TRANSPORT ACROSS CACO2 MONOLAYERS

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TRANSPORT ACROSS CaCO2 MONOLAYERS: BIOLOGICAL, PHARMACEUTICAL AND ANALYTICAL CONSIDERATIONS:

PREPARED BY : Kunal Vanpariya Sem -I, P’ceutics 2011-12 Roll no. 17 Anand Pharmacy College TRANSPORT ACROSS CaCO 2 MONOLAYERS: BIOLOGICAL, PHARMACEUTICAL AND ANALYTICAL CONSIDERATIONS GUIDED BY : Vaishali T. Thakkar

INTRODUCTION: :

A major objective in the pharmaceutical industry is to develop new drugs with Good Oral Bioavailability . Good oral bioavailability occurs when the drug has Maximum Solubility and Maximum Permeability at the site of absorption. Thus the Assessment of Intestinal Permeability represents one essential part in the prediction of oral any new drug candidate. The intestinal permeability data have been also used in preformulation studies to evaluate the effects of various pharmaceutical excipients as co-solvents or absorption enhancers on drug permeation . INTRODUCTION :

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So far as a number of in-vitro methods for assessing the intestinal permeability of a given drug candidate have been developed and recently reviewed. In the last decade, the use of Caco-2 cell monolayer has gained in popularity as an in-vitro primary absorption screening tool in several pharmaceutical companies and several examples of successful correlation with human absorption have been reported.

METHODS TO ASSESS DRUG ABSORPTION:

IN VITRO METHODS :- Cell Culture Models Isolated Mucosal Cells Brush border Membrane Vesicles Everted Tissue Techniques Isolated Tissue Techniques IN SITU METHODS:- Closed Loop Studies Perfused Loop Studies Perfused Intestine- Liver Preparation IN SILICO METHODS (COMPUTATIONAL) Rule Of Five QMPRPlus TM (Quantitative Molecular Permeability Relationship) GASTROPlus TM iDEA TM (In Vitro Determination For The Estimation Of ADME) METHODS TO ASSESS DRUG ABSORPTION

IN VITRO METHODS :

CELL CULTURE MODELS:- There are well accepted cell culture models for drug absorption studies. These models are based on the assumption that passage of drugs across the intestinal epithelium is the main barrier for drugs to reach the circulation. Several human colon carcinoma cell lines, such as the caco-2, HT-29, SW116, SW-480, LS174T cell lines, have been proposed for drug absorption. To, the date caco-2 cell line has been the most thoroughly investigated in vitro model and we will discussed it in detail. IN VITRO METHODS

What is CaCO2? ---- Biological consideration:

The caco-2 cell culture model was introduced in the early 1990s and has become a widely used tool for the determination of the intestinal transport characteristics of drug candidates. Origin : Human colon Adenocarcinoma that differentiates spontaneously into enterocyte- like cells. Caco2 cell monolayers are used as an in vitro model to study intestinal absorption and for high throughput screening of drugs and excipients . What is CaCO 2 ? ---- Biological consideration

Characteristics of parental CaCO 2 cells:

Characteristics of parental CaCO 2 cells origin Human colorectal adenocarcinoma Growth in culture Monolayer epithelial cells Differentiation 14-21 days in std culture medium Morphology Polarized cells,with tight junctions apical,brush border Electrical parameters High electrical resitance Digestive enzymes Typical membreanous peptidase and disaccharidases of the small intestine Active transport Amino acids, sugars, vitamins, hormones Membrane ionic transport Na + /K + ATPase , H + /K + ATPase,Na + /H + exchange,apical Cl - channels Membrane non ionic transporters Permiability glycop [ rptein,multidrug resistant associated protein,lung cancer associated protein Receptors Vit B12, Vit D3,Glut receptors(GLUT1,GLUT2,GLUT3,GLUT5)

STEPS IN CACO-2 MODEL:

