1 = Response of n2 = Response of t = Conc. of Std.
X dilution factor
STANDARD PATTERN:
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S1
S2
S2
S1
S1
S2
CALCULATION & OBSERVATIONS: Drug: Stock Concentration: Bath capacity:
OBSERVATION TABLE: Concentration of the drug (g/ml) S1 S2 T Dose of Drug (ml) Height of Response of the drug (mm) I II III Mean
CALCULATION: n1 Conc. of Unknown= Cs X ---- X antilog t

T S1 n2 ---------- X log ---S2 S1 n1
X Dilution Factor
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Where,
n1 n2 t S1 S2 T Cs
= Lower std. dose = Higher std. dose = Test dose = Response of n1 = Response of n2 = Response of t = Conc. of Std.
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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. PRINCIPLE: Acetylcholine produces contractions of smooth muscle of rat ileum. In 3-point assay method, 2 doses of the standard acetylcholine (S1,S2) and one dose of the test acetylcholine(T) are used. The test dose is fixed in such a way that it gives the response between the responses produced by S1 & S2. S1 & S2 are the doses of standard that are 20% & 70% of the maximum response. Select two concentrations (A,B) of the standard drug, eliciting sub maximal responses (S1,S2) and bearing a dose ration 1:2 preferentially. 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. The higher volume of the test solution selected would be D such that the dose ratio B/A=D/C. All the four responses (S1,S2,T1,T2) due to the doses thus selected (A,B,C,D) must lie on the linear part of the standard (sigmoid) curve. 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. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g.) Drugs : Acetylcholine (10 g/ml, 100 g/ml, 1 mg/ml) Apparatus : Students physiograph, Mammalian isolated organ bath, organ tube, Thermostat, isotonic transducer & Coupler, aeration tube cum Tissue holder, haemostatic forceps, 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. 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, the ileocaecal junction exposed, the terminal ileum was cut after discarding 10 cm nearest to the ileocaecal junction because of the presence of
excitatory - adrenoreceptor near the ileocaecal junction. It was placed in a petridish containing tyrode solution maintained at 370C. The mesenteric attachment was cut as close to the gut as possible without injury for a distance of about 20-25 cm. The intestine was then cut across, 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). Undue stretching, 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. (If strips were to be kept for further use, 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.) A small segment (2-3 cm, 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. 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 tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min. For maximum sensitivity the lever was nearly balanced, and the friction at the writing surface reduced to a minimum by smooth point. Record graded response with the standard solution of acetylcholine until peak effect is obtained. Select two concentrations (A,B) of the standard drug, eliciting sub maximal responses (S1,S2) and bearing a dose ration 1:2 preferentially. 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. The higher volume of the test solution selected would be D such that the dose ratio B/A=D/C. All the four responses (S1, S2, T1, T2) due to the doses thus selected (A, B, C, D) must lie oh the linear part of the standard (sigmoid) curve. Standardize the tissue with concentration A. (Tissue is said to be standardized when it responds identically to the same concentration, when repeated). The sequence of responses was followed as per the Latin square method of randomization in order to avoid bias. Record four sets of responses due to A,B,C,D adding them to the organ bath in a randomized fashion. Any of the following latin squares may be used to ensure good randomization and to account for the fluctuating sensitivity of the tissue. 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, S2, T1, and T2). Calculate the potency ratio (M) using formula:
(T2-S2) + (T1-S1) Potency ratio = M = (x1/y1) X antilog -------------------- X log (x2/x1) (T2-T1) + (S2-S1) Where, x1 = Lower volume of std. Drug(A) x2 = Higher volume of std. 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), dilution factor for the test solution and the potency ratio (M).
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
B T 2
B T 2 S 2
D S 2
T 1 S 1 S 1
T 1
CALCULATION & OBSERVATIONS: Drug: Stock Concentration: Bath capacity:
OBSERVATION TABLE:
Concentration of the drug (g/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 ------------------- X log(x2/x1) (T2-T1) + (S2-S1) Where, x1 x2 y1 S&T
= Lower volume of std. Drug(A) = Higher volume of std. Drug(B) = Lower volume of test drug(C) = Represent mean response
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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. PRINCIPLE: Acetylcholine produces contractile responses on rat ileum through muscarinic receptors. Atropine is a muscarinic receptor blocker. Atropine has a very slow dissociation rate. Thus recovery is slow. One has to wait for a longer period of 10-15 min for the recovery to come. Also large dose of Ach would not speed up the removal of atropine. A blocking agent produces dose dependent competitive and reversible antagonist of acetylcholine. Hence graded responses of atropine in the form of inhibition of the fixed dose of acetylcholine can be determined. 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. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g.) Drugs : Acetylcholine (10 g/ml, 100 g/ml, 1 mg/ml), Atropine (1 g/ml, 10 g/ml, 100 g/ml) Apparatus : Students physiograph, Mammalian isolated organ bath, organ tube, Thermostat, isotonic transducer & Coupler, aeration tube cum Tissue holder, haemostatic forceps, 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. 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, the ileocaecal junction exposed, the terminal ileum was cut after discarding 10 cm nearest to the ileocaecal junction because of the presence of excitatory - adrenoreceptor near the ileocaecal junction. It was placed in a petridish containing tyrode solution maintained at 370C.
