José A.G. Morais

Intestinal drug transporters: an overview.

The importance of drug transporters as one of the determinants of pharmacokinetics has become increasingly evident. While much research has been conducted focusing the role of drug transporters in the liver and kidney less is known about the importance of uptake and efflux transporters identified in the intestine. Over the past years the effects of intestinal transporters have been studied using in vivo models, in situ organ perfusions, in vitro tissue preparations and cell lines. This review aims to describe up to date findings regarding the importance of intestinal transporters on drug absorption and bioavailability, highlighting areas in need of further research. Wu and Benet proposed a Biopharmaceutics Drug Disposition Classification System (BDDCS) that allows the prediction of transporter effects on the drug disposition of orally administered drugs. This review also discusses BDDCS predictions with respect to the role of intestinal transporters and intestinal transporter-metabolizing enzyme interplay on oral drug pharmacokinetics.

Ecotoxicological assessment of leachates from MSWI bottom ashes.

In this paper, chemical and ecotoxicological data of leachates from bottom ashes collected in different Municipal Solid Waste Incinerators (MSWI) are shown. The bottom ashes were collected in Belgium (three incinerators--samples B1 to B3), France, Germany, Italy and United Kingdom (one incinerator in each country--samples F1, D1, I1 and UK1, respectively). Both chemical and ecotoxicological characterizations of leachates were done on the framework of the European Directive 91/689/EEC and the European Council Decision 94/904/EC. This work was carried out under the European project called Valomat, which was supported by the European Commission through Brite-Euram III program. Twenty-one inorganic parameters were analyzed. The ecotoxicological assays were done under standard laboratory conditions, using the bacterium Photobacterium phosphoreum, the freshwater alga Pseudokirchneriella subcapitata, the crustacean Daphnia magna and the vegetable Lactuca sativa. Chemical data varied from sample to sample. Similar results were obtained in biological assays. The samples can be classified as ecotoxic/hazardous according to the French proposal for a Criterion and Evaluation Methods of Waste Ecotoxicity (CEMWE) and the German regulation on Hazardous Waste Classification (HWC). However, samples B1, B2, B3 and D1 comply the maximum limits for direct valorization category defined in the French Classification of Bottom Ashes based on their Polluting Potential (CBAPP). Sample B1 presented the lowest level of ecotoxicity, being considered as the most interesting to be used in the development of new materials for civil engineering works.

Prediction of the human oral bioavailability by using in vitro and in silico drug related parameters in a physiologically based absorption model

Estimates of the human oral absolute bioavailability were made by using a physiological-based pharmacokinetic model of absorption and the drug solubility at the gastrointestinal pH range 1.5-7.5, the apparent permeability (P(app)) in Caco-2 cells and the intrinsic clearance (Cl(int)) in human hepatocytes suspensions as major drug related parameters. The predictive ability of this approach was tested in 164 drugs divided in four levels of input data: (i) in vitro data for both P(app) and Cl(int); (ii) in vitro data for Cl(int) only; (iii) in vitro data for P(app) only and (iv) in silico data for both P(app) and Cl(int). In all scenarios, solubility was estimated in silico. Excellent predictive abilities were observed when in vitro data for both P(app) and Cl(int) were used, with 84% of drugs with oral bioavailability predictions within a±20% interval of the correct value. This predictive ability is reduced with the introduction of the in silico estimated parameters, particularly when Cl(int) is used. Performance of the model using only in silico data provided 53% of drugs with bioavailability predictions within a±20% acceptance interval. However, 74% of drugs in the same scenario resulted in bioavailability predictions within a±35% interval, which indicates that a qualitative prediction of the absolute bioavailability is still possible. This approach is a valuable way to estimate a fundamental pharmacokinetic parameter, using data typically collected in the drug discovery and early development phases, providing also mechanistic information of the limiting bioavailability steps of the drug.

Developing and advancing dry powder inhalation towards enhanced therapeutics.

Enhanced therapeutics are drug products derived from existing generic drugs that provide additional benefits to the patients and the healthcare system. Enhanced therapeutics are considered to be an important and relatively low risk source of innovation. Pulmonary drug delivery is the major delivery route to treat chronic respiratory diseases and has been proven as a potential delivery route for complex drugs that cannot be delivered orally. Development of dry powder inhalation systems targets the delivery of fine drug particles to the deep lung surface by a combination of drug formulation, primary packaging and a device, whereby each contributes to the overall performance. Various methodologies for the non-clinical and clinical performance testing of orally inhaled products have been proposed and applied with variable success. Regulatory pathways have been developed and applied since. Considerable efforts have been made during the past decade to understand and optimize pulmonary drug delivery including their efficient commercial manufacturing. Pulmonary drug delivery remains an area of future innovation in the effective treatment of pulmonary diseases as well as the systemic delivery of systemically active complex drugs.

The New European Medicines Agency Guideline on the Investigation of Bioequivalence

In this MiniReview, the main modifications made during the revision of the current Note for Guidance on the Investigation of Bioavailability and Bioequivalence are reviewed and justified. Several new features have been added to this guideline, as well as changes aimed at improving the clarity of the guidance provided. The first issue to be addressed was to limit the scope of the guideline to bioequivalence studies for immediate release dosage forms with systemic action. Therefore, the guideline refers to bioequivalence alone. Moreover, the new definition of Generic Medicinal Product has been incorporated. Clearer guidance covering more specific cases is now given on sections such as: fed/fasting conditions, use of metabolite data, enantiomers and strength to be used in the bioequivalence study. Steady-state design is now restricted and other designs, such as parallel group design, replicate design and two-stage design, are now incorporated in a more explicit form. New practical guidance on Highly Variable Drug Products and Narrow Therapeutic Index Drugs has been incorporated. The possibility for a biowaiver based on the Biopharmaceutics Classification System is now more explicit for Class I drugs and can be extended to Class III drugs under restricted conditions. We are aware that the initial goal of providing a very specific and clear guidance on these issues has not been entirely achieved, mainly because it is almost impossible to cover all individual cases and predict every possible situation that may arise. Demonstration of bioequivalence will still require in many instances a case by case approach.