Intestinal permeability assay consist of three phases. cell culturing transport experiments Sample analysis STEPS IN CACO-2 MODEL

1. CELL CULTURE:

The cell culture phase consists of growing of cells in flasks and cultivating the cell monolayer on semi porous membranes . The cells are cultivated in high glucose Dulbecco’s Modified Eagle’s medium(DMEM) 1. CELL CULTURE Ingredients Quantity Glucose 4.5 g/lit L-glutamine NaHCO3 supplemented with heat inactivated Fetal Bovine Serum(FBS) 10% Streptomycin 0.1 mg/ml Penicillin 100 units HEPES buffer 10 mM Non essential amino acids (NEAA) Culture conditions Temperature 37 0 C Relative humidity 95% Air 5% CO 2 Medium should be exchanged every 48-72 hours

2. Transport experiments:

Used to check the caco2 cell integrity . Prior to starting transport studies, the integrity of cell monolayer is assessed by different methods like TEER ( T rans E pithelial E lectrical R esistance) measurements or using hydrophobic markers like mannitol, Lucifer yellow or sodium fluorescein . 2. Transport experiments

TEER (Trans Epithelial Electrical Resistance) measurements: :

Easy and noninvasive method. TEER can be measured by measuring the electrical resistance across the monolayer using, Millicell ®-ERS system ohmmeter The STX-100M electrode or EVOM Epithelial Voltammeter . TEER value → 450-650Ω cm 2 Monolayers with TEER values outside this range are discarded. Disadvantage : TEER value is indicative for the transjunctional flux of ions that are much smaller than the tight junction dimensions. Therefore, TEER values alone are not sufficient to assess integrity of monolayers when considering drug transport. TEER ( T rans Epithelial Electrical Resistance ) measurements :

Using markers::

The permeability coefficient P app of passive paracellular flux markers such as mannitol, sodium fluorescein, Lucifer yellow, PEG and dextran are more sensitive than TEER values. Generally radiolabeled mannitol is used but when the use of the radiolabeled material is undesirable , Lucifer yellow –a fluorescent passive paracellular permeability marker is used for the monolayer integrity detection. The acceptable Trans epithelial flux rate of Lucifer yellow is less than 4*10 -7 cm/sec . Using markers :

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Sodium fluorescien (M.W.=376) was chosen to assess the paracellular permeability and the tightness of the monolayers . The use of sodium fluorescein is advantageous because after the drug transport experiments, the cells can be rinsed and reused. The sodium fluorescien allows one to monitor the effect of drugs on the integrity of the cell monolayers . Determination of cell integrity by sodium fluorescein :- After an initial period of incubation with the test compound, the cell monolayers were rinsed and incubated with sodium fluorescien for 60 min at 37 o C, on a vibrax shaker at 80 rpm. The diffusion of sodium fluorescien into the basal compartment was determined by measuring the fluorescence integrity (excitation 485 nm, emission 535nm).

3. CACO-2 PERMEABILITY ASSAY:

Earlier the permeability assays were carried out in 12 or 24 well transwell plates. Procedure: Cell monolayer grown on PC(polycarbonate) filter are washed & bathed in a transport buffer . The pH of the transport buffer is adjusted depending upon the drug solubility. To determine the rate of drug transport, compound dissolved in transport solution are dosed in the A/B or B/A direction . Then the transwell plates are incubated at 37 0 c with appropriate agitation to reduce the thickness of the UWL. At the predetermined time point, the samples are collected from the receiver chambers & replenished with blank transport buffer. To determine the % recovery, sample is collected from the donor chamber at the end of the experiment . 3. CACO-2 PERMEABILITY ASSAY

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Disadvantage : Slow process & labor intensive. Require 3 weeks (21days) to form monolayer of fully differentiated cells. Advancement: 24 well plates are replaced with 96 transwell plates , which provides automation & allow measurement of drug transport in high throughput format.