The mesenteric attachment was cut as close to the gut as possible without injury for a distance of about 20-25 cm. The intestine was then cut across, 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). Undue stretching, 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. (If strips were to be kept for further use, 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.) A small segment (2-3 cm, 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. 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 tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min. For maximum sensitivity the lever was nearly balanced, and the friction at the writing surface reduced to a minimum by smooth point. Two equipotent responses to sub maximal doses of acetylcholine were recorded. The drum was moved for 30 seconds and the lowest dose of Atropine was added in bath. After 2 minutes, responses to the same dose of acetylcholine were taken in the presence of atropine. The 5-minute cycle was followed as usual. The responses to acetylcholine were taken after every 5 minutes till the recovery to the control height was achieved. The response to acetylcholine in presence of higher dose of atropine was taken. At least 4 such dose dependent inhibitions were recorded. 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. In the same fashion the responses to acetylcholine were produced in presence of unknown solution of atropine at least twice. 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 = ------------------ X 100 control The percent inhibition was plotted on log graph and the concentration of unknown was then calculated out. Dose of STD. ----------------Dose of TEST CALCULATION & OBSERVATIONS: Concentration of unknown =
X Conc. of STD. x Dilution Factor
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Drug: Agonist Antagonist Stock Concentration: Agonist Antagonist Bath capacity: OBSERVATION TABLE: Sr. No. Concentration of the antagonist (g/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. 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. ----------------Dose of TEST X Conc. of STD. x dilution factor
Date: PRACTICAL-7
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. Histamine produces a dose dependent contraction of rat fundus smooth muscle. Graded responses of acetylcholine are taken and two equipotent responses to unknown sample are taken. In simpler design, 5-6 response of the graded doses of the standard are taken and then two equi-active response of the test sample are taken. The height of the contraction is measured and Log-dose-response curve is plotted. 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. Advantages: Most simple method. Chances of error are less if the sensitivity of the preparation is not changed. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g.) Drugs : Histamine (10 g/ml, 100 g/ml, 1 mg/ml) Apparatus : Students physiograph, Mammalian isolated organ bath, organ tube, Thermostat, isotonic transducer & Coupler, aeration tube cum Tissue holder, haemostatic forceps, 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. 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, and the stomach dissected out and placed in warm salt solution. 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 fundus was then cut open along the lesser curvature and spread on a cork mat soaked in salt solution. Alternative transverse cuts were then made to preserve the longitudinal muscle. 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. 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 tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min. 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, the responses of histamine 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 histamine (It is better to start with the diluted unknown solution). The height of contraction was measured and plotted against the log dose. 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 STD. ----------------Dose of TEST
RESULT: The concentration of given unknown sample of the drug- _______________ is _____________ g/ml.
DISSCUSSION : It is suitable for assay of 5-Hydroxytryptamine, being very sensitive to its action. It is 10 times less sensitive to acetylcholine, the effect of which can be blocked by hyoscine, while it is over 1000 times less sensitive to histamine. It is also used for the assay of PGE2.
Teachers sign
- 30 -
Date: PRACTICAL-7 Bioassay of agonist-histamine by Graphical Method using rat fundus preparation CALCULATION & OBSERVATIONS: Drug: Stock Concentration: Bath capacity: OBSERVATION TABLE: Sr. No. Concentration of the drug (g/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. 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
