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Methods for studying drug uptake

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Methods for studying drug uptake

  1. 1. Presented by, Pratiksha C Chandragirivar M pharma 1st sem Dept. of pharmaceutics HSK COP Bagalkot Facilitated to, Mr. J.N.HIremath Asso.Professor Dept. of pharmaceutics HSK COP Bagalkot 1Advanced biopharmaceutics and pharmacokinetics
  2. 2. CONTENTS: 1. Methods of studying drug uptake 2. References 2Advanced biopharmaceutics and pharmacokinetics
  3. 3. METHODS OF STUDYING THE DRUG UPTAKE Methods of studying drug uptake In vitro In situ In vivo In silico 3Advanced biopharmaceutics and pharmacokinetics
  4. 4. 1. IN VITRO METHOD : As the name suggests the “in vitro” these methods are conducted/performed outside the living organisms. These type of methods involves the study of transport of drug through different types of membranes or biological materials. 4Advanced biopharmaceutics and pharmacokinetics
  5. 5. Experiments are conducted by using Diffusion cells Segments of GIT of laboratory animals Everted sac technique Everted sac modification Circulations techniques Everted ring or slice techniques Cell cultures of epithelium Example: Caco-2 cells 5Advanced biopharmaceutics and pharmacokinetics
  6. 6. A. DIFFUSION CELL METHOD: ( ref .1) 6Advanced biopharmaceutics and pharmacokinetics
  7. 7. Method : Diffusion cells consists of two compartments: 1. Donor compartment: It contains the drug solution and the lower end of which contains the synthetic or natural GIT membrane that interferes with the compartment. 2. Receptor compartment: It contains the buffer solution. Hence the amount of drug uptake is studied or determined by the measurement of rate of drug arrival in the receptor compartment. 7Advanced biopharmaceutics and pharmacokinetics
  8. 8. Advantages: 1. Can be predicted for almost all the dosage forms. 2. Easily handled Disadvantages: 1. Tedious 2. Some of the semi permeable membranes used are costly. 8Advanced biopharmaceutics and pharmacokinetics
  9. 9. B. SEGMENTS OF GIT OF LABORATORY ANIMALS I ) EVERTED SAC TECHNIQUE:(EVERTED INTESTINE SAC TECHNIQUE) (ref 1 and 2) 9Advanced biopharmaceutics and pharmacokinetics
  10. 10. Method: laboratory animal is taken i.e., rat About 3cm of segment of small intestine of rat isolated Then intestine is inverted Sac is filled with the small volume of drug free buffer solution 10Advanced biopharmaceutics and pharmacokinetics
  11. 11. Both ends of the segment are tied off Sac is immersed in an Erlenmeyer flask containing a larger volume of buffer solution that contains the drug The flask and contents are then oxygenated and maintained at 37℃ for a specified period of time and shaken mildly At predetermined time intervals, the sac is removed, serosal fluid is assayed for drug content 11Advanced biopharmaceutics and pharmacokinetics
  12. 12. Advantages: 1. The epithelial cells of the mucosal surface are exposed directly to the oxygenated mucosal fluid. 2. Prolongs the viability and integrity of the preparation after removal from the animal. 3. Convenience and accuracy with respect to drug analysis. Disadvantages: 1. Difficulty in obtaining more than one sample per intestinal segment. 12Advanced biopharmaceutics and pharmacokinetics
  13. 13. II). EVERTED SAC MODIFICATION:(CRANE AND WILSON MODIFICATION) Here the essential features of simple sac method are retained. The intestine is tied to cannula from which frequent serosal samples may be obtained. 13Advanced biopharmaceutics and pharmacokinetics Ref.2
  14. 14. Method: The test animal is fasted for 20 -24 hrs Water is allowed ad libitum (as directed or as desired) The animal is killed by a blow on the head or anaesthetized with ether or chloroform The entire small intestine is everted 14Advanced biopharmaceutics and pharmacokinetics
  15. 15. From intestine about 5 to 15cm region is selected and segments are made Distal end of the intestine is tied and proximal end is attached to the cannula The segment is suspended in the drug containing mucosal solution about 40 – 100ml 15Advanced biopharmaceutics and pharmacokinetics
  16. 16. The mucosal solution is aerated The determination of rate drug transfer is done by taking the serosal solution at each time with the help of syringe and replaced with fresh buffer solution The amount of drug that permeates the intestinal mucosa is plotted against time which describes the absorption profile of drug at any specific pH 16Advanced biopharmaceutics and pharmacokinetics
