Chapter-1; Bioassay (Part-3).pptx

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1. “Pharmaceutical Quality Management” PHR-619 “Bio Assay ” Part-3 By : Dr. Amjad Khan Assistant Professor, Department of Pharmacy, KUST

2. Outlines Assay Biological Assay (Bio Assay) Principal of Bio Assay Techniques for Bio Assay Topic: Quality Control Charts during Pharmaceutical Manufacturing Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected]

3. Bioassay of Insulin Injection Insulin is a hypoglycemic agent i -e it lowers the blood glucose level. Principle of the Bioassay: The potency of insulin injection (expressed in International Units/ml) is estimated by comparing the hypoglycemic effect of the test sample with that of the standard preparation of insulin. Standard preparation and unit: It is pure, dry and crystalline insulin supplied in ampoules containing approximately 110-125mg of insulin. 1mg contains 22 Units of insulin; 1 Unit is equivalent to 45.5 microgram (0.0455 milligram) of insulin. 1mg= 22 Units of Insulin. 1 Unit = 0.0455mg of Insulin . Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

4. Bioassay of Insulin Injection (Preparation of Standard and Sample Solutions) Take a specific quantity of insulin and dissolve it in normal saline. Acidify it with HCl to pH 2.5. Add 0.5% phenol as preservative. Add 1.4% to 1.8% glycerin. Final volume should contain 20 units/ml. Store the solution in a cool place (2-10 ºC) and use it within six months. Preparation of test sample solution: The solution of the test sample is prepared in the same way as the standard solution described above. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

5. Bioassay of Insulin Injection (Suggested Methods) Mouse Convulsion Method Rabbits Blood Sugar Method Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

6. Bioassay of Insulin Injection (Mouse Convulsion Method) Principle : Mice show characteristic hypoglycemic convulsions after S.C. injection of insulin at elevated temperatures. The percentage convulsions produced by the test and standard preparations are compared. Experimental Conditions: Minimum 100 mice weighing between 18-22 grams of the same strain are used. They should be maintained on a constant and an adequate diet. They should be fasted 2 hrs prior to the experiment. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

7. Mouse Convulsion Method (Standard and Sample Dilution) Two dilutions of the standard solution are prepared with sterile saline solution acidified with HCl to pH 2.5, so as to contain 0.030 units/ml (standard dilution-I) and 0.060 units/ml (Standard dilution-II). Similarly, two dilutions of test sample solution are also prepared. Dose: Suitable doses of insulin for mice weighing about 20g are 0.015 Units and 0.030 Units. Dilutions of standard and test solutions are prepared in such a way that the respective doses are contained in equal volumes not greater than 0.5ml. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

8. Mouse Convulsion Method (Procedure) 100 Mice are divided into 4 groups each containing 25 mice and insulin is injected S.C. to each group. A 4-point assay is performed by injecting 2 doses (dilutions) of the standard solution (S1 & S2) and 2 doses (dilutions) of the test solution (T1 & T2) into four groups of mice. Mice are then kept at a constant temperature (29-35°C) in an air incubator with a transparent font and observed for 1.5 hours following insulin injection. The numbers of mice which convulse or die are recorded. These mice usually convulse severely but failure of the animal to upright itself when placed on its back, should as well be considered as convulsion. Convulsive mice may be saved by an inj. of 0.5 ml. of 5% dextrose solution. Those animals which survive may be used again for another experiment after an interval of one week. The death or convulsion in the test animals are due to the rapid onset of hypoglycemia . Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

9. Mouse Convulsion Method (Procedure) Two dilutions of the standard solution are prepared with sterile saline solution acidified with HCl to pH 2.5, so as to contain 0.030 units/ml (standard dilution-I) and 0.060 units/ml (Standard dilution-II). Similarly, two dilutions of test sample solution are also prepared. Dose: Suitable doses of insulin for mice weighing about 20g are 0.015 Units and 0.030 Units. Dilutions of standard and test solutions are prepared in such a way that the respective doses are contained in equal volumes not greater than 0.5ml. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

10. Mouse Convulsion Method (Procedure) Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

11. Mouse Convulsion Method (Results) Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay Percentage convulsions produced by the test sample are compared with those of the standard sample. The potency of the test preparation is determined by a specific statistical method based on the direct proportional relationship between % animals showing convulsion and the potency of the preparation.

