BIO-ASSAY OF OXYTOCIN

By

S.Rohith Yadav

Roll.No:1008-10-885-012

M-Pharmacy 1st Year 1st Semester

Pharmaceutical Analysis & Quality Assurance

University College Of Technology,

Osmania University,

Hyderabad.

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CONTENTS

Introduction to Oxytocin harmone

Physiological effects of Oxytocin

Control of Oxytocin secretion

BIO-ASSAY OF OXYTOCIN(I.P)

Different Formulations of oxytocin

Description

Identification

Tests

Assay for determination of Potency

References

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Oxytocin

Oxytocin is a mammalian harmone that acts primarily as a neuro-modulator in the brain. Also known as alpha-hypophamine (α–hypophamine), oxytocin has the distinction of being the very first polypeptide hormone to be sequenced and synthesized biochemically, by Vincent du Vigneaud et al. in 1953.

Oxytocin in a nine amino acid peptide that is synthesized in hypothalamic neurons and transported down axons of the posterior pituitary for secretion into blood. Oxytocin is also secreted within the brain and from a few other tissues, including the ovaries and testes. Oxytocin differs from antidiuretic hormone in two of the nine amino acids. Both hormones are packaged into granules and secreted along with carrier proteins called neurophysins.

Physiologic Effects of Oxytocin

In years past, oxytocin had the reputation of being an "uncomplicated" hormone, with only a few well-defined activities related to birth and lactation. As has been the case with so many hormones, further research has demonstrated many subtle but profound influences of this little peptide, particularly in regards to its effects in the brain. Oxytocin has been implicated in setting a number of social behaviors in species ranging from mice to humans. For example, secretion or administration of oxytocin in humans appears to enhance trust and cooperation within socially-close groups, while promoting defensive aggression toward unrelated, competing groups.

Oxytocin has been best studied in females where it clearly mediates three major effects:

Stimulation of milk ejection (milk letdown): Milk is initially secreted into small sacs within the mammary gland called alveoli, from which it must be ejected for consumption or harvesting. Mammary alveoli are surrounded by smooth muscle (myoepithelial) cells which are a prominant target cell for

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oxytocin. Oxytocin stimulates contraction of myoepithelial cells, causing milk to be ejected into the ducts and cisterns.

Stimulation of uterine smooth muscle contraction at birth: At the end of gestation, the uterus must contract vigorously and for a prolonged period of time in order to deliver the fetus. During the later stages of gestation, there is an increase in abundance of oxytocin receptors on uterine smooth muscle cells, which is associated with increased "irritability" of the uterus (and sometimes the mother as well). Oxytocin is released during labor when the fetus stimulates the cervix and vagina, and it enhances contraction of uterine smooth muscle to facilitate parturition or birth.

In cases where uterine contractions are not sufficient to complete delivery, physicians and veterinarians sometimes administer oxytocin ("pitocin") to further stimulate uterine contractions - great care must be exercised in such situations to assure that the fetus can indeed be delivered and to avoid rupture of the uterus.

Establishment of maternal behavior: Successful reproduction in mammals demands that mothers become attached to and nourish their offspring immediately after birth. It is also important that non-lactating females do not manifest such nurturing behavior. The same events that affect the uterus and mammary gland at the time of birth also affect the brain. During parturition, there is an increase in concentration of oxytocin in cerebrospinal fluid, and oxytocin acting within the brain plays a major role in establishing maternal behavior.

Evidence for this role of oxytocin come from two types of experiments. First, infusion of oxytocin into the ventricles of the brain of virgin rats or non-pregnant sheep rapidly induces maternal behavior. Second, administration into the brain of antibodies that neutralize oxytocin or of oxytocin antagonists will prevent mother rats from accepting their pups. Other studies support the contention that this behavioral effect of oxytocin is broadly applicable among mammals.

While all of the effects described above certainly occur in response to oxytocin, doubt has recently been cast on its necessity in parturition and

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maternal behavior. Mice that are unable to secrete oxytocin due to targeted disruptions of the oxytocin gene will mate, deliver their pups without apparent difficulty and display normal maternal behavior. However, they do show deficits in milk ejection and have subtle derangements in social behavior. It may be best to view oxytocin as a major facilitator of parturition and maternal behavior rather than a necessary component of these processes.

Control of Oxytocin Secretion:

The most important stimulus for release of hypothalamic oxytocin is initiated by physical stimulation of the nipples or teats. The act of nursing or suckling is relayed within a few milliseconds to the brain via a spinal reflex arc. These signals impinge on oxytocin-secreting neurons, leading to release of oxytocin.

If you want to obtain anything other than trivial amounts of milk from animals like dairy cattle, you have to stimulate oxytocin release because something like 80% of the milk is available only after ejection, and milk ejection requires oxytocin. Watch someone milk a cow, even with a machine, and what you'll see is that prior to milking, the teats and lower udder are washed gently - this tactile stimulation leads to oxytocin release and milk ejection.

A number of factors can inhibit oxytocin release, among them acute stress. For example, oxytocin neurons are repressed by catecholamine’s, which are released from the adrenal gland in response to many types of stress, including fright. As a practical endocrine tip - don't wear a gorilla costume into a milking parlor full of cows or set off firecrackers around a mother nursing her baby.

