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We describe a novel strategy to achieve high affinity recognition for the specific, cerebrovascular large neutral amino acid transporter (LAT1) isoform by covalent coupling of small molecules to the amino acid, L-cysteine (L-Cys). L-Cys (as the carrier) was covalently attached via a disulfide bond to either 6-mercaptopurine or 2-methyl-1-propanethiol (IBM) to form the brain-targeted drug delivery systems (BTDS). BTDS were designed for high affinity recognition by LAT1 at the cerebrovasculature. Using an in situ rat brain perfusion technique, competition between BTDS and the radiotracer [14C]L-Leu demonstrated significant inhibition of [14C]L-Leu brain uptake. BTDS possess affinity for cerebrovascular LAT1 in many distinct brain compartments, and the recognition of BTDS by LAT1 is influenced by hydrophobicity of the side-chain in BTDS. Thus, the BTDS strategy may be utilized for rapid shuttling of various neuropharmaceuticals into brain.

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By derivatization at the N-terminus of amino acid-based anticancer agents (e.g. melphalan and acivicin) to form a drug delivery system (TDDS), we demonstrate a change in the mechanism of brain uptake from the large neutral amino acid transporter (LAT) pathway to passive. An in situ rat brain perfusion technique was used to determine the brain capillary permeability±surface area (PA) product for [ 14 C]L-Leu as control (5.18^0.32 Â 10 22 mL/s/g), which was inhibited competitively (to 7±18% of control) by an excess concentration of the amino-acid-containing anticancer agents, acivicin and melphalan. However, TDDS did not compete for LATmediated brain uptake of the radiotracer [ 14 C]L-Leu. Brain uptake of TDDS was determined after in situ brain perfusion followed by RP-HPLC along with LC-MS/MS detection of the analytes in brain samples. The PA product for CH 3 -TDDS containing melphalan (5.09^2.0 Â 10 22 mL/s/g) shows that these agents rapidly cross the blood±brain barrier. Furthermore, competition studies of CH 3 -TDDS with [ 3 H]verapamil suggest that the TDDS interacts signi®cantly with the multidrug resistant ef¯ux system (P-glycoprotein) at the blood± brain barrier. Therefore, TDDS were shown to lack LATmediated brain uptake. The drug delivery systems, however, showed uptake predominantly via the passive route along with recognition by the multidrug resistant ef¯ux protein at the cerebrovasculature. Keywords: anticancer agents, blood±brain barrier, drug delivery systems, large neutral amino acid transporter, melphalan, P-glycoprotein.Brain drug delivery is of high clinical relevance for many CNS diseases and disorders (e.g. brain tumors, Alzheimer's disease). The blood±brain barrier, effectively separating brain parenchyma from blood, restricts brain penetration for many potential CNS active agents because of the presence of tight-junctions, high metabolic capacity, low pinocytic vesicular traf®c and ef®cient ef¯ux mechanisms (Brightman and Reese 1969;Minn et al. 1991;Jette et al. 1993). However, ef®cient endogenous carrier systems at the blood± brain barrier have been discovered, for example the large neutral amino acid transporter (Oldendorf 1971), now known as LAT (Christensen et al. 1994). Recent reports have identi®ed two isoforms of LAT (designated LAT1 and LAT2) which differ in substrate speci®city and af®nity (Kanai et al. 1998;Pineda et al. 1999;Segawa et al. 1999). There are con¯icting reports as to whether LAT1 (Boado et al. 1999) or LAT2 (Segawa et al. 1999) is responsible for LAT activity at the blood±brain barrier. Here the acronym LAT without isoform designation is used throughout. Cerebrovascular LAT shows af®nity for l-amino acids containing a relatively large and lipophilic R-group . Some endogenous amino acids which have been shown to be substrates for LAT include l-Phe and l-Leu .Simple molecular modi®cations on the N-terminus of amino acid-based anticancer agents have been shown to prevent the carrier-mediated blood±brain barrier transport of the parent, anticancer agents (Chikha...

The large neutral amino acid (LNAA) transporter at the blood-brain barrier (BBB) mediates brain uptake of amino acid-based anticancer agents (e.g., melphalan and acivicin). In this study, we blocked the amino acid terminus of the anticancer agents using a bioreductive drug delivery system (TDDS). This molecular modification of the anticancer agents is expected to prevent LNAA carrier-mediated transport across the BBB. In this study, we demonstrate that the parent amino acid containing anticancer agents are substrates for the LNAA transporter at the BBB, whereas the TDDS is not recognized by the LNAA transporter. An in situ rat brain perfusion technique was used to determine competition for LNAA carrier-mediated transport at the BBB using [14C]L-leucine. The BBB capillary permeability-surface area (PA) product for the radiotracer [14C]L-leucine (control) was determined to be 5.18 +/- 0.32 x 10(-2) ml/s/g (100%). The control PA value for [14C]L-leucine was competitively inhibited (down to 7-18% of control) by excess L-phenylalanine as well as by excess concentration of the anticancer amino acids, melphalan and acivicin, showing competition for the LNAA transporter at the BBB. In contrast, brain perfusion of [14C]L-leucine in presence of excess TDDS resulted in no competition for brain uptake of [14C]L-leucine via the LNAA transporter. Thus, bioreversible derivatization of the parent anticancer amino acids resulted in blocking the amino acid functional group, thereby leading to loss of recognition for the cerebrovascular LNAA transporter at the BBB.

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We report a new two-wire atherectomy technique for side branch protection. Newer, more resilient wire designs are capable of withstanding cutting forces of the Simpson atherocath device. This technique expands atherectomy application to lesions previously excluded from atherectomy as high risk lesions.

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