12. Bioassay of Insulin Injection (Rabbit Blood Sugar Method) Principle: Insulin lowers blood sugar level, and the degree of blood sugar lowering is proportional to the potency of the preparation. The potency of a test sample is estimated by comparing the hypoglycemic effect (average reduction in the blood glucose level) of the sample with that of the std. preparation of insulin. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

13. Bioassay of Insulin Injection (Rabbit Blood Sugar Method) Experimental Conditions: 12 healthy rabbits (1.5 to 2 kg) are used. They should then be maintained on uniform diet but are fasted for 18 hrs before assay. Standard and sample dilutions: Two dilutions of the standard solution are prepared with sterile saline solution acidified with HCl to pH 2.5, so as to contain 1 unit/ml (standard dilution-I) and 2 units/ml (Standard dilution-II). Similarly, two dilutions of test sample solution are also prepared. Dose: Suitable doses of insulin for rabbits is about 0.5 units/kg and 1 unit/kg body weight of the rabbit. Dilutions of standard and test solutions are prepared in such a way that the respective doses are contained in equal volumes not greater than 1ml. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

14. Rabbit Blood Sugar Method (Procedure) 12 Rabbits are divided into 4 groups each containing 3 rabbits. Blood samples are withdrawn from the marginal ear vein of each rabbit and the average concentration of blood glucose in mg/dl is determined in each group (initial blood sugar). Then insulin is injected S.C. to each group. A 4-point assay is performed by injecting 2 doses (dilutions) of the standard solution (S1 & S2) and 2 doses (dilutions) of the test solution (T1 & T2) into four groups of rabbits. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

15. Rabbit Blood Sugar Method (Procedure) Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

16. Rabbit Blood Sugar Method (Procedure) Following insulin injection, blood samples are withdrawn from the marginal ear vein every hour for 5 consecutive hours and the average concentration of blood glucose in mg/dl is determined in each group (final blood sugar ). Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

17. Rabbit Blood Sugar Method (Results) For each dose of the test and standard insulin, the reduction in blood sugar level is calculated and compared and the potency of the test insulin is determined by a suitable statistical method. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

18. Bioassay of Prepared Digitalis BIOASSAY OF PREPARED DIGITALIS Digitalis is a drug that increases the force of myocardial contraction and is used to treat cardiac failure. It is also known as cardio-tonic as it increases the efficiency of failure heart. In higher doses it causes stoppage of the heart (cardiac arrest) and death. Principle of the Bioassay: The Potency of prepared digitalis is estimated by comparing the action of the test sample of prepared digitalis on the cardiac muscles with that of the standard preparation of prepared digitalis . Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

19. Bioassay of Prepared Digitalis Standard preparation and unit: The standard preparation is a mixture of dried and powdered digitalis purpurea leaves supplied in vials containing approximately 2.5g of dried powdered leaves. 1 unit is equivalent to 76 mg; 1mg contains 0.01316 units of standard preparation of digitalis. 1mg= 0.01316 Units of Digitalis. 1 Unit = 76mg of Digitalis. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

20. Bioassay of Prepared Digitalis (Preparation of Extract) Preparation of standard extract: Standard extract of digitalis is prepared by the following procedure: The contents of a vial containing standard preparation of digitalis are rapidly transferred to a stoppered weighing bottle and weighed. The contents are added to a stoppered glass container of at least 50ml capacity. 10ml of alcohol (80%) is added for each g of the powder. The container is stoppered and is shaken for 24 hrs at 20-30 ºC or for 48hrs at 10-20 ºC. After shaking the mixture is immediately centrifuged or filtered through a sintered glass filter with precautions to avoid evaporation of the solvent. The final extract is transferred to a dry, hard glass bottle which is tightly closed. The extract is stored at a temperature between 5 ºC and -5 ºC. The extract should be used within one month . Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