Both the production of oxytocin and response to oxytocin are modulated by circulating levels of sex steroids. The burst of oxytocin released at birth seems to be triggered in part by cervical and vaginal stimulation by the fetus, but also because of abruptly declining concentrations of progesterone. Another well-studied effect of steroid hormones is the marked increase in synthesis of uterine (myometrial) oxytocin receptors late in gestation, resulting from increasing concentrations of circulating estrogen.

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Bio-Assay of Oxytocin

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-GlyNH2C43H66N12O12S2 {Mol. Wt.1007.2}

Oxytocin is a cyclic nonapeptide hormone obtained by a process of fractionation from the posterior lobe of the pituitary gland of healthy oxen or other mammals or by synthesis that has the property of stimulating contraction of the uterus and milk ejection in receptive animals. It may be presented as a solid or as a solution in a solvent containing an appropriate antimicrobial preservative such as 0.2 per cent w/v solution of chlorbutol.

If it is derived from animal species, Oxytocin contains not less than 90.0 per cent and not more than 111.0 per cent of the stated number of Units of oxytocic activity. If it is a synthetic product presented as a solid, it contains not less than 560 Units per mg, calculated with reference to the peptide content and when presented as a liquid, it contains not lessthan 150 Units per ml.

Description. When presented as a solid, a white or almost white powder. When presented as a liquid, a clear colourless liquid.IdentificationGives rise to an appropriate response when administered as directed under one of the methods described for Assay.TestsPeptide. 90.0 to 110.0 per cent of the stated amount of oxytocin, C43H66N12O12S2

expressed per mg for the solid, and in mg per ml for the liquid,Determine by liquid chromatography (2.4.14).Test solution. Dissolve 3.5 mg of the substance under examination in sufficient of a 1.56 per cent w/v solution of sodium dihydrogen phosphate to produce 10.0 ml or use the liquid preparation as appropriate.Reference solution. Dissolve 3.5 mg of oxytocin RS in sufficient of a 1.56 per cent w/v solution of sodium dihydrogen phosphate to produce 10.0 ml.

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Chromatographic system– a stainless steel column 12 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 μm),– mobile phase: appropriate proportions of a 1.56 per cent w/v solution of sodium dihydrogen phosphate (mobile phase A) and a mixture of equal volumes of acetonitrile and water (mobile phase B),– flow rate. 1 ml per minute,– spectrophotometer set at 220 nm,– a 20 μl loop injector.Equilibrate the column with a mixture of 70 volumes of mobile phase A and 30 volumes of mobile phase B and record the chromatograms as follows. Operate by gradient elution increasing continuously and linearly the proportion of mobile phase B by 1.0 per cent v/v per minute for 30 minutes. Finally elute using the same mixture for 15 minutes to re-equilibrate the column.Calculate the content of the peptide, C43H66N12O12S2.Assay.The potency of oxytocin is determined by comparing its activity with that of the Standard Preparation of oxytocin under the conditions of a suitable method of assay.Standard PreparationThe Standard Preparation is the 4th International Standard for Oxytocin, established in 1978, consisting of freeze-dried synthetic oxytocin peptide with human albumin and citric acid (supplied in ampoules containing 12.5 Units), or any other suitable preparation the potency of which has been determined in relation to the International Standard.NOTE — Any of the following methods may be followed.Method A. By depression of the blood pressure in chicken —Anaesthetise a young healthy adult cockerel weighing 1.2 to 2.3 kg with an anaesthetic that will maintain a prolonged and constant high blood pressure. Expose the gluteus primus muscle in one thigh and cut and retract it to reveal the popliteal artery and crural vein. Cannulate the popliteal artery and record the blood pressure on a suitable recorder calibrated for useover a linear range. Cannulate the crural or brachial vein. Immediately before use prepare a solution of the Standard Preparation in saline solution so that the volume to be injected is between 0.1 ml and 0.5 ml. Record the blood pressure responses to the injection into the cannulated vein of two doses of this solution; the doses should be such as to produce clearly discriminated, precipitous, submaximal decreases in blood pressure; the required doses normally lie between 20 and 100 milli Units. The

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interval between injections should be constant and lie between 3 and 10 minutes depending on the rate at which the blood pressure returns to normal.Immediately before use dilute the preparation being examined with saline solution so as to obtain responses similar to those obtained with the Standard Preparation. The ratio between the two doses of the preparation under examination should be the same as that between the two doses of the StandardPreparation and this ratio should be kept constant throughout the assay. The two doses of the Standard Preparation and the two doses of the preparation under examination should be given according to a randomised block or a Latin square design and at least six responses to each should be recorded.If the animal rapidly becomes insensitive to the repeated injections of the solutions another animal must be used. Measure all the responses and calculate the result of the assay by standard statistical methods.Method B. By contraction of the rat uterus — Inject 100 μg of oestradiol benzoate intramuscularly into a female rat weighing 120 to 200 g 18 to 24 hours before the assay. Immediately before the assay confirm by vaginal smear that the rat is in oestrus or preoestrus. Kill the rat and suspend one horn of the uterus in a bath containing a solution of the following composition.