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SAMPLE ANALYSIS The drug concentration can be detected by various analytical methods. Early the radio labeled compounds are used and can be detected by radioactivity measurement by scintillation counters. For the detection of drug concentration, HPLC MS/MS LC-MS/MS Systems are used. Calculation Of Apparent Permeability: dt Papp d θ 1 C o A WHERE, Papp: apparent permeability coefficient (cm/s) dθ / dt : rate of drug transport ( ng /s) C o : initial concentration in apical chamber ( ng /ml) A: surface area of the monolayer (cm 2 )

MODEL VALIDATION :

HIGH ABSORPTION INTERMEDIATE ABSORPTION LOW ABSORPTION Isopropanol (reference) Cimetidine (reference) Atenolol (reference) Ethylene glycol (chemical) Paraquat (pesticide) Cupric sulphate (chemical) Sodium Valproate (drug) Paracetamol (drug) Colchicine (drug) Acetylsalicylic acid (drug) MODEL VALIDATION Acc . to FDA guidelines, validation of Caco-2 permeation assay was performed using reference drugs known as low, moderate or high absorbers . 10 reference chemicals

APPLICATION/ADVANTAGES OF CaCO2 MODEL:

In Drug Discovery : To test the absorption profiles of the new molecular entities in the lead optimization state . In pre-clinical drug development : US FDA recognizes Caco-2 to measure permeability as part of the bioequivalence waiver process . To evaluate effect of pharmaceutical excipients that increase intestinal permeability through an increase in solubility. Thus caco-2 cells are used to assess formulation strategies designed to enhance membrane permeability . Caco-2 cells cultured for sufficient time to express the various transporters which either aid or limit transepithelial transport, so caco-2 cell are used to study transport mechanism for many compounds In drug metabolism & toxicity effects . Others like regulation of protein expression; genetics study . APPLICATION/ADVANTAGES OF CaCO 2 MODEL

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Application Examples Examine the factors affecting the transport of the drug Investigate the effect of pH, glucose, temperature, inhibitors on the transport of alfa – methyl dopa Examine the effect of mannitol concentration on transport Investigate the effect of temperature on various transport mechanisms Examine the influence of the peptide structure on transport across the epithelial cell Study drug transport mediated by various carriers Characterize vitamin D receptor Identify bile acid carrier Investigate the transporter mediated mechanism for glucose Examine the role of gamma glutamyl transpeptidase in amino acid transport Explore the carrier mediated transport for large neutral amino acids- phenylalanine. proline Invastigate metabolism reactions Phase 2 metabolism Sulfation of p- nitrophenol Glucoronidation of p- nitro phenol Investigate vitamin A metabolism Study the metabolism of oxygenated derivatives of arachidonic acid

DISADVANTAGES OF CACO-2 CELLLINES:

High cost per assay. Time consuming, requires 21 days for full cell differentiation. Requires regular maintenance feeding for the preparation of stable monolayers. Relatively low throughput . The necessity of LC / MS or HPLC for quantification. Cells have a tumoral origin . Variable expression of transport and metabolizing proteins . The Caco-2 cell cultures are known to over express the P-glycoprotein efflux system, it may give rise to underestimation of the absorption of some compounds. The drug concentration at which Caco-2 is typically performed does not accurately model in vivo dosing . DISADVANTAGES OF CACO-2 CELLLINES

INTRINSIC LIMITATION OF THE CaCO2 CELL CULTURE SYSTEM :

Limitation Alternative s Absence of mucus Mucus producing cellines , cocultures (Co-cultures of HT29- MTX and Caco-2 cells) No cellular heterogeneity Cocultures No CYP3A4 Transfection or Upregulated by adding 1 α ,25-dihydroxy vitamin-D3 to the growth medium. Inability to study regional difference Using chambers, perfused intestinal segments Thickness of unstirred water layer shaking INTRINSIC LIMITATION OF THE CaCO 2 CELL CULTURE SYSTEM

MODIFICATION OF CaCO 2 CELL LINES::

