yathish M.methods of Determining absorption

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A SEMINER ON METHODS OF DETERMINING ABSORPTION PRESENTED BY YATHISH M

Methods For Studying The Absorption:

Methods For Studying The Absorption The method used for studying the absorption can classified in to IN VITRO METHOD IN SITU METHOD IN VIVO METHOD

IN VITRO METHOD:

IN VITRO METHOD In vitro method are carried out outside of the body & use to determine the permeability of drug using live animal tissues. In vitro model have introduced to assess the major factor involve in absorption process& predict the rate &extent of drug absorption. Here, the intestine of lower experimental animal such as rats, guinea pigs, rabbits are taken for the study.

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The different in vitro methods are: (a) Everted small intestinal sac technique (b) Everted sac modification (c) Circulation techniques (d) Everted intestinal ring or slice techniques

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Serosal side Buffer solution Mucosal side ligature Serosal side Mucosal side Everted small intestinal sac technique

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EVERTED SMALL INTESTINAL SAC TECHNIQUES This method involves isolating a small segment of a laboratory animal such as rat, & inverting the intestine & filling with small volume of drug free buffer solution. Both the ends of the segment are tied off & immersed in an ERLENMEYER FLASK containing a large volume of buffer solution that contain the drug. The flask and its content are oxygenated & agitated at 37 ºC for a specific period of time. After incubation, The serosal fluid is assayed for drug content.

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ADVANTAGE The epithelial cell of the mucosal surface are exposed directly to the oxygenated mucosal surface. Prolongs the viability and integrity of the preparation after removal from the animal. Convenience & accurate DISADVANTAGE Difficulty in obtaining more than one sample per intestinal segment.

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EVERTED SAC MODIFICATION In this method, The test animal is fasted for a period of 20-24hrs & water is allowed. The animal is killed & entire small intestine is everted. Segment, 5-15 cm in length are cut from a specific region of the intestine. The distal end of the segment is tied & the proximal end is attached to the cannula. The segments is suspended in mucosal solution which contain the drug. A drug free buffer solution is placed in the serosal compartments.

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For determining the rate of drug transfer, the entire volume of serosal solution is removed from the sac at each time interval with help of syringe & replace with buffer solution The amount of drug that permeates the intestinal mucosa is plotted against time to describe profile of the drug at specific pH ADVANTAGE A number of different solution may be tested with a single segment of the intestine Simple & reproducible. It distinguishes between active & passive absorption.

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It determines the region of the small intestine where absorption is optimal, in the case of active transport. Method used to study of pH , surface active agents, complexation & enzymatic hydrolysis. DISADVANTAGE The intestinal preparation is removed from the animal as well as from its normal blood supply. under this condition , the permeability characteristics of the membrane are altered. The rate transport of drug as determined from the everted sac technique, is slower.

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CIRCULATION TECHNIQUES In this method, small intestine may or may not be everted. This involve s isolating either the entire small intestine of small lab animal or a segment & circulating oxygenated buffer containing the drug through the lumen. Drug free buffer is circulated on the serosal side of the intestine membrane. Absorption rates from the lumen to the outer solution are determined by sampling both the fluid circulating through the lumen and outside.

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ADVANTAGES Method is applicable to kinetic studies of the factors affecting drug absorption. Both surface are oxygenated. Eversion is not necessary.

EVERTED INTESTINAL RING OR SLICE TECHNIQUE:

EVERTED INTESTINAL RING OR SLICE TECHNIQUE In this techniques, the entire small intestine is isolated from the fasted experimental animal & cut with a scalpel or scissors into ring like slices. The slice wash with a buffer and dried by blotting with filter paper. The dried ring are transferred to stoppered flasks containing the desired volume of buffer containing the drug. The content are continuously agitated and aerate.

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At selected time interval, The tissue slices are assayed for drug content. ADVANTAGE Simple & reproducible. Kinetic studies can be performed. DISADVANTAGE Process of cutting the intestine into rings may expose highly permeable areas of cut or damage the tissue to the medium.

IN VIVO METHOD:

IN VIVO METHOD In vitro and in situ techniques gives us an idea about absorption, but in vivo method gives us an idea about some important factor that influence absorption such as gastric emptying, intestinal motility, & the effects of drugs on the GIT can be determined. The in vivo method can be classified in to DIRECT METHOD IN DIRECT METHOD

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DIRECT METHOD The drug level in blood or urine is determine, For this, sensitive reproducible analytical procedure should be develop to determine the drug in the biological fluid that is sample. In this method, blank urine or blood sample is taken from the test animal before the experiment. The test dosage form is administered to the animal & at the appropriate interval of the time the blood or urine sample are collected &assayed for the drug content. From the data, we determine the rate & extent of the drug absorption.

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In this method ,the experimental animal chosen should bear some resemble to man. It is reported that pigs most closely resemble to man but are not used due to the handling problems. The other animal that can be used are rabbits, & rats.

