In vivo intestinal perfusion

Methodological Aspects on in vivo Intestinal Perfusion Techniques... 

Methodological Aspects on In-vivo Intestinal Perfusion Techniques j 163... 

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... 

Absolute bioavailability studies In vivo intestinal perfusion... 

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. 

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... 

Figure 2. Outline of in vitro digestibility and in vitro (everted sac) or in vivo (intestinal perfusion) uptake experiments. Amino acid analysis of everted sac contents or of original and final intestinal perfusate allows uptake determination.

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... 

Tannergren C, Knutson T, Knutson L and Lennernas H (2003) The Effect of Ketoconazole on the In Vivo Intestinal Permeability of Fexofenadine Using a Regional Perfusion Technique. BrJ Clin Pharmacol 55 pp 182-190. 

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%... 

Tannergreen, C., Knutson. T. Knutson, L., and Lennernas, H. (2003) The effect of ketoconazole on the in vivo intestinal permeability of fexofenadine using a regional perfusion technique. British Journal of Clinical Pharmacology, 55, 182-190. 

These automated assays can be used for high-throughput ADME screening in early drug discovery. The double-sink PAMPA permeability assay mimics in vivo conditions by the use of a chemical sink in the acceptor wells and pH gradient in the donor wells. The use of the pION gut-box integrated on the deck has shortened the PAMPA assay incubation time to 30 minutes. The permeability coefficient and rank order correlate well with data obtained using the in vitro Caco-2 assay and in vivo permeability properties measured in rat intestinal perfusions. 

In practice, estimation of Laq requires information on the rate of solute removal at the membrane since aqueous resistance is calculated from experimental data defining the solute concentration profile across this barrier [7], Mean /.aq values calculated from the product of aqueous diffusivity (at body temperature) and aqueous resistance obtained from human and animal intestinal perfusion experiments in situ are in the range of 100-900 pm, compared to lumenal radii of 0.2 cm (rat) and 1 cm (human). These estimates will necessarily be a function of perfusion flow rate and choice of solute. The lower Laq estimated in vivo is rationalized by better mixing within the lumen in the vicinity of the mucosal membrane [6],... 

The CAT model considers passive absorption, saturable absorption, degradation, and transit in the human small intestine. However, the absorption and degradation kinetics are the only model parameters that need to be determined to estimate the fraction of dose absorbed and to simulate intestinal absorption kinetics. Degradation kinetics may be determined in vitro and absorption parameters can also be determined using human intestinal perfusion techniques [85] therefore, it may be feasible to predict intestinal absorption kinetics based on in vitro degradation and in vivo perfusion data. Nevertheless, considering the complexity of oral drug absorption, such a prediction is only an approximation. 

A direct in vivo assessment of the quantitative importance of gut wall metabolism and transport of drugs and metabolites in humans is difficult and consequently has been attempted only rarely [3, 6, 11, 12, 15, 16, 23, 25-32, 34, 35, 81]. The most direct in vivo approach to investigating these processes in drugs with variable and incomplete bioavailability was intestinal perfusion by single-pass per-... 

A direct in vivo assessment was carried out with the single-pass perfusion approach in the human jejunum by using the Loc-I-Gut technique with R/S-verapamil (log D6 5 2.7, octanol/water pH 7.4 MW 455 Da) as the model compound for CYP 3A4 and P-gp-mediated local intestinal kinetics [2, 34, 35, 122] (see Figs. 7.7 and 7.9). The Peff for both enantiomers at each of the concentrations (4.0, 40, 120, and 400 mg L-1) was 2.5 x 10 4, 4.7 x 105.5 x 104 and 6.7 x 104 cm s-1, respectively (Fig. 7.15) [34, 35], A luminal concentration of 400 mg L 1 is expected to be achieved in the upper part of the small intestine after oral administration of a 100-mg dose of verapamil in an immediate-release dosage form [1, 34, 35], The three other perfusate concentrations represent fractions of the dose when 30%, 10%, and 1%, respectively are left to be absorbed [34, 35], The increased in vivo jejunal Peff of R/S-vcrapamil, along with its increased luminal perfusate concentration, is in accordance with a saturable efflux mechanism mediated by... 

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