  17. 17. Advantages: 1. A number of different solutions may be tested with a single segment of intestine. 2. It is simple and reproducible. 3. It distinguishes between active and passive absorption. 4. It determines the region of the small intestine where the absorption is optimal, particularly in the case of active transport. Disadvantages: 1. Permeability is less. 2. Time consuming. 17Advanced biopharmaceutics and pharmacokinetics
  18. 18. III). CIRCULATION TECHNIQUES: In this method, small intestine may or may not be everted. This method involves the isolation of either the entire small intestine of lab animal or a segment. Oxygenated buffer containing drug is circulated through the lumen. Drug free buffer solution is also circulated on the serosal side of the intestinal membrane and oxygenated. Absorption rates from the lumen to the outer solutions are determined by sampling both the fluid circulating through the lumen and outside. 18Advanced biopharmaceutics and pharmacokinetics
  19. 19. IV). EVERTED RING OR SLICE TECHNIQUES: Method: Appropriate section of small intestine of rat is isolated and everted Dissected into slices or rings of thickness 1 and 3 mm ,with length 0.1 to 0.5 cm length 19Advanced biopharmaceutics and pharmacokinetics
  20. 20. Then slices are placed an oxygenated isotonic drug solution at 37℃ Incubated for a predetermined period of time with gentle shaking After incubation ring are washed, dried and placed in pre weighed scintillation vials Each vial is reweighed to determine the wet tissue weight, and then sample is analysed for drug 20Advanced biopharmaceutics and pharmacokinetics
  21. 21. Advantages: 1. This method is reproducible. 2. Kinetics studies can be performed. Disadvantages: 1. Process of cutting intestine into rings may expose highly permeable areas of cut or damaged tissue the medium. 21Advanced biopharmaceutics and pharmacokinetics
  22. 22. C. CELL CULTURES OF GUT EPITHELIUM EX: CACO-2 CELLS Method: Differentiated cells of the intestine, originating from Caco-2 cells i.e., cells of carcinoma of colon These placed on previously treated polycarbonate with collagen(which on incubation aids reproduction of cells while not retarding drug permeation characteristics) 22Advanced biopharmaceutics and pharmacokinetics
  23. 23. Solution of the drug is placed in this layer of cultured cells The system is place in a bath or receptor compartment of buffer solution The drug that reaches the latter compartment is sample and analyzed periodically 23Advanced biopharmaceutics and pharmacokinetics
  24. 24. 2. IN SITU METHOD The term in situ refers to the methods where animal blood supply intact. They just mimics the in vivo models. The rate of absorption is determined based on the perfusion of a segment of GIT by drug solution and the amount of drug diffused through it. Advanced biopharmaceutics and pharmacokinetics 24
  25. 25. In situ Absorption from the intestine Doluisio method Perfusion technique intestinal loop technique Absorption from the stomach 25Advanced biopharmaceutics and pharmacokinetics
  26. 26. A. ABSORPTION FROM THE INTESTINE 1. DOLUISIO METHOD: (1) 26Advanced biopharmaceutics and pharmacokinetics
  27. 27. Method: Upper and lower part of the small intestine of anesthetised and dissected Dissected rats are connected by means of tubing to syringes of capacity 10-30 ml The intestinal segment is washed with normal saline 27Advanced biopharmaceutics and pharmacokinetics
  28. 28. The syringe is filled with a solution of radio labelled drug and a non-absorbable marker which is used as indicator of water-flux during perfusion Part of the content of the syringe containing drug is delivered to the intestinal segment which is then collected in the second syringe and analysed for drug 28Advanced biopharmaceutics and pharmacokinetics
  29. 29. 2. SINGLE PASS PERFUSION TECHNIQUE: 29Advanced biopharmaceutics and pharmacokinetics Ref.1
  30. 30. In this method, the drug solution passes through the intestinal segment just once. This is superiors to Doluisio method 30Advanced biopharmaceutics and pharmacokinetics
  31. 31. 3. INTESTINAL LOOP TECHNIQUE: Method: Adult male rats are fasted, water also being withheld for 1-2 hr before the experiment Under anaesthesia an abdominal midline incision is made and the small intestine is exposed Both proximal and distal ends are closed by ligature 31Advanced biopharmaceutics and pharmacokinetics
  32. 32. The drug solution is introduced in the lumen of the loop by means of syringe which is secured by proximal ligature After injection, syringe is removed and proximal suture is tightened The loop is placed into abdominal cavity and the incision is closed After a predetermined period of time animal is sacrificed, the intestinal loop is excised and homogenized and the amount of drug unabsorbed is determined 32Advanced biopharmaceutics and pharmacokinetics
