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The purpose was to develop a general mathematical model for estimating passive permeability and efflux transport parameters from in vitro cell culture experiments. The procedure is applicable for linear and non-linear transport of drug with time, <10 or >10% of drug transport, negligible or relevant back flow, and would allow the adequate correction in the case of relevant mass balance problems. A compartmental kinetic approach was used and the transport barriers were described quantitatively in terms of apical and basolateral clearances. The method can be applied when sink conditions are not achieved and it allows the evaluation of the location of the transporter and its binding site. In this work it was possible to demonstrate, from a functional point of view, the higher efflux capacity of the TC7 clone and to identify the apical membrane as the main resistance for the xenobiotic transport. This methodology can be extremely useful as a complementary tool for molecular biology approaches in order to establish meaningful hypotheses about transport mechanisms.

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The relevant parameters for predicting rate and extent of access across the blood-brain barrier (BBB) are fu,plasma (unbound fraction in plasma), Vu,brain (distribution volume in brain) and Kp,uu,brain (ratio of free concentrations in plasma and brain). Their estimation still requires animal studies and in vitro low throughput experiments which make difficult the screening of new CNS candidates. The aim of the present work was to develop a new whole in vitro high throughput method to predict drug rate and extent of access across the BBB. The system permits estimation of fu,plasma, Vu,brain and Kp,uu,brain in a single experimental system, using in vitro cell monolayers in different conditions. From the ratios of the apparent permeability values (Papp) with the adequate mathematical analysis the relevant parameters can be estimated. Papp of ten model compounds has been obtained in MDCKII and MDCK-Mdr1cell monolayers in the absence and presence of albumin and brain homogenate. The ratio of Papp in the absence and presence of albumin allows estimation of in vitro fu,plasma. Papp in the presence of brain homogenate is used to estimate fu,brain and Vu,brain. Kp,uu,brain is estimated from the apical to basal versus basal to apical clearances. The BBB parameters obtained with the new method were predictive of the in vivo behavior of candidates. In vitro fu,plasma, Kp,uu,brain and Vu,brain (calculated with Papp from MDCKII cell line) presented a good correlation with in vivo fu,plasma, Kp,uu,CSF and Vu,brain published values (r=0.92; r=0.85; and r=0.99 respectively). Despite its simplicity the predictive performance is fairly good considering the reduced number of tested compounds with different physicochemical and transport properties. Further experimental modifications could be checked to optimize the method, but the present data support its feasibility. As other in vitro cell culture models, the system is suitable for miniaturization and robotization to allow high throughput screening of CNS candidates.

Absorption of cefadroxil in a selective intestinal absorption area (the proximal third of the small intestine) of the anaesthetized rat, at seven initial perfusion concentrations, ranging from 0.01 to 10.0 mg mL-1, is shown to be a non-linear transport mechanism. With the aid of computer-fitting procedures based on differential and integrated forms of Michaelis-Menten equation, Vm and Km values of 36.7-37.3 mg h-1 and 12.0-13.0 mg, respectively, were found. The statistical parameters were better than those obtained both for first-order and for combined Michaelis-Menten and first-order kinetics. There is no evidence for substantial passive diffusion processes. The results reported here, together with allometric considerations and literature data analysis, may help to explain some particular non-linear features of plasma level curves associated with the administration of fairly high oral doses of cefadroxil to humans.