MODIFICATION OF CaCO 2 CELL LINES :

ASSAY DEVELOPMENT:

The preparation time of a fully functional Caco-2 monolayer can be shortened to 3 days using a modified system “ biocoat intestinal epithelium differentiation environment(BIEDE) ” ASSAY DEVELOPMENT

CACO-2: PHARMACEUTICAL CONSIDERATIONS…:

CACO-2: PHARMACEUTICAL CONSIDERATIONS…

EFFECT OF COMMON EXCIPIENTS ON CACO2 TRANSPORT OF LOW PERMEABILITY DRUGS:

Lactose monohydrate, HPMC, EDTA, propylene glycol, PEG 4oo, anhydrous cherry flavor : no effect on permeability SLS: moderately increases the permeability of all most all drugs. Tween80 : increased the apical to basolateral direction permeability of furosemide, cimetidine, and hydrochlorothiazide, presumably by inhibiting their active efflux . EFFECT OF COMMON EXCIPIENTS ON CACO2 TRANSPORT OF LOW PERMEABILITY DRUGS

ADSORPTION AND NON SPECIFIC BINDING:

Adsorption to the culture device and non specific binding into the cell mono layer can lead to under estimation of the apparent permeability coefficient ( P app ) of the compounds as well as to poor recovery of the compound and a low mass balance. Minimized adsorption include Pretreatment of the device with albumin Post experiment wash step by acetonitrile,methanol , etc…. The calculation of the apparent permeability coefficient based on the disappearance of the compound from the donor compartment . ADSORPTION AND NON SPECIFIC BINDING

SINK CONDITION :

For low solubility compounds , maintenance of sink conditions avoids the saturation of the acceptor compartment For the sink condition: We can change the acceptor solvent more frequently at well defined time points To use a medium in the acceptor compartment that is able to decrease the free concentration of the drug. SINK CONDITION

THE LIMITED SOLUBILITY OF HIGHLY LIPOPHILIC DRUGS:

Sr no. Excipients Drugs 1 Cyclodextrin Mefenamic acid, phenytoin 2 Dimethyl acetamide Hydrochorothiazide 3 Ethanol Taxol , quercetin 4 Propylene glycol Frusemide , atenolol 5 Polysorbate 20 metfomin 6 Sodium docusate Frusemide , atenolol 7 SLS Frusemide , atenolol 8 tween 80 Frusemide , atenolol THE LIMITED SOLUBILITY OF HIGHLY LIPOPHILIC DRUGS

Influence of lipid content of a liposome formulation used for solubilization of a poorly water-soluble drug on absorption through Caco-2 monolayers. :

The caco-2 cell monolayers are also used to determine the influence of liposome which are used to solubilize poorly water soluble drugs on transport processes. In one of the study, the influence of different lipid concentrations investigated with progesterone as a passively absorbed model drug with a high permeability. Progesterone was incorporated in phosphatidylcholine liposomes produced by film method and extraction through polycarbonate filters. The drug absorption was determined in a bi-directional Caco-2 assay, using purely aqueous drug solution as reference. The cells were grown on transwell inserts for 19-21 days. The drug quantification was performed by HPLC/MS. Influence of lipid content of a liposome formulation used for solubilization of a poorly water-soluble drug on absorption through Caco-2 monolayers .

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A mathematical model was developed that took into account transport between the apical, the cellular and basal compartments and expressed passive cell membrane permeation by the apparent permeability coefficient. Conclusion :- For the liposomal formulations, P app strongly decrease with increasing lipid concentration . This may be explained by the assumption that only unbound drug can pass the cell monolayer. The majority of progesterone is distributed into the liposome membrane because of high lipophilicity of this drug. With increasing lipid content in the formulation, the concentration of free progesterone decreases which leads to the decreasing P app values.

