V.G. Casabó

A modelistic approach showing the importance of the stagnant aqueous layers in in vitro diffusion studies, and in vitro-in vivo correlations

Abstract The present study deals with the role of the aqueous diffusion layers on the in vitro penetration of xenobiotics across artificial lipoidal membranes, and their ability to reproduce biophysical absorption models when in vivo results are to be simulated from the in vitro tests. The aqueous boundary layers which are invariably formed on artificial lipoidal membranes can be optionally preserved or disrupted, according to the type of absorption site which should be simulated, a condition which could reasonably lead to a better correspondence between in vitro and in vivo results; in practice, disruption of water layers can be easily achieved by a synthetic surfactant solution at its critical micelle concentration, in contact with the desired membrane side. This approach is illustrated by using a dimethylpolysiloxane artificial membrane, 4-alkylanilines as permeant test compounds, and the nonionic polysorbate 80 as model surfactant, in a series of experiments developed with a Franz-type diffusion cell apparatus. Experimental designs simulating different types of biological absorbing membranes, merely by changing the number of aqueous boundary layers, are described and analyzed. Apart from the effects of the water layers, it is shown that perfect sink conditions at the receptor side must be strictly maintained in order to obtain reliable results.

Non‐linear Intestinal Absorption Kinetics of Cefadroxil in the Rat

Abstract— 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.

Kinetic modelling of passive transport and active efflux of a fluoroquinolone across Caco-2 cells using a compartmental approach in NONMEM

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.

Intestinal transport of cefuroxime axetil in rats: absorption and hydrolysis processes.

Studies were performed using three cefuroxime axetil solutions (11.8, 118 and 200 microM) in three selected intestinal segments and one cefuroxime axetil solution (118 microM) in colon of anaesthetized rats. First-order absorption rate pseudoconstants, k(ap) and effective permeability coefficients, P(eff), were calculated in each set. Absorption of cefuroxime axetil can apparently be described as a carrier-mediated transport, which obeys Michaelis-Menten and first order kinetics in the proximal segment of the small intestine and a passive diffusion mechanism in the mean and distal segments. The absorption kinetic parameters for cefuroxime axetil were obtained: Vm=0.613 (0.440) microM min-1; Km=31.49(28.31) microM and ka=0.011(0.003) min-1. Parameters characterizing degradation of the prodrug were obtained in each intestinal segment: proximal segment k(dp)=0.0049(0.0003) min-1, mean segment, k(dm)=0.0131(0.0007) min-1 and distal segment k(dd)=0.019(0.0009) min-1. Therefore, in situ intestinal absorption of cefuroxime axetil in the proximal segment of the rat in the presence of variable concentrations of cefadroxil has been investigated in order to examine the inhibitory effect of cefadroxil on cefuroxime axetil transport. The data suggest that cefadroxil and cefuroxime axetil share the same intestinal carrier.

Influence of polyunsaturated fatty acids on Cortisol transport through MDCK and MDCK-MDR1 cells as blood-brain barrier in vitro model.

Transport across the blood-brain barrier is a relevant factor in the pharmacological action of many drugs and endogenous substances whose action site is located in brain. An overactive P-gp has been suggested to be of relevance for the resistance of the HPA system to be suppressed by glucocorticoids, which is one of the best described biological abnormalities in certain types of depression. PUFA acids have shown clinical efficacy in depressed patients and the hypothesis is that these compounds are able to reduce HPA axis activity as this effect has been shown in animal models of depression. The objective of the present work was (1) to characterize Cortisol transport through MDCK and MDCK-MDR1 cell lines (as in vitro models of the BBB) to confirm its transport mechanism as substrate of P-gp and (2) to evaluate the effect of PUFA acids as enhancers of Cortisol transport in the BBB model and explore the enhancement mechanism. Transport studies of Cortisol were performed in both directions, from apical-to-basolateral and from basolateral-to-apical sides. The in vitro experiments showed that Cortisol transport is concentration dependent and it is affected by several transporters (absorption and secretion processes). The results indicate that PUFA acids increase Cortisol transport in the BBB models but not through the inhibition of P-gp efflux but thanks to membrane fluidification and some effect on tight junction integrity.

Kinetic modelling of the intestinal transport of sarafloxacin. Studies in situ in rat and in vitro in Caco-2 cells

The absorption kinetics of sarafloxacin, as a model of fluoroquinolone structure, were studied in the rat small intestine and in Caco-2 cells. The objective of the study was to investigate the mechanistic basis of the drugs intestinal transport in comparison with other members of the fluoroquinolone family and to apply a mathematical modelling approach to the transport process. In the rat small intestine, sarafloxacin showed dual mechanisms of intestinal absorption with a passive diffusional component and an absorptive carrier-mediated component. The characteristics of the animal study design made it suitable for population analysis, thus allowing the accurate estimation of transport parameters and their inter and intra-individual variances. The transport system in the rat model was ATP-dependent, as sodium azide was able to decrease the absorption rate constant in a concentration-dependent fashion. The inhibition mechanism of sodium azide was modelled based on its ATP depletion capacity. The rationale of this approach was to consider the inhibitor-carrier interaction as a concentration- dependent response. This interaction was accurately described by a non-competitive mechanism. In Caco-2 cells, sarafloxacin showed a concentration dependent permeability in both directions apical to basal, and basal to apical. The permeability values and ratios of permeability values at different concentrations suggested the presence of two carriers (absorption and efflux carriers). The passive diffusion component in both systems was compared to that predicted by the absorption–partition correlation, previously established for two series of fluoroquinolones. The discrepancy between the experimental and predicted value suggested the presence of an efflux mechanism similar to that already described for other fluoroquinolones. The differences and similarities of the in situ and the in vitro results are discussed as well as the usefulness of the modelling approach.