Composition (per cent w/v)Sodium chloride 0.662

Potassium chloride 0.045Calcium chloride 0.007

Sodium bicarbonate 0.256 Disodium hydrogen phosphate 0.029 Sodium dihydrogen phosphate 0.003

Magnesium chloride 0.010 Dextrose 0.050

Maintain the bath at a temperature of 32º or at some other suitable temperature at which spontaneous contractions of the uterus are abolished and the preparation maintains its sensitivity. Oxygenate the solution with a mixture of 95 per cent of oxygen and 5 per cent of carbon dioxide and record the contractions of the muscle using a suitable instrument giving a linear response (for example an isotonic lever with a load not exceeding 2 g). Record the contractions produced bythe addition to the bath of two doses of the Standard Preparation suitably diluted with the above solution. The doses should be such as to produce clearly

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discriminated, submaximal contractions; the required doses normally lie between 10 and 50 micro Units per ml of bath liquid. When maximal contraction has been reached, replace the bath liquid by a fresh solution. The doses should be added at regular intervals of 3 to 5 minutes depending upon the rate of recovery of the muscle. Dilute the preparation under examination so as to obtain responses on the addition of two doses similar to those obtained with the Standard Preparation. The ratio between the two doses of the preparation under examination should be the same as that between the two doses of the Standard Preparation and this ratio should be kept constant throughout the assay. The two doses of Standard Preparation and the two doses of the preparation under examination should be given according to a randomised block or a Latin square design and at least six responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods.

Method C. By measurement of milk-ejection pressure in a lactating rat— Select a lactating rat, in the third to twentyfirst day after parturition and weighing about 300 g, separate it from the litter and 30 to 60 minutes later anaesthetise (for example, by the intraperitoneal injection of a solution of Pentobarbitone Sodium). Tie the rat to an operating table, maintained at 37º, by its hind legs leaving the front legs free. Cannulate the trachea with a short polyethylene tube of internal diameter about 2.5 mm in such a manner so as to ensure a free airway; apply artificial respiration only if necessary. Cannulate an external jugular or femoral vein with a polyethylene tube of internal diameter about 0.4 mm whichis filled with saline solution and closed with a pin. Shave the skin surrounding the inguinal and abdominal teats and excise the tip of one teat, preferably the lower inguinal teat. Insert a polyethylene tube of internal diameter about 0.3mm and external diameter about 0.6 mm, to a depth sufficient to obtain appropriate measurement of pressure (3 to 10 mm depth), into the primary teat duct which opens onto the cut surface and tie firmly in place with a ligature. Connect this cannula with a suitable strain gauge transducer (such as that used for recording arterial blood pressure in the rat) and fill the whole system with a 3.8 per cent w/v solution of sodium citrate or saline solution containing 50 Units of heparin sodium per ml to prevent clotting of milk. After cannulation, inject a small volume (0.05 to 0.2 ml) of this solution into the teat duct through the transducer to clear the milk from the tip of the cannula. (This procedure may be repeated during the assay should obstruction arise from milk ejected into the cannula). Clamp the strain gauge so that a slight tension is applied to the teat and its natural

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alignment is preserved and connect the gauge to a potentiometric recorder adjusted to give full-scale deflection for an increase in milk-ejection pressure of about 5.3 kPa. Inject all solutions through the venous cannula using a 1-ml syringe graduated in 0.01 ml and wash them in with 0.2 ml of saline solution. Prepare a solution of the Standard Preparation and a solution of the preparation under examination in saline solution so that the volume to be injected is between 0.1 ml and 0.4 ml.Choose two doses of the Standard Preparation such that the increase in milk-ejection pressure is about 1.35 kPa for the lower dose and about 2.7 kPa for the higher dose. As an initial approximation, a lower dose of between 0.1 and 0.4 milliUnit and an upper dose of 1.5 to 2 times this amount may be tried. Choose two doses of the preparation under examination with the same inter-dose ratio, matching the effects of the doses of the Standard Preparation as closely as possible. Inject the four doses (two doses of the Standard Preparation and two doses of the preparation being examined) at intervals of 3 to 5 minutes. The two doses of Standard Preparation and the two doses of the preparation under examination should be given according to a randomised block or a Latin square design and at least four responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods.The estimated potency is not less than 90 per cent and not more than 111 per cent of the stated potency. The fiducial limits of error are not less than 80 per cent and not more than 125 per cent of the stated potency.Oxytocin of natural origin obtained by extraction and purification complies with the following additional requirement.