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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. Serotonin produces a dose dependent contraction of rat fundus smooth muscle. Graded responses of acetylcholine are taken and two equipotent responses to unknown sample are taken. In simpler design, 5-6 response of the graded doses of the standard are taken and then two equi-active response of the test sample are taken. The height of the contraction is measured and Log-dose-response curve is plotted. 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. Advantages: Most simple method. Chances of error are less if the sensitivity of the preparation is not changed. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g.) Drugs : Serotonin (10 g/ml, 100 g/ml, 1 mg/ml) Apparatus : Reservoir, tubing, Mammalian isolated organ bath, organ tube, heating coil, Thermostat, isotonic frontal writing lever, recording drum, aeration tube cum Tissue holder, haemostatic forceps, sketch pen tip, 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. 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, and the stomach dissected out and placed in warm salt solution. 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 fundus was then cut open along the lesser curvature and spread on a cork mat soaked in salt solution. Alternative transverse cuts were then made to preserve the longitudinal muscle. 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. 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 tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min. 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, the responses of serotonin 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 serotonin (It is better to start with the diluted unknown solution). The height of contraction was measured and plotted against the log dose. 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 STD. ----------------Dose of TEST
RESULT: The concentration of given unknown sample of the drug - _______________ is _____________ g/ml.
DISSCUSSION : It is suitable for assay of 5-Hydroxytryptamine, being very sensitive to its action. It is 10 times less sensitive to acetylcholine, the effect of which can be blocked by hyoscine, while it is over 1000 times less sensitive to histamine. It is also used for the assay of PGE2. Teachers sign
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Date: PRACTICAL-8 Bioassay of agonist-serotonin by Graphical Method using rat fundus preparation CALCULATION & OBSERVATIONS Drug: Stock Concentration: Bath capacity:
OBSERVATION TABLE: Sr. No. Concentration of the drug (g/ml) Dose of Drug (ml) Log dose of drug Height of Response of the drug (mm) Percentage height of contraction (%)
Date: PRACTICAL-9
Bioassay of agonist- 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. PRINCIPLE: This method is based on the assumption of the dose-response relationship. Dopamine produces a dose dependent contraction of rat vas deferens smooth muscle. Graded responses of acetylcholine are taken and two equipotent responses to unknown sample are taken. In simpler design, 5-6 response of the graded doses of the standard are taken and then two equi-active response of the test sample are taken. The height of the contraction is measured and Log-dose-response curve is plotted. 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. ADVANTAGES: Most simple method. Chances of error are less if the sensitivity of the preparation is not changed. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g.) Drugs : Dopamine (10 g/ml, 100 g/ml, 1 mg/ml) Apparatus : Reservoir, tubing, Mammalian isolated organ bath, organ tube, heating coil, Thermostat, isotonic frontal writing lever, recording drum, aeration tube cum Tissue holder, haemostatic forceps, sketch pen tip, 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. 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, and two vas deference dissected out and placed in warm salt solution.
- 35 -
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 tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min. 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, 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). The height of contraction was measured and plotted against the log dose. 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 STD. Concentration of unknown = ----------------X Conc. of STD. x dilution factor Dose of TEST RESULT: The concentration of given unknown sample of the drug - _______________ is _____________ g/ml. DISSCUSSION : In comparison with other smooth muscle like aorta, trachea, vas-deference is more easy to dissect. As it is available in pairs the control and the test preparation can be done from the same animal. It is good preparation to study sympathetic nerve trunk and its relations with drug. The vas-deference arises from the caudal epididymis which is situated at the posterior end of testes, leads back through the inguinal canal and crosses the ureter before joining the urethra. They are 5 to 7 cm in length covered by a thin layer of connective tissues and surrounded by fat cells. The vas-deference of rat is supplied with hypo gastric nerve. The muscle contains dense plexus of catecholamine neurons as adrenergic nerves extensively innervate it. 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. Thus some of the increase in sensitivity to adrenaline and nor adrenaline Teachers sign found after denervation can be attributed to this basis.
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Date: PRACTICAL-9 Bioassay of agonist- Dopamine by Graphical Method using rat vas deferens CALCULATION & OBSERVATIONS Drug: Stock Concentration: Bath capacity: OBSERVATION TABLE: Sr. No. Concentration of the drug (g/ml) Dose of Drug (ml) Log dose of drug Height of Response of the drug (mm) Percentage height of contraction (%)
Date: PRACTICAL-10
Bioassay of agonist- 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. Oxytocin produces a dose dependent contraction of rat uterine horns smooth muscle. Graded responses of acetylcholine are taken and two equipotent responses to unknown sample are taken. In simpler design, 5-6 response of the graded doses of the standard are taken and then two equi-active response of the test sample are taken. The height of the contraction is measured and Log-dose-response curve is plotted. 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. ADVANTAGES: Most simple method. Chances of error are less if the sensitivity of the preparation is not changed. REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g.) Drugs : Oxytocin (1-100 IU) Apparatus : Reservoir, tubing, Mammalian isolated organ bath, organ tube, heating coil, Thermostat, isotonic frontal writing lever, recording drum, aeration tube cum Tissue holder, haemostatic forceps, sketch pen tip, 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. A virgin female rat was injected 100 g/100 gms body weight of Diethylstilbestrol, 24 hours before it was sacrificed. 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 The abdominal cavity was quickly
