Intestinal perfusion method

According to the FDA BCS guideline, measurements of the permeability and fraction dose absorbed of a drug can be made by mass balance, absolute bioavailability or intestinal perfusion methods. The intestinal permeability of a drug can be determined by ... 

Many variations of intestinal perfusion methods have been used as absorption models over the years. In situ methods offer advantages over in vitro models. Although the animal has been anaesthetised and surgically manipulated, neural, endocrine, lymphatic, and mesenteric blood supplies are intact and therefore all the transport mechanisms present in a live animal should be functional. As a result absorption rates from these methods may be more realistic in magnitude than those determined from in vitro techniques. 

The advantages of the in situ techniques include an intact blood supply multiple samples may be taken, thus enabling kinetic studies to be performed. A fundamental point regarding the in situ intestinal perfusion method is that the rat model has been demonstrated to correlate with in vivo human data [46 19], Amidon et al. [36] have demonstrated that it can be used to predict absorption for both passive and carrier-mediated substrates. However, the intestinal luminal concentrations used in rat experiments should reflect adequately scaled and clinically relevant concentrations to ensure appropriate permeability determinations [50], There are limitations of the in situ rat perfusion models. The assumption involved in derivation of these models that all drug passes into portal vein, that is drug disappearance reflects drug absorption, may not be valid in some circumstances as discussed below. 

Salphati L, Childers K, Pan L, Tsutsui K and Takahashi L (2001) Evaluation of a Single-Pass Intestinal-Perfusion Method in Rat for the Prediction of Absorption in Man. J Pharm Pharmacol 53 pp 1007-1013. 

With the difficulties associated with accurate estimation of permeability based only on physicochemical properties, a variety of methods of measuring permeability have been developed and used, among which are (l)cul-tured monolayer cell systems, such as Caco-2 or MDCK ( 2 diffusion cell systems that use small sections of intestinal mucosa between two chambers (3) in situ intestinal perfusion experiments performed in anesthetized animals such as rats and (4)intestinal perfusion studies performed in humans (40,54-62). All of these methods offer opportunities to study transport of drug across biological membranes under well-controlledconditions. Caco-2 mono-layer systems in particular have become increasingly commonly used in recent years and human intestinal perfusion methods are also becoming more commonly available. Correlations between Caco-2 permeability and absorption in humans have been developed in several laboratories (63-72). As shown in Fig. 

It should be noted however that it is almost impossible to predict fully the in vivo dissolution rate due to the many factors involved, of which several have not yet been completely characterized. The introduction of new study techniques to directly follow drug dissolution in vivo in the human intestine should therefore be of importance [30, 31]. For example, in vivo dissolution studies discriminated between the dissolution rates of the two different particle sizes of spironolactone, based on the intestinal perfusate samples. In addition, dissolution rates of carba-mazepine obtained in vitro were significantly slower than the direct in vivo measurements obtained using the perfusion method. The higher in vivo dissolution rate was probably due to the efficient sink conditions provided by the high permeability of carbamazepine [30, 31]. 

This technique, also referred to as the auto-perfused method after experiments by Windmuller and Spaeth [67] involves cannulation and drainage of a vein from an intestinal segment and donor blood replacement via a sustainable blood vessel (e.g. jugular vein). The most commonly reported site of cannulation is the mesenteric vein. Cannulation is performed as follows. A midline incision of 4 cm is made and an 8-12-cm segment of the ileum is located to... 

To facilitate a standardisation of inter-laboratory results of permeability, it is now common practice to include a range of model drugs as internal standards in initial validation (i.e. method suitability) of intestinal perfusion techniques [116]. A list of 20 model drugs has been reported by the FDA for the standardisation of the in situ intestinal perfusion experiment, whereas six drugs are recommended for human studies. Once the method has been... 

Sutton SC, Rinaldi MT and Vukovinsky KE (2001) Phenol Red and Comparison of the Gravimetric 14c-Peg-3350 Methods to Determine Water Absorption in the Rat Single-Pass Intestinal Perfusion Model. AAPS Pharm Sci 3 pp 1-5. 

Animal methods In vivo intestinal perfusion In situ intestinal perfusion... 

The permeability class of a drug substance can be determined in human subjects using mass balance, absolute BA, or intestinal perfusion approaches. Recommended methods not involving human subjects include in vivo or in situ intestinal perfusion in a suitable animal model (e.g., rats), and/or in vitro permeability methods using excised intestinal tissues, or monolayers of suitable epithelial cells. In many cases, a single method may be sufficient (e.g., when the absolute BA is 90%... 

The following methods can be used to determine the permeability of a drug substance from the gastrointestinal tract (1) in vivo intestinal perfusion studies in humans (2) in vivo or in situ intestinal perfusion studies using suitable animal models (3) in vitro permeation studies using excised human or animal intestinal tissues or (4) in vitro permeation studies across a monolayer of cultured epithelial cells. 

Sinko, P.J., et al. 1996. Analysis of intestinal perfusion data for highly permeable drugs using a numerical aqueous resistance—Nonlinear regression method. Pharm Res 13 570. 

Some efforts have been made to determine the effect P-gp has on its substrates by use of in situ perfusion methods, including intestinal perfusion, liver perfusion, kidney perfusion, and brain perfusion. These experiments allow the researcher to study the transport of compounds in a physiologically relevant environment in which the integrity of the organ is preserved with regards to cell polarity and representation of all cell types seen in the organ. Furthermore, the reduction in complexity of in situ models versus in vivo studies facilitates the conduct of complex studies and allows more definitive conclusions to be made regarding the role P-gp may play in disposition. 