Vasopressor activity. Not more than 0.5 Unit per 20 Units of oxytocic activity, when assayed by the biological assay for vasopressor activity described below.The vasopressor activity is estimated by comparing the activity of the preparation under examination with that of the Standard Preparation of arginine vasopressin under the conditions of a suitable method of assay.Standard PreparationThe Standard Preparation is the Ist International Standard for Arginine vasopressin, established in 1978, consisting of freeze-dried synthetic arginine vasopressin peptide acetate with human albumin and citric acid (supplied in ampoules containing 8.20 Units), or another suitable preparation the potency of which has been determined in relation to that of the International Standard.Inject slowly into the tail vein of a male albino rat weighing about 300 g a solution of a suitable a-adrenoceptor blocking agent, for example 10 ml per kg of body weight

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of a solution prepared by dissolving 5 mg of phenoxybenzaminehydrochloride in 0.1 ml of ethanol (95 per cent), adding 0.05 ml of 1 M hydrochloric acid and diluting to 5 ml with saline solution. After 18 hours, anaesthetise the rat with an anaesthetic that will maintain a prolonged and uniform blood pressure. After 45 to 60 minutes, tie the rat on its back to the operating table by its hind legs. Cannulate the trachea with a short polyethylene tube of external diameter about 2.5 mm and dissect a carotid artery ready for cannulation. Then cannulate the femoral vein close to the inguinal ligament. Retract the abdominal muscles to expose the inguinal ligament. Retract the superficial pudendal vein to one side and dissect the femoral vein towards the inguinal ligament from the corresponding artery. When dissecting, a deep branch reaching the femoral vein must be found and tied off to prevent bleeding during cannulation. Tie a short polyethylene cannula of external diameter about 1 mm into the femoral vein by two ligatures and join by a short piece of flexible tubing to a 1-ml burette with an attached thistle funnel containing saline solution at about 37º. Firmly fix a wet absorbent cotton swab to the thigh so as to cover the incision and cannula. At thisstage inject through the venous cannula 200 Units of heparin, dissolved in saline solution, per 100 g of body weight. Then tie in a carotid cannula of external diameter about 1 mm and connect by a column of saline solution containing heparin with a suitable pressure measuring device such as a mercury manometer of internal diameter about 2 to 3 mm.The central and peripheral nervous system including both vagus and associated sympathetic nerves is left intact. No artificial respiration is necessary. Taking care that no air is injected, inject all solutions through the venous cannula by means of a 1-ml syringe graduated in 0.01 ml and wash in with 0.2 ml of saline solution from the burette. Dilute the extract of the Standard Preparation and the preparation under examination with saline solution so that the volume to be injected is between 0.1 ml and 0.5 ml.Choose two doses of the Standard Preparation such that the elevation of the blood pressure is about 4 kPa for the lower dose and about 7 kPa but always submaximal for the higherdose, the ratio of low to high dose being determined by the response and usually being 3 to 5. As an initial approximation doses of 3 and 5 milliUnits may be tried. Choose two doses of the preparation under examination with the same inter-doseratio, matching the effects of the dose of the Standard Preparation as closely as possible. Inject doses at intervals of 10 to 15 minutes.The two doses of the Standard Preparation and the two doses of the preparation under examination should be given in a randomised block or a Latin square design and four to five

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responses to each should be recorded. Measure all the responses and calculate the result of the assayby standard statistical methods.Oxytocin intended for use in the manufacture of parenteral preparations without a further procedure for the removal of bacterial endotoxins.complies with the following additional requirement.Bacterial endotoxins (2.2.3). Not more than 100 Endotoxin Units per 200 Units of oxytocin.Oxytocin intended for use in the manufacture of parenteral preparations without a further sterilisation procedure complies with the following additional requirement.Sterility (2.2.11). Complies with the test for sterility.Storage. Store protected from moisture. If the substance is intended for use in the manufacture of parenteral preparations, the container should be sterile, tamper-evident and sealed so as to exclude micro-organisms.Labelling. The label states (1) the number of Units of oxytocic activity per mg (for solid) or per ml (for liquid); (2) either the animal species from which it is obtained or whether it is synthetic, as appropriate; (3) whether or not the contents are intended for use in the manufacture of parenteral preparations.

Oxytocin InjectionOxytocin Injection is a sterile solution of Oxytocin in Water for Injections. Oxytocin Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated number of Units of oxytocin activity.Description. A clear , colourless liquid.IdentificationGives rise to an appropriate response when administered as directed under one of the methods described for Assay.TestspH (2.4.24). 3.5 to 4.5.Other Tests. Complies with the tests stated under Parenteral Preparations (Injections).Bacterial endotoxins (2.2.3). Not more than 100 Endotoxin Units per 200 Units of oxytocin.Assay.The potency of oxytocin is determined by comparing its activity with that of the Standard Preparation of oxytocin under the conditions of a suitable method of assay.Standard Preparation

