Absorption perfusion methods

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. 

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. 

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). 

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. 

Both perfusion methods can use different evaluation systems for testing drug absorption, using the difference between in and out concentrations in the perfusion solutions, and/or disappearance and appearance on both sides of the membrane, and also by analysing the drug concentration on the blood side. The permeability, usually called the P is calculated from the following equation,... 

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. 

According to investigations carried out by Krebs et al. (5) who adopted our experimental perfusion methods and extended them, lactate is among the most adaptable substrates for gluconeogenesis. So it is noteworthy that with glucagon absorption of L-Lactate from the medium is tripled. 

L. Celesti, C. Murratzu, M. Valoti, G. Sgaragli, and P. Corti. The single-pass perfused rabbit ear as a model for studying percutaneous absorption of clonazepam. I. General characteristics. Methods Find. Exp. Clin. Pharmacol. 14 701-709 (1992). 

An additional consideration when using in situ techniques is the volume of the luminal drug solution as water absorption and secretion during the perfusion may introduce errors in the lumenal concentrations and therefore in the calculated absorption. Various water flux correction methods have been published [42] including the co-perfusion of a non-absorbed marker such... 

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. 

Although this section deals mainly with the advantages of excised tissues with respect to nasal drug delivery studies, it is important to highlight some important attributes of nasal in situ perfusion model. Although this method does not provide data on systemic absorption, it enables study of the interactions of nasal mucosal enzymes, peptide substrates, and metabolic inhibitors and their implications for nasal drug absorption [13], It also enables the rate of nasal drug absorption to be determined. 

French MC, Wishart GN (1985) Isolated perfused rabbit lung as a model to study the absorption of organic aerosols. J Pharmacol Methods 13 241-248. 

An improved gas-uptake model should incorporate the features of the DuBois and Rogers model and the McJilton et al. model. As shown in Figure 7-2 the model for gas uptake in the airways should include separate layers for mucous-serous fluid epithelial tissue and blood. Development of such a model awaits reliable data and methods for predicting the coefficient of diffusion of pollutant gases in tissue and information on the rates of local perfusion of blood and lymph in the bronchial epithelium. Experimental data from humans and animals on the rate of sulfur dioxide absorption in blood could be used to make improved estimates of the tissue-diffusion coefficients in vivo. 

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