Perfusion Methods

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]. 

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. 

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... 

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. 

S. Fulton and D. Vanderburgh, The Busy Researcher s Guide to Biomolecule Chromatography (1996), PerSeptive Biosystems (Framingham, MA). Practical guide to chromatography of biomolecules with emphasis on perfusion methods. 

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. 

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. 

Capone, D. G., and Carpenter, E. J. (1982b). A perfusion method for assaying microbial activities in estuarine sediments. Applicability to studies of N2 (C2H2) reduction. Appl. Environ. Microbiol. 43,... 

The isolation and subsequent study of hepatocytes in in vitro conditions was first transformed by Berry and Friend (1), who developed a collagenase liver perfusion method, allowing the isolation of large numbers of cells with high viability. 

When submicrometer seed crystal is added to the CET solution, the CET solution must be saturated or supersaturated and the resulting suspension must maintain the metastable state. In order to prepare the supersaturated solution, special equipment has been invented. This includes a high-performance solvating machine used as a heat exchange perfusion method, in which CET is dissolved by... 

Adhesion of horse platelets to subendothelial collagen has been investigated using the Baumgartner perfusion method (Weiss et al. 1990). Bodi the total surfiice area of subendolhelium covered by platelets and the surfece area covered by platelet thrombi were... 

TAFq = tissue attenuation factor using perfusion method, cpm/pCi... 

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,... 

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