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Other cell models…

1) PARALLEL ARTIFICIAL MEMBRANE PERMEABILITY ASSAY (PAMPA):

PAMPA offers a fast and robust tool for screening permeability of drugs in early discovery phase. This method was introduced in 1998 and it uses a phospholipid -coated filter separating two aqueous compartments to mimic the passive transport of small molecules. Because of its speed, low cost, and versatility, it is a particularly helpful complement to cellular permeability models, such as Caco-2. 1) PARALLEL ARTIFICIAL MEMBRANE PERMEABILITY ASSAY (PAMPA)

PAMPA technique :

The donor solutions, were varied in pH, while the acceptor solution had the same pH 7.4 . The buffers are automatically prepared by the robotic system. The p ION Gut Box™ (PN 110205) was used to effect stirring and enable environmental control. The plate sandwich was formed and allowed to incubate in the Gut- BoxTM at 23±1 ºC for 30 min in an atmosphere saturated in humidity, and scrubbed free of oxygen and carbon dioxide. Each donor well of the plate had its own stirrer. The speed setting dial on the magnetic stirring device had been calibrated in units of the thickness of the expected unstirred water layer (UWL). After the short incubation time, the sandwich plates were separated, and both the donor and acceptor compartments were assayed for the amount of material present, by comparison with the UV spectrum (225–500 nm) obtained from reference standards. Mass balance was used to determine the amount of material remaining in the membrane barrier PAMPA technique

ENLARGE DIAGRAM OF ONE CHAMBER: :

ENLARGE DIAGRAM OF ONE CHAMBER : Artificial membrane

Comparision Of PAMPA & Caco-2 Permiability Assay Characteristics :

PAMPA MODEL CACO-2 CELL MODEL There is no cell culture involved , so do not required long planning. Membrane composition comprise of phospholipids in alkane . This method is based on cell culture , so required planning High throughput & low cost Specific requirements are needed For HT in this method like 96- well plats & compatible detection system. the method is Useful only for passive transcellular permeability and not for compounds transported through an active mechanism or via the paracellular rought . For paracellular & transcellular permeability Useful tool in early drug discovery to assess the permeability potential of large no. of compounds. Method is more suitable during lead optimization or preclinical development stages, where true transepithelial permeability is needed. Comparision Of PAMPA & Caco-2 Permiability Assay Characteristics

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PAMPA & caco-2 should not be considered as competing permeability methods. Good correlation between PAMPA & caco-2 data for a compound indicates a predominance of passive diffusion in its permeation. Lack of correlation PAMPA <<Caco-2 ;absorptive (active, paracellular ,gradient effect for acids) transport or PAMPA>>Caco-2 ; secretary (efflux, gradient effect for bases) permeation mechanism

2) THE MADIN-DARBY CANINE KIDNEY (MDCK) CELL MODEL :

One of the commonly used cell monolayer systems to assess the human intestine barrier . MDCK cell lines can reach full differentiation in 3-7days and are therefore relatively easy for cell culturing and assay maintenance in comparison to other cell lines such as caco-2. DISADVANTAGE MDCK cell lines originate from dog kidney . The expression of transporters is quite different from human intestine . 2) THE MADIN-DARBY CANINE KIDNEY (MDCK) CELL MODEL

3) Isolated mucosal cells:

Procedure for isolating mucosal cells include mechanical agitation, scraping, hydrolytic collagenase enzymes or chelating agents such as EDTA . Such isolated cell suspensions have been used to study enzyme activity, drug transport, and cellular metabolism . 3) Isolated mucosal cells

Disadvantage:-:

Isolated mucosal cells lack a submucosa and muscle layer that may present a diffusional barrier to transfer absorbed substances. The use of mucosal cells in drug absorption and transport studies is limited due to rapid autolysis. Isolation process requires opening the tight junctions, which destroys cell polarity , making it impossible to determine the specific membrane domain involved in cellular uptake. Disadvantage :-

4) Brush border membrane vesicles (BBMV):