Various modifications are reported with respect to the experimental setup (single pass or recirculated intestinal perfusion) as well as the site of blood collection, e.g. mesenteric vessels for estimation of the intestinal absorption rate (DeGraw RT, Anderson BD 2004). vs. peripheral veins for estimation of systemic availability of the candidate compound. This method is widely used for investigation of intestinal absorption of nutrients by using radioactive tracers (e.g. cholesterol, glucose) and their interference with the candidate compound (Arts et al. 2004). In addition the secretion of the candidate compound into the intestine can be studied by peripheral administration of the compound into a peripheral vein and subsequent determination of the appearance of the candidate compound in the intestinal perfusate (Merino et al. 2003 Berggren et al. 2004). Also variations are reported using chronically isolated intestinal loops in rats (Poelma et al. 1992). 

Other attempts to define the site of absorption were other kinds of vehicles as well as catheters. Three main perfusion methods have been employed in the small intestine (i) a triple lumen tube including a mixing segment, (ii) a multilumen tube with a proximal occluding balloon, (iii) a multilumen tube with two balloons occluding a 10 cm long intestinal segment. 

Lennemas et al. have developed a method for measuring human effective permeability (H-Peff) using a regional intestinal perfusion technique. In this method, a perfusion apparatus consisting of a multichannel tube with two inflatable balloons (10 cm apart) is swallowed by the patient and eventually located in the proximal jejunum. Dilute solutions of the test drag are introduced at the inlet located at the center of the 10 cm section, and the loss of drag is determined from the concentration in the outlet intestinal perfusate. In such a fashion, the H-Peff for 22 carefully selected drug molecules has been determined and a theoretical model of H-Peff has been developed. " The small size of the published H-Peff database is most likely due to the expense of the human measurement. 

Systemic bioavailability is the product of fraction of dose absorbed (/a), fraction of dose escaping gut metabolism (/g), and fraction of dose escaping first-pass metabolism (F ). Permeability class is based upon /a, which may be estimated either in vivo or in vitro by direct measurement of mass transfer across human intestinal epithelium. In vivo methods include (i) mass balance studies using unlabeled, stable-isotope labeled, or a radiolabeled drug substance (ii) oral bioavailability using a reference intravenous dose or (iii) intestinal perfusion studies either in humans or an acceptable animal model. Suitable in vitro methods involve the use of either excised human/animal intestinal tissues or cultured epithelial monolayers. All of these methods are deemed appropriate for drugs whose absorption is controlled by passive mechanisms. 

Permeability can be assessed by pharmacokinetic studies (for example, mass balance studies), or intestinal permeability methods, e.g. intestinal perfusion in humans, animal models, Caco 2 cell lines or other suitable, validated cell lines. In vivo or in situ animal models or in vitro models (cell lines) are only considered appropriate by HHS-FDA for passively transported drugs. It should be noted that all of these measurements assess the fraction absorbed (as opposed to the bioavailability, which can be reduced substantially by first-pass metabolism). 

Experimental animals also efficiently absorb selenium compounds from the gut independent of the level of selenium exposure. Several studies have reported absorption of 80-100% in rats given dietary selenium administered as sodium selenite, sodium selenate, selenomethionine, or selenocystine (Furchner et al. 1975 Thomson and Stewart 1973). Other animal species also readily absorb orally administered selenium compounds. Furchner et al. (1975) estimated that over 90% of an oral dose of selenious acid was absorbed in mice and dogs, although monkeys absorbed less of the administered dose (amount unspecified). Using an in vivo perfusion method in which selenite was added directly to the duodenal end of the small intestine, the absorption of selenite was found to be linear (slope=0.0386) over the concentration range of 1-200 pM (Chen et al. 1993). 

Methods primarily used are in situ perfusions of the rat gut, regionally cannulated/fistulated rats and dogs, bioavailability models in different animals, intestinal perfusions in man (Loc-i-Gut , Lennernas et al. 1992) (Figure 4.15) and triple-lumen perfusions (Gramatte et al. 1994) and bioavailability studies in man. 

In the closed segment procedure, referred to above, the extent of absorption is calculated on the basis of disappearance of the test substance from both the lumen and the intestinal tissue. With the closed segment method, at the end of the absorption period, the entire segment, both intestinal wall and contents, is assayed quantitatively for the amount of the test substance remaining. A disadvantage to this technique when compared with perfusion methods is that sequential samples cannot be taken from a single animal. However, this method is readily used in small animals and can therefore be relatively economical. 

In addition to cell-based models, tissue-based models such as the Ussing chamber technique, the everted gut sac approach, and perfused isolated intestinal segments are also used, but only when it is important to understand the absorption processes in more detail. Unlike Caco-2, tissue-based models have the correct physiological levels of transporters and the presence of an apical mucus layer. Also, in situ and isolated organ perfusion methods exist for the gut, liver, lungs, kidneys, and brain and can provide data not directly obtainable in vitro. The isolated perfused liver is particularly useful since it allows an assessment of first-pass hepatic clearance, the quantitative distribution of metabolites in liver, blood, and bile, the effects of binding to plasma proteins and intracellular sites, and cellular uptake processes. 

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