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Invitro invivo insitu method

This document discusses various in vitro, in vivo, and in situ methods used to study drug absorption. It describes several in vitro techniques like partition coefficient studies, artificial membrane models, and cell culture models. It also explains various in vivo methods like direct blood/urine sampling and indirect pharmacological response studies in animals. Finally, it covers in situ techniques like intestinal perfusion that better simulate in vivo conditions compared to in vitro models. The document provides detailed descriptions of specific methods like the everted sac technique, diffusion cell method, and stomach perfusion studies in rats.

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Presented by GURUPRASAD S I-M.PHARM(PHARMACEUTICS) JKK NATRAJAH COLLEGE OF PHARMACY KOMARAPALAYAM
“Absorption is defined as the process of movement of unchanged drug from the site of administration to the systemic circulation”
A) In vitro method B) In Vivo Method C) In situ method
 In vitro method are carried out outside of the body and are used to determine the permeability of drug using live animal tissues.  In vitro models have been introduced to assess the major factors involved in the absorption process and predict the rate and extent of drug absorption.  Here, the intestine of lower experimental animals such as rats,guinea pigs, rabbits are taken for the study.
 Physico chemical methods 1) Partition coefficient 2) Artificial membrasnes 3) Chromatographic retention indices 4) Brush border membrane vesicles(BBMV) 5) Isolated intestinal cells 6) Tissues techniques a)Everted small intestinal sac technique b) Everted sac modification c) Circulation techniques d) Everted intestinal ring or slice techniques 7) Diffusion cell method 8) Cell culture techniques
 Partition coefficient between an oil and water phase, log P, is one of the easiest property of a drug molecule that can be determined.  It provides a measure of the lipophilicity of a molecule and can be used to predict to what extent it will cross the biological membrane. Eg. Octanol is selected as an oil phase as it has similar properties to biological membranes.  It’s important to note that Log P does not take the degree of ionization into consideration and hence log D is used.
 An artificial membrane, or synthetic membrane, is a synthetically created membrane which is usually intended for separation purposes in laboratory or in industry.  Synthetic membranes have been successfully used for small and large-scale industrial processes since the middle of twentieth century.  A wide variety of synthetic membranes is known.
 They can be produced from organic materials such as polymers and liquids, as well as inorganic materials. The most of commercially utilized synthetic membranes in separation industry are made of polymeric structures. They can be classified based on their surface chemistry, bulk structure, morphology, and production method. The chemical and physical properties of synthetic membranes and separated particles as well as a choice of driving force define a particular membrane separation process.  The most commonly used driving forces of a membrane process in industry are pressure and concentration gradients. The respective membrane process is therefore known as filtration. Synthetic membranes utilized in a separation process can be of different geometry and of respective flow configuration. They can also be categorized based on their application and separation regime.
 The best known synthetic membrane separation processes include water purification, reverse osmosis, dehydrogenation of natural gas, removal of cell particles by microfiltration and ultrafiltration, removal of microorganisms from dairy products, and Dialysis.
 In gas chromatography, Kovats retention index (shorter Kovats index, retention index; plural retention indices) is used to convert retention times into system- independent constants.  The index is named after the Hungarian- born Swiss chemist Ervin Kováts, who outlined this concept during the 1950s while performing research into the composition of the essential oils.
 The retention index of a certain chemical compound is its retention time normalised to the retention times of adjacently eluting n- alkanes. While retention times vary with the individual chromatographic system (e.g. with regards to column length, film thickness, diameter, carrier gas velocity and pressure, and void time), the derived retention indices are quite independent of these parameters and allow comparing values measured by different analytical laboratories under varying conditions.
 Tables of retention indices can help identify components by comparing experimentally found retention indices with known values
 A brush border (striated border or brush border membrane) is the microvilli-covered surface of simple cuboidal epithelium and simple columnar epithelium cells found in certain locations of the body.  Microvilli are approximately 100 nanometers in diameter and their length varies from approximately 100 to 2,000 nanometers in length.  Because individual microvilli are so small and are tightly packed in the brush border, individual microvilli can only be resolved using electron microscopes
 Here, the small intestine is perfused with enzyme solutions that release the cells are treated with chelating agents or enzymes  The freshly isolated cells are suspended in buffer solution  At the time of experiment, the cells are separated, resuspended in buffer containing the drug under O2/CO2 and shaken well.  After a specific period of time, the cells are separated by filtration, extracted and drug absorbed is determined.