21. Bioassay of Prepared Digitalis (Preparation of Sample Extract) Preparation of test sample extract: The extract of the test sample is prepared in the same way as the standard extract described above. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

22. Bioassay of Prepared Digitalis (Suggested Methods) Guinea–pig Method Pigeon Method Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

23. Guinea Pig Method (Principle) Principle : At higher doses digitalis causes stoppage of the heart (cardiac arrest) and death of guinea pigs, which indicates the end point response of digitalis. The potency of the test sample is estimated by comparing the lethal dose of the test sample to that of the standard preparation of digitalis. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

24. Guinea Pig Method (Experimental Conditions) Experimental Conditions: 12 guinea pigs (200-600 g) of the same strain are used. The weight of the heaviest and the lightest animals should not differ by more than 100 g. Later on when the guinea pigs are divided into 2 groups each containing 6 guinea pigs, the mean body weights of the two groups should not differ by more than 10 %. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

25. Guinea Pig Method (Standard and Sample Dilution) Standard and sample dilutions: The standard and test sample extracts are diluted with saline solution until the concentration of alcohol are the same and do not exceed 10 % v/v. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

26. Guinea Pig Method (Procedure) Procedure: 12 guinea pigs are divided in 2 groups each containing 6 guinea pigs. One group is used for standard preparation and one for the test sample. The guinea pig is anaesthetized with urethane and the diluted standard extract is injected at a slow and uniform rate into the vein of a guinea pig. The injection is continued until the heart is arrested (this may conveniently be determined from an electrical recording). The amount (volume) of extract required to produce this effect is taken as the lethal dose of the extract. Another set of 5 animals of the same species are used for this experiment and the average lethal dose (ml per kg of the body weight) of 6 guinea pigs is determined for standard extract. The average lethal dose of the test sample is determined in a similar way using 6 guinea pigs of the same strain. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

27. Guinea Pig Method (Results) The potency of the test sample is calculated in relation to that of the standard preparation by dividing the average lethal dose of the sample to the test and expressed as units per gram. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

28. Guinea Pig Method (Results) Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

29. Bioassay of Prepared Digitalis (Pigeon Method) 2. Pigeon Method: Principle: At higher doses digitalis causes stoppage of the heart (cardiac arrest). In pigeons, stoppage of heart is associated with a characteristic vomiting response called „Emesis‟. This may be taken as the end point response of digitalis. The potency of the test sample is estimated by comparing the lethal dose of the test sample to that of the standard preparation of digitalis. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

30. Pigeon Method (Experimental Conditions) 12 pigeons are used. They should be free from gross evidence of disease, obesity or emaciation (To become extremely thin, especially as a result of starvation or disease). The weight of the heaviest pigeon should not exceed twice the weight of the lightest pigeon. Food is withheld 16-28 hours before the experiment. Later on when the pigeons are divided into 2 groups each containing 6 pigeons, the mean body weights of the two groups should not differ by more than 30 %. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

31. Pigeon Method (Standard and Sample Dilution) The standard and test sample extracts are diluted with saline solution so that the estimated lethal dose per kg of the body weight is contained in 15ml. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

32. Pigeon Method (Procedure) 12 pigeons are divided in 2 groups each containing 6 pigeons. One group is used for standard preparation and one for the test sample. The pigeons are lightly anaesthetized with anesthetic ether and the diluted standard extract is injected through cannula at a slow and uniform rate into the alar vein of a pigeons. The dose is 1ml per kg of the body weight and is administered within few seconds & repeated at 5 minutes interval until the heart is arrested which is associated with emetic response. The amount (volume) of extract required to produce this effect is taken as the lethal dose of the extract. Another set of 5 pigeons are used for this experiment and the average lethal dose (ml per kg of the body weight) of 6 pigeons is determined for standard extract. The average lethal dose of the test sample is determined in a similar way using 6 pigeons. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