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opened through a midline incision, and the uterine horns were dissected out. They was placed in a petridish containing de-Jalon solution maintained at 370C. 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 0.5-1 g. The tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min. 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, the responses of Oxytocin 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). The height of contraction was measured and plotted against the log dose. 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 STD. ----------------Dose of TEST
RESULT: The concentration of given unknown sample of the drug - _______________ is _____________ g/ml. DISSCUSSION : The rat uterus is chiefly used for the assay of Oxytocin, 5-hydroxytryptamine and adrenaline. On this tissue Acetylcholine, 5-Hydroxytryptamine, Oxytocin , Barium Chloride and Potassium chloride produces a contractile effect (spasmogenic effect ). Hence, such drugs are known as Spasmogenics. While histamine and adrenaline decreases responses of Potassium chloride and also relax the preparation previously contracted with a spsasmogen. Such drugs are known as Spasmolytics. Generally blockers act as spasmolytics but it is interesting to note spasmolytic property of histamine and adrenaline. Excitatory -adrenoreceptors have been shown to exist in the rat uterus only under certain conditions such as after estrogen treatment, during natural oestrus, in the late pregnancy, and for five to six days after partuation. The adrenoreceptors are temperature sensitive, and the initial excitation phase produced by several sympathomimetic amines is greatly reduced or even abolished by lowering the bath temperature to 25C. For the estimation of oxytocic activity , female rats are kept separated from males because pregnant animals are not suitable.
The rhythmic contractions normally present are abolished by using de-Jalons solution . To have relatively quiescent but sensitive uterus for routine assays, virgin rats are injected with stilbesterol 24 hours before sacrificing. For 5-hydroy tryptamine assay a relatively large dose of stilbestrol (0.25 mg/ 100 gm) is injected intra-peritoneally for three days before sacrificing. Histamine by acting on H2 receptors releases nor adrenaline which in turn relaxes uterine muscles. It is very sensitive to stimulation by posterior pituitary extract , bradykinin, substance P and adenosine compounds, and to inhibition of adrenaline and noradrenaline. Rats in natural oestrus may also be selected by microscopic examination of the vaginal smear. The uterus is generally still suitable when kept at 4C for 24 hours.
Teachers sign
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Date: PRACTICAL-10 Bioassay of agonist- Oxytocin by Graphical Method using rat uterine horns CALCULATION & OBSERVATIONS: Drug: Stock Concentration: Bath capacity:
Calculation: Dose of STD. ----------------Dose of TEST
Date:
PRACTICAL-11 Bioassay of agonist- 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. PRINCIPLE: Acetylcholine produces a dose dependent contraction of rat trachea smooth muscle. Graded responses of acetylcholine are taken and two equipotent responses to unknown sample are taken. The responses can be plotted against log dose of standard and the amount of standard, producing the same response as produced by unknown, is directly read from the graph and the concentration of unknown is determined by the formula.
REQUIREMENTS: Animals : Rat (of either sex weighing between 200-250g.) Drugs : Acetylcholine (10 g/ml, 100 g/ml, 1 mg/ml) Apparatus : Reservoir, tubing, Mammalian isolated organ bath, organ tube, heating coil, Thermostat, isotonic frontal writing lever, recording drum, aeration tube cum Tissue holder, haemostatic forceps, sketch pen tip, 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. A rat fasted over night was anaesthetized by chloroform and sacrificed by cutting neck blood vessels and bleeding the animal to death. The neck portion opened through a midline incision, the trachea dissected out. It was placed in a petridish containing Krebss solution maintained at 370C. A transverse cut between the segment of cartilage is given to get a number of rings of tracheal chain. Rings are tied together with cotton thread to form a chain. Preparation can also obtained by cutting tracheal chain into zigzag fashion. 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 0.5-1 g. The tissue was allowed to stabilize for 30 minutes during which period washing was given at an interval of 10 min.
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, the responses of Acetylcholine 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). The height of contraction was measured and plotted against the log dose. 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 STD. ----------------Dose of TEST
RESULT: The concentration of given unknown sample of the drug _______________is _____________ g/ml.
Teachers sign
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Date: PRACTICAL-11 Bioassay of agonist- Acetylcholine by Graphical Method using rat tracheal chains CALCULATION & OBSERVATIONS: Drug: Stock Concentration: Bath capacity:
- 44 -