a) Everted small intestinal sac technique:  This method involves isolating a small segment of the intestine of a laboratory animal such as rat, inverting the intestine and filling sac with a small volume of drug free buffer solution.  Both the segments are tied off and the sac is immersed in an ERLENMEYER FLASK containing a large volume of buffer solution that contains the drug.  The flask and its contents are they oxygenated and the whole preparation is maintained at 37 degree celsius and shaken mildly.  At predetermined time intervals, the sac is removed and the concentration of drug in the serosal fluid is determined/assayed for drug content.
b) EVERTED SAC MODIFICATION  In this method, the test animal is fasted for a period of 20-24 hr and water is allowed.  The animal is killed and the entire small intestine is everted. Segments,5-15 cm in length are cut from a specific region of the intestine.  The distal end of the segment is tied and the proximal end is attached to the cannula. The segment is suspendedin a mucosal solution which contains the drug.  A drug free buffer is then placed in the serosal compartment.
c) CIRCULATION TECHNIQUES  In this method, small intestine may or may not be everted.  This involves isolating either the entire small intestine of small lab animal or a segment and circulating oxygenated buffer containing the drug through the lumen.  Drug free buffer is circulated on the serosal side of the intestinal membrane and oxygenated  Absorption rate from the lumen to the outer solution are determined by sampling both the fluid circulating through the lumen and outside
d) EVERTED INTESTINAL RING OR SLICE TECHNIQUE  In this technique, the entire small intestine is isolated from the fasted experimental animal and washed with saline solution and dried by blotting with filter paper.  The segment is tied at one end and by placing on glass rod it is carefully everted and cut into small rings.  The everted intestinal rings are then incubated in drug containing buffer maintained at 37 degree celsius with constant oxygenation.  Under optimal conditions, rings remain viable for up to 2 hrs and the transport of drug is stopped by rinsing the rings with ice cold buffer and drying them.
 In this method, small segments of small intestine are mounted between two glass chambers filled with buffer at 37 degree celsius.  Diffusion cell consist of two compartments: i. Donar compartment-which contains the drug solution and the lower end of which contains the synthetic or natural GI membrane that interface with the receptor compartment. ii. Receptor compartment- which contain the buffer solution.
 Cell culture is the complex process by which cells are grown under controlled conditions, generally outside their natural environment.  In this technique, differentiated cells of the intestine, originating from CaCo2 cells (cells or carcinoma of colon )are placed on synthetic polycarbonated membrane previously treated with an appropiate material such as collagen which on incubationaids reproduction of cells while not retarding drug permeation characteristics  These models are based on the assumption that passage of the drugs acrossd the intestinal epithelium is the main barrier for drugs to reachthe circulation.
 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 motility and the effects of drugs on the GIT Can be determined.  The IN VIVO method can be classified into: 1. Direct method 2. Indirect Method
 The drug levels in blood or urine is determined as a function of time. For this, a suitable sensitive reproducible analytical procedure should be developed to determine the drug in the biological fluid.  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 and at appropriate intervals of time the blood or urine samples are collected and assayed for the drug content.  From the data, we can determine the rate and extent of drug absorption.
 In this method, the experimental animal chosen should bear some resembles 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 dogs,rabbits and rats.
 When the measurement of drug concentration in the blood or urine is difficult or not possible, but a sensitive method is available to test the activity, then absorption studies can be done by this indirect method.  In this method, pharmacological response of the drug is related to the amount of the drug in the body.  The response is determined after the administration of a test dosage form,LD 50 appears to be dependent on the rate of the absorbing of drug.
 It stimulates the in vivo conditions for drug absorption and are based on perfusion of a segment of GIT by drug solution and determination of amount of drug diffused through it.  In situ refers to those method in which the animals blood supply remains intact in which the rate of absorption determined from these methods may be more realistic than those determined from in vitro techniques  These models are powerful tools to study the mechanistic aspects of these important process.  Acts as a bridge between in vivo and in vitro methods.
 Absorption of drug from small intestine is described in two methods: 1. Perfusion technique:  Doluisio method  Single pass perfusion method 2. Intestinal loop techniques
II- Absorption from the stomach  Fasted adult male rats are anaesthetized, stomach is exposed and the cardiac end is ligated.  An incision is made in the pylorus in which the cannula is introduced and ligated.  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 and after 1 hour, the solution is removed from the gatric pouch and assayed for drug content.
Invitro invivo insitu method