Intestinal perfusion studies, drug

The permeability of the drug substance can be determined by different approaches such as pharmacokinetic studies in humans (fraction absorbed or mass balance studies) or intestinal permeability studies (in vivo intestinal perfusion studies in humans or suitable animal models or in vitro permeation studies using excised intestinal tissue or epithelial cell culture monolayers like CaCo-2 cell line). In order to avoid misclassification of a drug subject to efflux transporters such as P-glycoprotein, functional expression of such proteins should be investigated. Low- and high-permeability model... 

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. 

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. 

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. 

Single-pass intestinal perfusion studies For evaluatton of intestinal prermeability and lymphatic uptake Predict the exact mechanism of absorption, that is, passive absorption, carrier mediated absorption or active transport Evaluate the P-gp efflux and role of transporters (MRP, BCRP2) in reducing the oral bioavailability of drugs... 

In situ perfusion studies assess absorption as lumenal clearance or membrane permeability and provide for isolation of solute transport at the level of the intestinal tissue. Controlled input of drug concentration, perfusion pH, osmolality, composition, and flow rate combined with intestinal region selection allow for separation of aqueous resistance and water transport effects on solute tissue permeation. This system provides for solute sampling from GI lumenal and plasma (mesenteric and systemic) compartments. A sensitive assay can separate metabolic from transport contributions. 

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]. 

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. 

Lennernas s group at Uppsala has performed extensive studies to confirm the validity of this in vivo experimental set-up at assessing the rate and the extent of drug absorption. Recovery of PEG 4000 (a non-absorbable marker) is more than 95%, which indicates that the absorption barrier is intact. In addition, maintenance of functional viability of the mucosa during perfusion has been demonstrated by the rapid transmucosal transport of D-glucose and L-leucine. Estimation of absorption half-lives from the measured Pefr agree well with half-lives derived from oral dose studies in humans (i.e. physiologically realistic half-lives). Human Peff estimates are well correlated with the fraction absorbed in humans, and served as the basis for BCS development, and hence the technique is ultimately the benchmark by which other in situ intestinal perfusion techniques are compared. The model has been extensively used 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... 

Clinical studies of Peff, secretion, and metabolism of various compounds such as drugs, environmental pollutants, and nutrients are rarely performed in vivo in humans even if experimental techniques are available (Figures 9.2-9.4) [3, 11, 13, 16, 17, 24—31]. Direct measurements of compound transport and metabolism in mesenteric and portal veins in humans are not possible for obvious reasons. Perfusion techniques, however, present great possibilities to measure intestinal processes. Over the past 70 years, different in vivo intestinal perfusion techniques have been developed and the importance of this work has been clearly demonstrated [3, 5, 6,11,13-16, 25-31]. The fundamental principle of an in vivo intestinal perfusion experiment is that Peff is calculated from the rate at which the compound disappears... 

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). 

Figure 4.14 Schematic drawing of the intestinal perfusion technique. The intestine of the animal is catheterised in both ends and a flow of buffer solution of 37°C is perfused using a pump. For in vitro studies in which only the absorption over the intestine is to be measured, the vascular support can be cannulated, and a separate buffer solution can be perfused through the intestine. If the influence of the liver on drug absorption is to be studied, an in situ system with an intact anaesthetised animal can be used by only perfusion of the intestine, keeping the blood flow of the vascular support intact.

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