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The Standard Preparation is the 4th International Standard for Oxytocin, established in 1978, consisting of freeze-dried synthetic oxytocin peptide with human albumin and citric acid (supplied in ampoules containing 12.5 Units), or any other suitable preparation the potency of which has been determined in relation to the International Standard.NOTE — Any of the following methods may be followed.Method A. By depression of the blood pressure in chicken —Anaesthetise a young healthy adult cockerel weighing 1.2 to 2.3 kg with an anaesthetic that will maintain a prolonged and constant high blood pressure. Expose the gluteus primus muscle in one thigh and cut and retract it to reveal the popliteal artery and crural vein. Cannulate the popliteal artery and record the blood pressure on a suitable recorder calibrated for use over a linear range. Cannulate the crural or brachial vein. Immediately before use prepare a solution of the Standard Preparation in saline solution so that the volume to be injected is between 0.1 ml and 0.5 ml. Record the blood pressure responses to the injection into the cannulated vein of two doses of this solution; the doses should be such as to produce clearly discriminated, precipitous, submaximal decreases in blood pressure; the required doses normally lie between 20 and 100 milliUnits. The interval between injections should be constant and lie between 3 and 10 minutes depending on the rate at which the blood pressure returns to normal. Immediately before use dilute the preparation being examined with saline solution so as to obtain responses similar to those obtained with the Standard Preparation. The ratio between the two doses of the preparation under examination should be the same as that between the two doses of the Standard Preparation and this ratio should be kept constant throughout the assay. The two doses of the Standard Preparation and the two doses of the preparation under examination should be given according to a randomised block or a Latin square design and at least six responses to each should be recorded. If the animal rapidly becomes insensitive to the repeated injections of the solutions another animal must be used.Measure all the responses and calculate the result of the assay by standard statistical methods.

Method B. By contraction of the rat uterus — Inject 100 μg of oestradiol benzoate intramuscularly into a female rat weighing 120 to 200 g 18 to 24 hours before the assay. Immediately before the assay confirm by vaginal smear that the rat is in oestrus or preoestrus. Kill the rat and suspend one horn of the uterus in a bath containing a solution of the following composition.

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Composition (per cent w/v) Sodium chloride 0.662 Potassium chloride 0.045 Calcium chloride 0.007 Sodium bicarbonate 0.256

Disodium hydrogen phosphate 0.029 Sodium dihydrogen phosphate 0.003

Magnesium chloride 0.010 Dextrose 0.050Maintain the bath at a temperature of 32º or at some other suitable temperature at which spontaneous contractions of the uterus are abolished and the preparation maintains its sensitivity. Oxygenate the solution with a mixture of 95 per cent of oxygen and 5 per cent of carbon dioxide and record the contractions of the muscle using a suitable instrument giving a linear response (for example an isotonic lever with a load not exceeding 2 g). Record the contractions produced bythe addition to the bath of two doses of the Standard Preparation suitably diluted with the above solution. The doses should be such as to produce clearly discriminated, submaximal contractions; the required doses normally lie between 10 and 50 micro Units per ml of bath liquid. When maximal contraction has been reached, replace the bath liquid by a fresh solution. The doses should be added at regular intervals of 3 to 5 minutes depending upon the rate of recovery of the muscle. Dilute the preparation being examined so as to obtain responses on the addition of two doses similar to those obtained with the Standard Preparation. The ratio between the two doses of the preparation under examination should bethe same as that between the two doses of the Standard Preparation and this ratio should be kept constant throughout the assay.The two doses of Standard Preparation and the two doses of the preparation under examination should be given according to a randomised block or a Latin square design and at least six responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods.

Method C. By measurement of milk-ejection pressure in a lactating rat — Select a lactating rat, in the third to twentyfirst day after parturition and weighing about 300 g, separate it from the litter and 30 to 60 minutes later anaesthetise (for example, by the intraperitoneal injection of a solution of Pentobarbitone Sodium). Tie the rat to an operating table, maintained at 37º, by its hind legs

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leaving the front legs free. Cannulate the trachea with a short polyethylene tube of internal diameter about 2.5 mm in such a manner so as toensure a free airway; apply artificial respiration only if necessary. Cannulate an external jugular or femoral vein with a polyethylene tube of internal diameter about 0.4 mm which is filled with saline solution and closed with a pin. Shave the skin surrounding the inguinal and abdominal teats and excise the tip of one teat, preferably the lower inguinal teat. Insert a polyethylene tube of internal diameter about 0.3 mm and external diameter about 0.6 mm, to a depth sufficient to obtain appropriate measurement of pressure (3 to 10 mm depth), into the primary teat duct which opens onto the cut surface and tie firmly in place with a ligature. Connect this cannula with a suitable strain gauge transducer (such as that used for recording arterial blood pressure in the rat) and fill the whole system with a 3.8 per cent w/v solution of sodium citrate or saline solution containing 50 Units of heparin sodium per ml to prevent clotting of milk. After cannulation, inject a small volume (0.05 to 0.2 ml) of this solution into the teat duct through the transducer to clear the milk from the tip of the cannula. (This procedure may be repeated during the assay should obstruction arise from milk ejected into the cannula). Clamp the strain gauge so that a slight tension is applied to the teat and its natural alignment is preserved and connect the gauge to a potentiometric recorder adjusted to give full-scale deflection for an increase in milk-ejection pressure of about 5.3 kPa. Inject all solutions through the venous cannula using a 1-ml syringe graduated in 0.01 ml and wash them in with 0.2 ml of saline solution. Prepare a solution of the Standard Preparation and a solution of the preparation under examination in saline solution so that the volume to be injected is between 0.1 ml and 0.4 ml. Choose two doses of the Standard Preparation such that the increase in milk-ejection pressure is about 1.35 kPa for the lower dose and about 2.7 kPa for the higher dose. As an initial approximation, a lower dose of between 0.1 and 0.4 milliUnit and an upper dose of 1.5 to 2 times this amount may be tried. Choose two doses of the preparation under examination with the same inter-dose ratio, matching the effects of the doses of the Standard Preparation as closely as possible. Inject the four doses (two doses of the Standard Preparation and two doses of the preparation under examination at intervals of 3 to 5 minutes. The two doses of Standard Preparation and the two doses of the preparation under examination should begiven according to a randomised block or a Latin square design and at least four responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods. The estimated potency is not less than 90