Methods to isolate BBMV include disruption of the cellular structure followed by density centrifugation, immunoabsorbent chromatography, or precipitation of the nonbrush border enzymes with calcium or magnesium . The calcium or magnesium precipitation method seems to be most widely used. Brush border membrane vesicles have been used extensively to study mucosal uptake process , especially to investigate factors that influence mucosal uptake without interference of intracellular metabolism 4) Brush border membrane vesicles (BBMV)

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Advantage:- The ability to investigate the luminal and contra luminal sides separately Studying different compositions of the intra and extra vascular spaces. The unstirred layer is small compared to tissue preparations Para cellular mechanism do not contribute to transport. Disadvantage:- The isolation procedure may either damage the membrane or may be incomplete, resulting in contamination of the preparation with other membranes. Since the cells are homogenized, active transport cannot be investigated. BBMV have demonstrated variability and time dependent changes that also limit their use in drug absorption and transport studies.

Applications of BBMV:

Applications of BBMV Applications Examples Investigate transport Study transport of bicarbonate, biotin, and riboflavin Study transport systems Identify intestinal peptide transport system Examine the specificity of peptide transport Characterize Na+ dependent bile acid transport system Examine factors affecting transport Role of insulin in glucose absorption Investigate the effect of renal failure on biotin transport Examine age related modifications of leucine uptake and D- glucose transport Determine the effect of stereochemistry on uptake of beta lactam antibiotics

5) EVERTED TISSUE TECHNIQUES:

Everted Sac Technique:- To prepare an everted sac, a small length of the intestine is excised, turned inside out, filled with a fluid and ligated at both ends. The sac is immersed in an oxygenated solution that contains drug. The fluid inside the sac is assayed for drug and the rate of drug transfer across the membrane provides an estimate of the drug permeability. 5) EVERTED TISSUE TECHNIQUES

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Advantages :- The everted sac technique is simple, inexpensive, eliminates variation due to blood flow and permits sampling from mucosal and serosal sides. Everted sacs have been used to investigate questions regarding intestinal metabolism, uptake rates, and the permeability in different regions of the intestine.

Disadvantage:- :

Alteration of the data by experimental variables such as method of oxygenation, serosal sampling, and fluid loss, or damaging the tissue while removing it. Since the tissue is removed from the animal and no longer associated with the blood supply, there is also always the potential of obtaining unrealistic rate and extent of absorption. Disadvantage:-

6) Everted Intestinal Rings:

In everted intestinal rings, the musculature is intact as whole intestinal segments are used . Briefly, intestinal segments are quickly excised, usually from rats, and flushed with saline solution 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 0 c with constant oxygenation. Under optimal conditions, rings remain viable for up to 2 hrs. The transport of the dug is stopped by rinsing the rings with ice cold buffer and drying them. The drug content is assayed and expressed as mol/gm/time . 6) Everted Intestinal Rings

Advantage: :

Method is relatively simple and quick. Method is fast and inexpensive Regional differences in drug absorption can be studied Mechanism of drug absorption can be studied by changing the experimental conditions. Advantage:

Disadvantage:- :

Extreme care is needed to maintain the viability of the tissue throughout the experiment. Tissue needs to be disrupted completely for the determination of drug contents, which complicates the assay procedure. Polarity of the absorption cannot be assayed. Disadvantage:-

7) ISOLATED TISSUE TECHNIQUES:

Isolated sheets of intestinal mucosa are generally obtained by cutting the intestine into strips. After removal of musculature the sheet is mounted in diffusion chamber or an ussing chamber filled with buffers. In this technique small intestine sheets are mounted between two compartments ( ussing chambers). The mucosal and serosal compartments are usually supplied with krebs ringer bicarbonate buffer. The integrity of the tissue is confirmed by transepithelial resistance measurement . The system is maintained at 37 o c with constant stirring and supply of oxygen and carbon dioxide mixture (o 2 :co 2 = 95:5) 7) ISOLATED TISSUE TECHNIQUES