  33. 33. Advantages: 1. The method is relatively simple and reproducible. Disadvantages: 1. Only one sample can be obtained from the experimental animal. 33Advanced biopharmaceutics and pharmacokinetics
  34. 34. B. ABSORPTION FROM THE STOMACH Method: Adult male rats which are fasted are anaesthetized Cardiac is ligated An incision is made in pylorus in which cannula is inserted and ligated 34Advanced biopharmaceutics and pharmacokinetics
  35. 35. The lumen is washed several times with saline and subsequently with 0.1 N HCl solution containing 0.15M NaCl The drug solution of known concentration is introduced into the stomach After 1hr,the solution removed from the gastric pouch and assayed for drug content The percentage of drug absorbed in 1hr can be calculated Advanced biopharmaceutics and pharmacokinetics 35
  36. 36. 3. IN VIVO METHOD These are the actual method. Even though in situ method mimic the in vivo method, most of the factors like gastric emptying, intestinal motility and effects of the drug on the Git are only predicted by in vivo method Advanced biopharmaceutics and pharmacokinetics 36
  37. 37. Advanced biopharmaceutics and pharmacokinetics 37
  38. 38. DIRECT METHOD: This method involves the study of drug uptake is done by determining the drug present in urine or blood. For this suitable sensitive analytical procedure are developed to assay the intact drug in the biological fluid that is sampled. If the drug undergoes metabolism extensively, specific assay is developed for the one of the therapeutically active metabolite, hence concentration of metabolite can also ne determined. Before conducting experiments on animal it should be pre clinically trialled. Advanced biopharmaceutics and pharmacokinetics 38
  39. 39. The animal chosen should be resembalance to man in some extent. Most preferable is pig but it is not used because of handling problem. Then second preferred are dog. At last if no choice the rabbits and rats. Advanced biopharmaceutics and pharmacokinetics 39
  40. 40. METHOD: A blank urine or blood sample is taken from the test animal before the experiment Then test dosage form is administered to the animal At appropriate intervals of time, the blood or urine sample are collected Assayed for drug content From this data, we can determine the rate and extent of drug absorption Advanced biopharmaceutics and pharmacokinetics 40
  41. 41. INDIRECT METHOD: Here pharmacologic response is taken as the index of drug absorption. It is done when measurement of drug concentration in blood or urine is difficult or not possible but sensitive method is available to test the activity. Assumption is made that, when a test dosage form is administered in a body, the obtained pharmacologic response of a drug is related to the amount of drug in the body. Advanced biopharmaceutics and pharmacokinetics 41
  42. 42. LD50 appears to be dependent on the rate of absorption of the drug and hence on the rate of dissolution. A plot of log dose v/s response time is plotted Slope is: FKa 2.303 And intercept of is log d Where, F = bioavailability Ka = absorption rate constant d = threshold dose Advanced biopharmaceutics and pharmacokinetics 42
  43. 43. 4. IN SILICO METHOD In silico is an expression used to mean “performed on computer or via computer simulation”. Important model for determining the drug uptake by in silico method is PAMPA model. Advanced biopharmaceutics and pharmacokinetics 43
  44. 44. PAMPA MODEL(PARALLELARTIFICIAL MEMBRANE PERMEABILITYASSAY) It contains, hydrophobic filter material coated with a mixture of lecithin/phospholipids dissolved in an inert organic solvent such as dodecane creating an artificial lipid membrane that mimics the intestinal epithelium. The rate of permeation across the membrane barrier is correlated with the extent of drug absorption in humans. Advanced biopharmaceutics and pharmacokinetics 44
  45. 45. Advanced biopharmaceutics and pharmacokinetics 45
  46. 46. REFERENCES: (1).Biopharmaceutics and pharmacokinetics – A treatise by D.M.Brahmankar and Sunil.B.Jaiswal , 3rd edition published by - M K Jain for VALLABH PRAKASHAN,C- 5 SMA cooperative industrial estate, GT Karnal road, Delhi – 110033, Page number: 78-81. (2). Textbook of biopharmaceutics and pharmacokinetics by Dr. Shobha Rani.R.Hiremath,1st edition , published by PRISM BOOKS PVT.LTD,1865- 32nd cross BSK II stage Bangalore – 560070, Page number: 25-31. (3). Video was taken from, https://youtu.be/6xdaj6bOtLo Advanced biopharmaceutics and pharmacokinetics 46
  47. 47. Advanced biopharmaceutics and pharmacokinetics 47