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per cent and not more than 111 per cent of the stated potency. The fiducial limits of error are not less than 80 per cent and not more than125 per cent of the stated potency. Oxytocin Injection containing Oxytocin of natural origin obtained by extraction and purification complies with the following additional requirement.Vasopressor activity. Not more than 0.5 Unit per 20 Units of oxytocic activity, when assayed by the biological assay for vasopressor activity described below. The vasopressor activity is estimated by comparing the activity of the preparation under examination with that of the Standard Preparation of arginine vasopressin under the conditions of asuitable method of assay.Standard PreparationThe Standard Preparation is the Ist International Standard for Arginine vasopressin, established in 1978, consisting of freeze-dried synthetic arginine vasopressin peptide acetate with human albumin and citric acid (supplied in ampoules containing 8.20 Units), or any other suitable preparation the potency of which has been determined in relation to that of the International Standard. Inject slowly into the tail vein of a male albino rat weighing about 300 g a solution of a suitable a-adrenoceptor blocking agent, for example 10 ml per kg of body weight of a solution prepared by dissolving 5 mg of phenoxybenzamine hydrochloride in 0.1 ml of ethanol (95 per cent), adding 0.05 ml of 1 M hydrochloric acid and diluting to 5 ml with saline solution. After 18 hours, anaesthetise the rat with an anaesthetic that will maintain a prolonged and uniform blood pressure. After 45 to 60 minutes, tie the rat on its back to the operating table by its hind legs. Cannulate the trachea with a short polyethylene tube of external diameter about 2.5 mm and dissect a carotid artery ready for cannulation. Then cannulate the femoral vein close to the inguinal ligament. Retract the abdominal muscles to expose the inguinal ligament. Retract the superficial pudendal vein to one side and dissect the femoral vein towards the inguinal ligament from the corresponding artery. When dissecting, a deep branchreaching the femoral vein must be found and tied off to prevent bleeding during cannulation. Tie a short polyethylene cannula of external diameter about 1 mm into the femoral vein by two ligatures and join by a short piece of flexible tubing to a 1-ml burette with an attached thistle funnel containing saline solution at about 37º. Firmly fix a wet absorbent cotton swab to the thigh so as to cover the incision and cannula. At this stage inject through the venous cannula 200 Units of heparin, dissolved in saline solution, per 100 g of body weight. Then tie in a carotid cannula of external diameter about 1 mm and connect by a column of

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saline solution containing heparin with a suitable pressure measuring device such as a mercury manometer of internal diameter about 2 to 3 mm. The central and peripheral nervous system including both vagus and associated sympathetic nerves is left intact. No artificial respiration is necessary. Taking care that no air isinjected, inject all solutions through the venous cannula by means of a 1-ml syringe graduated in 0.01 ml and wash in with 0.2 ml of saline solution from the burette. Dilute the extract of the Standard Preparation and the preparation under examination with saline solution so that the volume to be injected is between 0.1 ml and 0.5 ml. Choose two doses of the Standard Preparation such that the elevation of the blood pressure is about 4 kPa for the lower dose and about 7 kPa but always submaximal for the higher dose, the ratio of low to high dose being determined by the response and usually being 3 to 5. As an initial approximationdoses of 3 and 5 milliUnits may be tried. Choose two doses of the preparation under examination with the same inter-dose ratio, matching the effects of the dose of the Standard Preparation as closely as possible. Inject doses at intervals of10 to 15 minutes. The two doses of the Standard Preparation and the two doses of the preparation under examination should be given in a randomised block or a Latin square design and four to five responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods.Storage. Store at temperature not exceeding 30º. Do not freeze.Labelling. The label states (1) the number of Units of oxytocin activity per ml; (2) either the animal spieces from which it is obtained or whether it is synthetic, as appropriate.

Oxytocin Nasal SolutionOxytocin Nasal Solution is a solution of Oxytocin in a suitable solvent containing an appropriate antimicrobial preservative. Oxytocin Nasal Solution contains not less than 85.0 per cent and not more than 120.0 per cent of the stated number of Units of oxytocic activity.Description. A clear, colourless solution.TestspH (2.4.24). 3.5 to 4.5.Other Tests. Complies with the tests stated under Nasal Preparations.Assay.The potency of oxytocin is determined by comparing its activity with that of the Standard Preparation of oxytocin under the conditions of a suitable method of assay.

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Standard PreparationThe Standard Preparation is the 4th International Standard for Oxytocin, established in 1978, consisting of freeze-dried synthetic oxytocin peptide with human albumin and citric acid (supplied in ampoules containing 12.5 Units), or any other suitable preparation the potency of which has been determined in relation to the International Standard.NOTE — Any of the following methods may be followed.