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The permiability can be measured by adding drug to the donor chamber and checking appearance of the drug in receiving chamber. Fluxes in both the directions i.e. mucosal to serosal and serosal to mucosal can be studied. Effect of pH can be studied simply by altering the pH of the buffer in appropriate chamber. For this technique mostly rat intestine is preferred as its permeability correlates well with that of human intestine

Advantage:- :

Permeability across different regions can be determined and thus regional differences in intestinal absorption can be studied. Determination of permeability in different animal models can be done by using different animal tissue . thus species differences with respect to intestinal absorption characteristics can be determined. Amount of drug needed is relatively small Advantage:-

Disadvantage:- :

The viability of intestinal tissue mounted in ussing chamber is quite controversial Dissection of epithelial tissue is quite difficult and serosal muscle layer can only be partially removed. Disadvantage:-

IN SITU METHODS TO ASSESS DRUG ABSORPTION… :

IN SITU METHODS TO ASSESS DRUG ABSORPTION… In situ absorption models permit the study of individual organ processes and site specific absorption. The tissue is maintained intact with blood flow to the organ. Samples of drugs solution from in situ loop experiments can be obtained to measure drug disappearance and metabolite formation .

Closed Loop Studies:

Two types of in situ loop studies have been repoted : An anesthesia recovery method The method of doluisio et al . Closed Loop Studies

AN ANESTHESIA RECOVERY METHOD:

To perform an anesthesia recovery closed loop study, ligatures are formed in an intestinal region, drug solution is introduced in to the intestinal loop, the abdominal area is sutured, and the animal is permitted to recover from the anesthesia. Multiple samples may be obtained to measure the appearance of the drug in the blood. At a specified time, the animal is sacrificed, the loop is excised, and the amount of unabsorbed drug is determined . In this technique, the effect of anesthesia on the absorption process is minimized. Limitations of the technique include volume fluctuations and the fact that only one data point can be obtained from each animal’s luminal content. AN ANESTHESIA RECOVERY METHOD

The Method Of Doluisio And Co Workers:

This is a technique that permits multiple sampling of the luminal content. In this technique, the animal is anesthesized and two cannulae are inserted into the small intestine. The drug solution is introduced into the cannulated segment and multiple samples are withdrawn from the luminal solution during the experimental period. The disadvantage of the technique include the use of an anesthetic that may alter blood flow to the intestine and the potential for volume fluctuations in the loop . The Method Of Doluisio And Co Workers

2) Perfused Loop Studies:

The perfused loop study design is more physiological than the closed loop design . The cannulation procedure is identical to the procedure detailed above. The drug solution is perfused into the loop and the flow rate can be regulated . The single pass technique can be adapted to permit recycling of the drug solution through the intestinal lumen. In addition, the venous blood supply can be collected to monitor the appearance of drug. 2) Perfused Loop Studies

3) Perfused Intestine-liver Preparations:

This technique uses a direct approach to examine the contribution of each organ/tissue in first pass metabolism and can be used to study the sequential processing of drug and metabolites by the intestine and liver. The model is flexible and permits manipulation of the flow, intestinal site, concentration and perfusate 3) Perfused Intestine-liver Preparations

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applications Examples Effect of absorption enhancers Effect of EDTA , sod.caprate , sod.glycocholate on the absorption on insulin Effect of enzyme inhibitors on absorption Effect of aprotinin on insulin absorption Site specific absorption Cyclosporin , carbamazepine and imidapril Examine the effect of chemical modification Study the intestinal absorption of thyrotropin releasing hormone by chemical modification with lauric acid Examine the effect of micelles Examine permeability of acyclovir across he intestine in mixed micelles Examine carrier mediated transport Baclofen , beta- lactam antibiotics Examine the site of stereo inversion Metabolic inversion of r(-) ibuprofen Effect of chelating agents on absorption Effect of citric acid, tartaric acid, penicillamine on the absorption of mercuric chloride

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