Prediction of the in vitro permeability determined in caco-2 cells by using artificial neural networks

Caco-2 cells are currently the most used in vitro tool for prediction of the potential oral absorption of new drugs. The existence of computational models based on this data may potentiate the early selection process of new drugs, but the current models are based on a limited number of cases or on a reduced molecular space. We present an artificial neural network based only on calculated molecular descriptors for modelling 296 in vitro Caco-2 apparent permeability (P(app)) drug values collected in the literature using also a pruning procedure for reducing the descriptors space. LogP(app) values were divided into a training group of 192 drugs for network optimization and a testing group of another 59 drugs for early stop and internal validation resulting in correlations of 0.843 and 0.702 and RMSE of 0.546 and 0.791 for the training and testing group, respectively. External validation was made with an additional group of 45 drugs with a correlation of 0.774 and RMSE of 0.601. The selected molecular descriptors encode information related to the lipophilicity, electronegativity, size, shape and flexibility characteristics of the molecules, which are related to drug absorption. This model may be a valuable tool for prediction and simulation in the drug development process, as it allows the in silico estimation of the in vitro Caco-2 apparent permeability.

Prediction of drug distribution within blood.

Drug distribution in blood, defined as drug blood-to-plasma concentration ratio (R(b)), is a fundamental pharmacokinetic parameter. It relates the plasma clearance to the blood clearance, enabling the physiological interpretation of this parameter. Although easily experimentally determined, R(b) values are lacking for the vast majority of drugs. We present a systematic approach using mechanistic, partial least squares (PLS) regression and artificial neural network (ANN) models to relate various in vitro and in silico molecular descriptors to a dataset of 93 drug R(b) values collected in the literature. The ANN model resulted in the best overall approach, with r(2)=0.927 and r(2)=0.871 for the train and the test sets, respectively. PLS regression presented r(2)=0.557 for the train and r(2)=0.656 for the test set. The mechanistic model provided the worst results, with r(2)=0.342 and, additionally, is limited to drugs with a basic ionised group with pKa<7. The ANN model for drug distribution in blood can be a valuable tool in clinical pharmacokinetics as well as in new drug design, providing predictions of R(b) with a percentage of correct values within a 1.25-fold error of 86%, 84% and 87% in the train, test and validation set of data.

Liposomal formulations of Cu,Zn-superoxide dismutase: physico-chemical characterization and activity assessment in an inflammation model

Different liposomal formulations of Cu,Zn-superoxide dismutase were studied. The effect of lipid composition, initial protein to lipid ratio and type of liposomes on the encapsulation parameters were studied. According to these parameters some liposomal formulations were selected for further tests: dehydration-rehydration vesicles (sDRV) and extruded vesicles (VET) made from PC:Chol:SA and PC:Chol:PI. After encapsulation in these vesicles, no significant loss of enzymatic activity of the enzyme was observed. Pharmacokinetic studies indicated that upon liposomal encapsulation of the enzyme, the terminal half-life of the enzyme after intravenous administration increased 5- to 10-fold. No acute toxicity was observed for the liposomal formulations. The therapeutic activity of the free and liposomal enzyme was assayed in Wistar rats with a chronic inflammation. All liposomal formulations tested showed a reduction of arthritis indexes: 25 to 50% of mean oedema regression was obtained with liposomal enzyme, while no regression with free enzyme and an increase of 50% of the oedema for the control animals were observed.

Liposomal palmitoyl-L-asparaginase: characterization and biological activity.

A new derivative ofL-asparaginase, palmitoyl-L-asparaginase (palmitoyl-L-ASNase), has been incorporated in liposomes. For this purpose we modified the dehydration-rehydration method and optimized the liposomal composition. The pharmacokinetics, toxicity, and in vivo antitumor activity against P1534 lymphoma of different liposomal palmitoyl-L-ASNase formulations were studied. Liposomal encapsulation of palmitoyl-L-ASNase as compared with free palmitoyl-L-ASNase resulted in a prolongation of the blood half-life (from 2.88 h to longer than 23.7 h), abrogation of acute toxicity, and preservation of in vivo antitumor activity.

An isocratic LC method for the simultaneous determination of vitamins A, C, E and β-carotene

An isocratic liquid chromatographic method for the separation and simultaneous determination of retinyl acetate, propionate or palmitate (esters of vitamin A), beta-carotene (pro-vitamin A), ascorbic acid (vitamin C) and DL-alpha-tocopherol (vitamin E) is described. Samples are analysed by means of a reversed-phase column (LiChrospher 100 RP-18), using methanol as mobile phase. The UV Vis detector used was set at a wavelength of 300 nm and switched to 450 nm at 17 min, allowing the determination of beta-carotene. These vitamins were separated within 25 min and the detection limits ranged from 7 (beta-carotene) to 65 ng ml(-1) (ascorbic acid).