Method A. By depression of the blood pressure in chicken —Anaesthetise a young healthy adult cockerel weighing 1.2 to 2.3 kg with an anaesthetic that will maintain a prolonged andconstant high blood pressure. Expose the gluteus primus muscle in one thigh and cut and retract it to reveal the popliteal artery and crural vein. Cannulate the popliteal artery and record the blood pressure on a suitable recorder calibrated for use over a linear range. Cannulate the crural or brachial vein. Immediately before use prepare a solution of the Standard Preparation in saline solution so that the volume to be injectedis between 0.1 ml and 0.5 ml. Record the blood pressure responses to the injection into the cannulated vein of two doses of this solution; the doses should be such as to produce clearly discriminated, precipitous, submaximal decreases inblood pressure; the required doses normally lie between 20 and 100 milliUnits. The interval between injections should be constant and lie between 3 and 10 minutes depending on the rate at which the blood pressure returns to normal. Immediately before use dilute the preparation being examined with saline solution so as to obtain responses similar to those obtained with the Standard Preparation. The ratio between the two doses of the preparation under examination should be the same as that between the two doses of the Standard Preparation and this ratio should be kept constant throughout the assay.The two doses of the Standard Preparation and the two dosesof the preparation under examination should be given according to a randomised block or a Latin square design and at least six responses to each should be recorded. If the animal rapidly becomes insensitive to the repeated injections of the solutions another animal must be used. Measure all the responses and calculate the result of the assay by standard statistical methods.

Method B. By contraction of the rat uterus — Inject 100 μg of oestradiol benzoate intramuscularly into a female rat weighing

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120 to 200 g 18 to 24 hours before the assay. Immediately before the assay confirm by vaginal smear that the rat is in oestrus or preoestrus. Kill the rat and suspend one horn of the uterus in a bath containing a solution of the following composition.

Composition (per cent w/v)Sodium chloride 0.662

Potassium chloride 0.045Calcium chloride 0.007

Sodium bicarbonate 0.256 Disodium hydrogen phosphate 0.029 Sodium dihydrogen phosphate 0.003

Magnesium chloride 0.010 Dextrose 0.050Maintain the bath at a temperature of 32º or at some other suitable temperature at which spontaneous contractions of the uterus are abolished and the preparation maintains its sensitivity. Oxygenate the solution with a mixture of 95 per cent of oxygen and 5 per cent of carbon dioxide and record the contractions of the muscle using a suitable instrument giving a linear response (for example an isotonic lever with a load not exceeding 2 g). Record the contractions produced bythe addition to the bath of two doses of the Standard Preparation suitably diluted with the above solution. The doses should be such as to produce clearly discriminated, submaximal contractions; the required doses normally lie between 10 and 50 micro Units per ml of bath liquid. When maximal contraction has been reached, replace the bath liquid by a fresh solution. The doses should be added at regular intervals of 3 to 5 minutes depending upon the rate of recovery of the muscle. Dilute the preparation being examined so as to obtain responses on the addition of two doses similar to those obtained with the Standard Preparation. The ratio between the two doses of the preparation under examination should be the same as that between the two doses of the Standard Preparation and this ratio should be kept constant throughout the assay.The two doses of Standard Preparation and the two doses of the preparation under examination should be given according to a randomised block or a Latin square design and at least six responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods.

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Method C. By measurement of milk-ejection pressure in a lactating rat — Select a lactating rat, in the third to twentyfirst day after parturition and weighing about 300 g, separate it from the litter and 30 to 60 minutes later anaesthetise (for example, by the intraperitoneal injection of a solution of Pentobarbitone Sodium). Tie the rat to an operating table, maintained at 37º, by its hind legs leaving the front legs free. Cannulate the trachea with a short polyethylene tube of internal diameter about 2.5 mm in such a manner so as to ensure a free airway; apply artificial respiration only if necessary. Cannulate an external jugular or femoral vein with a polyethylene tube of internal diameter about 0.4 mm which is filled with saline solution and closed with a pin. Shave the skin surrounding the inguinal and abdominal teats and excise the tip of one teat, preferably the lower inguinal teat. Insert a polyethylene tube of internal diameter about 0.3 mm and external diameter about 0.6 mm, to a depth sufficient to obtain appropriate measurement of pressure (3 to 10 mm depth), into the primary teat duct which opens onto the cut surface and tie firmly in place with a ligature. Connect this cannula with a suitable strain gauge transducer (such as that used for recording arterial blood pressure in the rat) and fill the whole system with a 3.8 per cent w/v solution of sodium citrate or saline solution containing 50 Units of heparin sodium per ml to prevent clotting of milk. After cannulation, inject a small volume (0.05 to 0.2 ml) of this solution into the teat duct through the transducer to clear the milk from the tip of the cannula. (This procedure may be repeated during the assay should obstruction arise from milk ejected into the cannula). Clamp the strain gauge so that a slight tension is applied to the teat and its natural alignment is preserved and connect the gauge to a potentiometric recorder adjusted to give full-scale deflection for an increase in milk-ejection pressure of about 5.3 kPa. Inject all solutions through the venous cannula using a 1-ml syringe graduated in 0.01 ml and wash them in with 0.2 ml of saline solution. Prepare a solution of the Standard Preparation and a solution of the preparation under examination in saline solution so that the volume to be injected is between 0.1 ml and 0.4 ml. Choose two doses of the Standard Preparation such that the increase in milk-ejection pressure is about 1.35 kPa for the lower dose and about 2.7 kPa for the higher dose. As an initial approximation, a lower dose of between 0.1 and 0.4 milliUnit and an upper dose of 1.5 to 2 times this amount may be tried. Choose two doses of the preparation under examination with the same inter-dose ratio, matching the effects of the doses of