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What are some important considerations for bioassays?

What are some important considerations for bioassays?

What are the different types of responses that can be measured in a bioassay?

What are the different types of responses that can be measured in a bioassay?

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

n1 T S1 n2 Conc. of Unknown= Cs X ---- X antilog ------------ X log ---X Dilution Factor t S2 S1 n1

M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

Nor adrenaline Acetylcholine

Curariform drugs e.g. d-tubocurarine Heparin

Antibiotics Vitamin D. Insulin

Vasopressin Growth hormone Gonadotrophin (FSH) Gonadotrophin

M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

(LH) Gonadotrophin (FSH & LH) *Prolactin

Hypophysectomized rat. 19 20 *Corticotrophin *Thyrotropin Hypophosectomized rats. Mice or rats.

Castrated capon Castrated male rat. Castrated male rats.

Rat or mouse (Female) Sexual immature rabbits

*Radioimmunoassay or radio receptor assay methods are also available

M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

X Conc. of STD. x dilution factor

M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

X Conc. of STD. x dilution factor

CALCULATION & OBSERVATIONS

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

CALCULATION & OBSERVATIONS: Drug: Stock Concentration: Bath capacity:

CALCULATION: n1 Conc. of Unknown= Cs X ---- X antilog t

T S1 n2 ---------- X log ---S2 S1 n1

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

CALCULATION & OBSERVATIONS: Drug: Stock Concentration: Bath capacity:

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

Concentration of the drug (g/ml) S1 S2 T1 T2

Dose of Drug (ml)

Height of Response of the drug (mm) I II III IIII Mean

M. Pharm II-semester (Pharmacology) ANAND PHARMACY COLLEGE

Hypophysectomized rat. 19 20 Corticotrophin Thyrotropin Hypophosectomized rats. Mice or rats.