33. Pigeon Method (Results) Results: The potency of the test sample is calculated in relation to that of the standard preparation by dividing the average lethal dose of the sample to the test and expressed as units per gram. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

34. Pigeon Method (Results) Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

35. Bioassay of Vitamin D Vitamin D is also known as anti-rachitic vitamin. The deficiency of vitamin D leads to rickets. Rickets is a disorder caused by a lack of vitamin D, calcium, or phosphate. It leads to softening and weakening of the bones thus potentially leading to fractures and deformity. Vitamin D helps the body to control calcium and phosphate levels. If the blood levels of these minerals become too low, the body may produce hormones that cause calcium and phosphate to be released from the bones. This leads to weak and soft bones. The deficiency of Vitamin D is the predominant cause of rickets. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

36. Bioassay of Vitamin D (Principle) The potency of vitamin D is determined by comparing the antirachitic activity of the test sample of vitamin D with that of the standard preparation of vitamin D. The antirachitic activity of vitamin D is measured in rats. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

37. Bioassay of Vitamin D (Standard Preparation and Unit) The standard preparation of vitamin D consists of activated crystalline 7- dehydrocholestrol supplied in bottles as solution in vegetable oils containing 1000 units per gram of the solution. 1 unit is equivalent to 0.000025 mg; 1mg contains 40000 units of pure vitamin D. 1mg= 40000 Units of vitamin D. 1 Unit = 0.000025mg of vitamin D. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

38. Bioassay of Vitamin D (Experimental Conditions) Minimum of 40 young rats of either gender, shortly after being weaned, are used for the test. About 10 litters (the offspring at one birth of a multiparous mammal- those which produce more than one at a birth) of 4 rats are selected. (Alternatively 5 litters of 8 rats can also be selected). 40 rats= 10 litters (groups) of 4 rats (10 ×4 = 40) Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

39. Bioassay of Vitamin D (Experimental Conditions) The weight of the heaviest rat in any litter should not exceed that of the lightest by more than 10g. All of the 40 rats are fed for about 21 days (3 weeks) on a rachitogenic diet, which consists mostly of carbohydrates, proteins and electrolytes and no fats. The development of the necessary degree of rickets may be determined in each rat under light anaesthesia by taking X-ray photographs of the proximal ends of the tibia or the distal ends of the ulna and radius. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

40. Bioassay of Vitamin D (Experimental Conditions) Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

41. Bioassay of Vitamin D (Procedure) 40 rats are divided into 4 groups, each group containing 10 rats. The groups are made in such a way that one rat from each litter set of 4 or two rats from each litter set of 8, being assigned to each group. The rats in two groups receive doses of x and nx units (where n is a suitable value such as 1, 2, 3 or 4), respectively, of standard preparation, while the rats of the other two groups receive, respectively, doses of test sample preparation in the same ratio as the doses of the standard preparation ( i -e x & nx doses). Suitable doses of the standard preparation may vary from 2 to 8 units (x=2 units; n=1, or 2, or 3, or 4). Each rat may receive the whole of its dose at once or the dose may be divided into 8 daily doses. Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

42. Bioassay of Vitamin D (Procedure) 10-14 days after receiving the doses of standard preparation or of the sample preparation if given in one amount (single dose), or 10-14 days after receiving the first fraction of the dose (if given in divided doses), the rats are killed and the extent to which rickets has been cured is estimated by means of X-ray photographs, or by examination of the bones after staining, in either instance by reference to a standard scale . Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

43. Bioassay of Vitamin D (Results) The extent to which rickets has been cured by the sample preparation of vitamin D is compared with that of standard preparation of vitamin D, and the potency of the sample preparation is calculated by standard statistical methods . Course: Pharmaceutical Quality Management, PHR-619- - Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

44. References British Pharmacopeia, 2012 United States Pharmacopeia (USP/NF), 2018 Course: Pharmaceutical Quality Management, PHR-619-- Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay

45. Thank you … Course: Pharmaceutical Quality Management, PHR-619-- Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: [email protected] Topic: Bio Assay