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the Standard Preparation as closely as possible. Inject the four doses (two doses of the Standard Preparation and two doses of the preparation under examination at intervals of 3 to 5 minutes. The two doses of Standard Preparation and the two doses of the preparation under examination should be given according to a randomised block or a Latin square design and at least four responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods.The estimated potency is not less than 90 per cent and not more than 111 per cent of the stated potency. The fiducial limits of error are not less than 80 per cent and not more than 125 per cent of the stated potency.Oxytocin Nasal Solution containing Oxytocin of natural origin obtained by extraction and purification complies with the following additional requirement.Vasopressor activity. Not more than 0.5 Unit per 20 Units of oxytocic activity, when assayed by the biological assay for vasopressor activity described below. The vasopressor activity is estimated by comparing the activity of the preparation under examination with that of the Standard Preparation of arginine vasopressin under the conditions of a suitable method of assay.Standard PreparationThe Standard Preparation is the Ist International Standard for Arginine vasopressin, established in 1978, consisting of freeze-dried synthetic arginine vasopressin peptide acetate with human albumin and citric acid (supplied in ampoules containing 8.20 Units), or any other suitable preparation the potency of which has been determined in relation to that of the International Standard. Inject slowly into the tail vein of a male albino rat weighing about 300 g a solution of a suitable a-adrenoceptor blocking agent, for example 10 ml per kg of body weight of a solution prepared by dissolving 5 mg of phenoxybenzamine hydrochloride in 0.1 ml of ethanol (95 per cent), adding 0.05 ml of 1 M hydrochloric acid and diluting to 5 ml with saline solution. After 18 hours, anaesthetise the rat with an anaesthetic that will maintain a prolonged and uniform blood pressure. After 45 to 60 minutes, tie the rat on its back to the operating table by its hind legs. Cannulate the trachea with a short polyethylene tube of external diameter about 2.5 mm and dissect a carotid artery ready for cannulation. Then cannulate the femoral vein close to the inguinal ligament. Retract the abdominal muscles to expose the inguinal ligament. Retract the superficial pudendal vein to one side and dissect the femoral vein towards the inguinal ligament from the corresponding artery. When dissecting, a deep branch

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reaching the femoral vein must be found and tied off to prevent bleeding during cannulation. Tie a short polyethylene cannula of external diameter about 1 mm into the femoral vein by two ligatures and join by a short piece of flexible tubing to a 1-ml burette with an attached thistle funnel containing saline solution at about 37º. Firmly fix a wet absorbent cotton swab to the thigh so as to cover the incision and cannula. At this stage inject through the venous cannula 200 Units of heparin, dissolved in saline solution, per 100 g of body weight. Then tie in a carotid cannula of external diameter about 1 mm and connect by a column of saline solution containing heparin with a suitable pressure measuring device such as a mercury manometer of internal diameter about 2 to 3 mm. The central and peripheral nervous system including both vagus and associated sympathetic nerves is left intact. No artificial respiration is necessary. Taking care that no air isinjected, inject all solutions through the venous cannula by means of a 1-ml syringe graduated in 0.01 ml and wash in with 0.2 ml of saline solution from the burette. Dilute the extract of the Standard Preparation and the preparation under examination with saline solution so that the volume to be injected is between 0.1 ml and 0.5 ml. Choose two doses of the Standard Preparation such that the elevation of the blood pressure is about 4 kPa for the lower dose and about 7 kPa but always submaximal for the higher dose, the ratio of low to high dose being determined by the response and usually being 3 to 5. As an initial approximationdoses of 3 and 5 milliUnits may be tried. Choose two doses of the preparation under examination with the same inter-dose ratio, matching the effects of the dose of the Standard Preparation as closely as possible. Inject doses at intervals of10 to 15 minutes.The two doses of the Standard Preparation and the two doses of the preparation under examination should be given in a randomised block or a Latin square design and four to five responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods.Storage. Store at a temperature not exceeding 30º.Labelling. The label states (1) the number of Units of oxytocic activity per ml; (2) either the animal species from which it is obtained or whether it is synthetic, as appropriate; (3) that the preparation is intended for intranasal administration only.

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REFERENCES

INDIAN PHARMACOPOEIA (VOL-3)

LINKS

http://en.wikipedia.org/wiki/Oxytocin

http://www.oxytocin.org/oxytoc

http://www.ncbi.nlm.nih.gov/pubmed/2488622

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