Evaluation of the aptitude to cross the BBB of a new dopamine aminoacidic prodrug

One of the most important factors limiting the development of new drugs for the CNS is the ability to cross the BBB which is a barrier that controls the entrance and exit of both endogenous and exogenous compounds. BBB expresses several transport systems that carry actively into the brain important nutrients (e.g. glucose and amino acids) and are able to import or export various xenobiotics including drugs and their metabolites. The content of active in the brain depends on the overall difference between the drug uptake and drug efflux processes [1]. Dopamine (DA) is a crucial neurotransmitter; its striatal depletion is responsible of clinical signs of Parkinson’s disease (PD). Owing to the high hydrophilicity, polarity and the absence of a specific transport system within the membrane of endothelial cells, DA is not able to cross the BBB, thus precluding its use in treatment of PD [2]. The prodrug approach could be considered as an alternative, non invasive way to enhance brain bioavailability of DA; it consists in covalent linking of actives with specific molecules able to modify the physico-chemical properties of drugs and/or interact with the BBB endogenous transporters, so promoting the CNS uptake. Previously we have described the synthesis of the potential prodrug L-phenylalanine-β-(3,4-dihydroxyphenyl) etilamide (DA-Phe) by covalent linking DA with the essential amino acid phenylalanine (L-Phe), seeing that L-Phe readily crosses the BBB with the so called large neutral amino acid (LNAA) carrier system [3]. The distribution coefficient, expressed as Log Papp, was determined as 0.76 thus suggesting that DA-Phe possesses adequate characteristics to permeate through biological membranes. Stability studies using rat brain homogenate have shown that DA-Phe behaves as a prodrug, undergoes cleavage by cerebral enzymes, and slowly and constantly releases DA [3]. In early discovery of new compounds, different models are used to evaluate the ability to cross the BBB. Over the past decade, the Parallel Artificial Membrane Permeability Assay (PAMPA) have been considered for prediction of passive transcellular drug transport through non-biological membranes. By modification of the lipid composition of artificial membranes, the system could be adapted to predict, with reasonable accuracy, the BBB permeability (PAMPA-BBB). By this system, compounds could be classified as permeable (CNS+) or non-permeable (CNS-) [4]. We experienced that the effective DA-Phe permeability (Peff), using the PAMPA-BBB, is not adequate to obtain sufficient drug amounts in the CNS. Since PAMPA-BBB model is useful in predicting only passive transport, the role of endothelial cell metabolism or active transport in the overall BBB permeability of a drug is not accounted. The active drug transport could be assessed by a bidirectional Caco-2 assay. This system is able to recognize not only the carrier-mediated pathways that facilitate drug entry into the brain, but also the major drug efflux mechanisms (e.g. the P-gp transporter, whose specificity is not well defined). In addition, a cell-based model offers the potential to verify transcellular and paracellular diffusional pathways. The apparent permeability value (Papp) using Caco-2 was determined. By combination of the permeability values calculated with the two models it was observed that the transport of DA-Phe through the cell line is 100 fold greater than that obtained using the PAMPA-BBB model. These results suggest that DA-Phe crosses the BBB by a combination of two different mechanisms: passive diffusion and carrier-mediated transport. Furthermore, the efflux ratio value of DA-Phe was determined as 0.13 and, as reported in the literature, being < 2 it indicates low affinity for the P-gp efflux protein [4]. [1] Pardridge W.M., Drug Disc Today, 12 (2007) 54-62 [2] Antolin I., et al., Brain Res, 943 (2002) 163-173 [3] Giannola L.I., et al., Pharmazie, 63 (2008) 704-710 [4] Mensch J., et al, Int J Pharm, 395 (2010) 182-197