IN DIRECT METHOD:

IN DIRECT METHOD When the measurement of drug concentration in blood or urine is difficult or not possible, but a sensitive method is available to test the activity,& absorption studies can be done by indirect method. In this method, pharmacological response of the drug is related to amount of a drug in the body determine. The response is determine after the administration of a test dosage form, LD 50 appears to be dependents on the rate of the drug absorption.

IN SITU METHOD:

IN SITU METHOD The term in situ refers to those method in which the animal’s blood supply remains intact, as result of which the rate of absorption determined from this method may be realistic than those determined from in vitro techniques. These in situ models are powerful tools to study the mechanistic aspects of these important process. They acts as bridge between the in vitro & in vivo methods .

ABSORPTION FROM SMALL INTESTINE:

ABSORPTION FROM SMALL INTESTINE Absorption of drug from small intestine describe by two techniques. (a) Perfusion techniques (b) Intestinal loop techniques. (a) PERFUSION TECHNIQUES. In this techniques, adult male rats are fasted for about 16 to 24 hrs before the experiment. The animal is anaesthetised & a midline abdominal incision is made and the small intestine is isolate & cannulated at the duodenal & ileal ends with polyethylene cannula.

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The stomach & cecum are close off by ligature & the intestine is replaced in the rat’s abdominal cavity. The incision is close & the duodenal cannula is attached to infusion pump. The intestine is cleared off using drug free buffer solution for 30 min. Then, drug containing buffer solution is perfused for 30 min. Sample at 10min. interval are collected from ileal cannula & assayed for the drug content & the relative rate of the absorption is calculated. This method was used to demonstrate the validity of the pH partition hypothesis in the absorption of weakly acidic & basic drugs.

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DOLUISION & COWERKER have reported a simple & reproducible method for studying intestinal absorption of drugs in rats. Here, a ret has been fasted overnight is anaesthetised, a midline abdominal incision is made, & the intestinal segment (jejunal) to be perfused is isolated. A L shape glass inlet cannula is secured into the segment and the outlet cannula is placed 15-50cm from the inlet cannula. These are secured with suture & the intestine is replaced in the abdominal cavity. A hypodermic syringe containing perfusion solution, is attached to the duodenal cannula & the first intestinal lumen is cleared off by introducing the solution from the syring.

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The syringe is filled with drug solution & 10ml volume is introduce into the intestine. At appropriate time interval, the solution in the intestine is pumped into either syringe,0.1ml sample is removed and assayed for drug content. The effective permeability (P) is calculated by the equation : C out = exp [ -p(2 π rl)/Q] C in here, r = radius of the gut lumen l = length of the gut lumen Q = perfusate flow rate. p = Permeability of the compound

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Cout and Cin represents the concentration of the drug leaving the perfused segment and concentration of the drug entering the segment respectively. b)Intestinal loop technique :- here single or multiple intestinal loops are used for the studying the absorption Adult male rats are fasted before the experiment under the anesthesia and abdominal midline incision is made and the small intestine is exposed. A proximal ligature is placed around the intestine from the pylorus and a distal ligature is siquared at the distance of 4 inches to the proximal ligature.

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The drug solution is introduce in to the lumen of loop by means of syringe which is secured by the proximal ligature. After the injection, the needle is removed, the loop is replaced in to the abdominal cavity and the incision is closed. After a specific period of time the animal is sacrificed , the intestinal loop is excised and homogenised and the amount of drug unabsorbed is determined. ADVANTAHGES Simple and reproducible DISADVANTAGES Only one sample can be obtain from the experimental animal.

ABSORPTION FROM THE STOMACH:

ABSORPTION FROM THE STOMACH Fasted adult male rats are anaesthetised , stomach is exposed and cardiac end ligated An incision is made in the pylorus in which a cannula is introduced and ligated. The lumen is washed several time with saline and 0.1 N HCL solution containing 0.15 M NaCl. Drug solution of a known concentration is introduced in to stomach. After 1 hour the solution is removed from the gastric pouch and analyse for the drug content.

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The percentage of drug absorbed in one hour may be calculated The gastric pouch may also be homogenised and analysed for a drug. IN ORDER TO OBTAIN THE NO OF SAMPLE AS A FUNCTION OF TIME, THE FOLLOWING MODIFICATION ISA DONE. Drug solution is introduced in to the gastric lumen through the cardiac cannula. A poly ethyline tubing connect to 2 ml syringe is attached to the duodenal L shape cannula At a specific interval the stomach contain are sampled by withdrowing the solution in syringe.

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DISADVANTAGES In situ techniques equate absorption with the lose of drug from the GI lumen. A drug is accumulated or metabolized in the gut wall , the one will get an overestimate of the amount of drug absorbed.

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29 Thank you