Pharmacokinetics and absolute bioavailability of oral cefuroxime axetil in the rat.

The objectives of this study were to determine the oral bioavailability of cefuroxime (C) and to evaluate the pharmacokinetic model that best describes the plasma concentration behaviour following single intravenous (IV), intraperitoneal (IP) and oral single doses. The same dose of C was administered by IV, IP and oral routes to three separate groups of rats (2.02 mg of cefuroxime axetil (CA) by the oral route or 1.78 mg of cefuroxime sodium (CNa) by IV and IP route). A two-compartment open model without lag time can predict the C disposition kinetics. The influence of the administration route on the pharmacokinetic parameters and AUC values was investigated by means of a one-way analysis of variance test. The results indicated that the first-pass effect in the intestine and liver reduce oral bioavailability when the drug is administered orally. Cefuroxime bioavailability after oral and IP administration estimated from the plasma levels was nearly 24 and 75%, respectively.

Intestinal absorption kinetics of amiodarone in rat small intestine

Amiodarone is a widely used antiarrhythmic agent with highly variable therapeutic effects. These seem to be related, at least in part, to the pharmacokinetics of the drug and particularly to some features of its gastrointestinal absorption process. The drug exhibits physico‐chemical properties highly suitable for diffusion across lipophilic absorbing membranes, but its low aqueous solubility can act as the rate limiting step for absorption, making the process erratic and variable. In order to gain an insight into the intestinal absorption mechanism of the drug and detect possible non‐linearities, a series of experiments using a classical rat gut in situ preparation were carried out with three amiodarone hydrochloride solutions (10, 75, and 200 μg mL−1). A synthetic non‐ionic surfactant, polysorbate 80, at supramicellar concentration (2 mM) was used as the drug solubilizer. Amiodarone was assayed in biological samples by HPLC using a rapid, sensitive technique that was validated. The amiodarone first‐order absorption rate constants obtained in these conditions were similar. No significant differences between ka values were found. Amiodarone absorption was clearly identified as a passive diffusion process.

Variability of permeability estimation from different protocols of subculture and transport experiments in cell monolayers

INTRODUCTION In vitro models with high predictive ability have been revealed as strong tools for pharmaceutical industry. However, the variability in permeability estimations complicates the comparison and combination of data from different laboratories and it makes necessary the careful validation of the model and the continuous suitability demonstration. The adequate standardization of pre-experimental, experimental and post-experimental factors might help to reduce the inter- and intra-laboratory variability in permeability values. METHODS The objective of this paper is the evaluation of the effect of passage number, experimental protocol, time after seeding and calculation method on the permeability values and their variability in transport experiments in Caco-2, MDCK and MDCK-MDR1 cells. Metoprolol, Lucifer yellow and Rhodamine-123 were used to check the performance of the cell lines. Protocols used differ mainly in the differentiation time and the filter support coating with collagen. Data was analyzed with sink and non-sink approaches. The final purpose was to explore pre-experimental, experimental and post-experimental conditions in order to select the best experimental scenarios for permeability assays. RESULTS Results indicated that for passive diffusion studies, coating helps cell differentiation in a more stable manner in all cell lines compared to protocol without coating which showed permeability changes with passages and more variable values. In both protocols the paracellular route became more restricted with higher passage numbers. Functionality of P-gp assessed with Rhodamine permeability did not change with passage number in Caco-2 cells with any of the protocols but increased in both protocols in MDCK and MDCK-MDR1 cells. Protocol without coating showed the less variable results in these cell lines. Rhodamine permeabilities increased with higher maturation times due to a higher expression of the transporter. Nevertheless for compounds absorbed by passive diffusion there was not a clear trend neither in permeability values nor in variability. DISCUSSION As a conclusion, we have confirmed the influence of maturation conditions and passage number in permeability values and in their variability. Based on our results protocol with coating would be more adequate for studies of compounds absorbed by passive diffusion but the protocol without coating gave us better results for studies about P-gp interactions.

Effects of polysorbate 80 on amiodarone intestinal absorption in the rat

Abstract Amiodarone is a widely used anti-arrhythmic agent which shows physico-chemical properties that are highly suitable for diffusion across lipophilic absorbing membranes, however,.its low aqueous solubility could represent the rate-limiting step for absorption, making it erratic and variable. In a previous paper, the influence of an anionic surfactant (sodium lauryl sulphate) at variable supramicellar concentrations was studied. The absorption rate constants of amiodarone decreased as the surfactant concentration increased, and absorption was unusually fast at lower surfactant concentrations. The previously proposed equations for interpreting the relationships between the amiodarone absorption rate constant and sodium lauryl sulphate concentration in the intestinal lumen are extended here to the absorption of the drug in the presence of a non-ionic surfactant (polysorbate 80) at different supramicellar concentrations (from 0.4 to 80 mM). In the same way, a modification of the previously reported equations for quantifying the absorption of micellized drug through the intestinal membrane is described. This modification has been applied to the results obtained for both surfactants, but does not improve the previously postulated biophysical model.