MCT1 Transporters Expressed in Blood Brain Barrier Cells

Abstract

MCT1 is consistently expressed at high levels in brain microvessel endothelial cells. Disclosed herein are assays for determining whether a test material/molecule is a substrate for, and/or is actively transported by, the MCT1 transporter, and therefore a candidate substrate for crossing the blood brain barrier. The assays are useful in screening for therapeutic, cytotoxic or imaging compounds used in the treatment or diagnosis of neurological diseases.

1 - 32 . (canceled) 
     
     
         33 . A method of delivering a conjugate to the brain of a patient, comprising administering to a patient a pharmaceutical composition comprising a neuropharmaceutical agent linked to a conjugate moiety to form the conjugate, wherein the conjugate has a higher V max  for the MCT1 transporter than the neuropharmaceutical agent alone, wherein the MCT1 transporter is the protein encoded by SEQ ID NO:1 or has at least 90% sequence identity to the protein encoded by SEQ ID NO:1, and the MCT1 transporter can transport lactic acid; and
 provided the neuropharmaceutical agent is not a cytotoxic agent.   
     
     
         34 . The method of  claim 33 , wherein the V max  for the MCT1 transporter of the conjugate is at least two-fold higher than that of the neuropharmaceutical agent alone. 
     
     
         35 - 38 . (canceled) 
     
     
         39 . The method of  claim 33 , wherein the V max  of the neuropharmaceutical agent or conjugate is determined by steps comprising:
 (a) providing a cell expressing the MCT1 transporter, wherein a nucleic acid molecule encoding the MCT1 transporter has been transfected or injected into the cell, the MCT1 transporter being situated in the plasma membrane of the cell;   (b) contacting the cell with the neuropharmaceutical agent or conjugate in vitro;   (c) determining the V max  of the neuropharmaceutical agent or conjugate; and   (d) determining whether the conjugate has a higher V max  for the MCT1 transporter than the neuropharmaceutical agent alone.   
     
     
         40 . The method of  claim 39 , wherein the cell is an oocyte. 
     
     
         41 . The method of  claim 39 , wherein the cell is a human embryonic kidney HEK-PEAK cell. 
     
     
         42 . The method of  claim 39 , wherein the determining the V max  is performed by a competition assay. 
     
     
         43 . The method of  claim 39 , wherein the determining the V max  is performed by a direct uptake assay. 
     
     
         44 . The method of  claim 39 , wherein the determining the V max  is performed by a pH assay. 
     
     
         45 . The method of  claim 39 , wherein the cell is transformed with an SV40 large T antigen that can be expressed in a temperature sensitive fashion. 
     
     
         46 . The method of  claim 39 , wherein the neuropharmaceutical agent or conjugate comprises a monocarboxylic acid. 
     
     
         47 . The method of  claim 46 , wherein the monocarboxylic acid is selected from the group consisting of lactic acid, pyruvic acid, nicotinic acid, 2-thiophene glyoxylate, pravastatin, butyric acid, salicylic acid, and carindicillin. 
     
     
         48 . The method of  claim 39 , wherein the ratio of the V max  for the MCT1 transporter to the V max  for at least one efflux transporter of the conjugate is greater than the ratio of the V max  for the MCT1 transporter to the V max  for the at least one efflux transporter of the neuropharmaceutical agent; wherein the at least one efflux transporter is selected from the group consisting of the P-glycoprotein transporter, the multidrug resistance protein transporter, and the breast cancer resistance protein transporter. 
     
     
         49 . The method of  claim 48 , wherein the V max  for the at least one efflux transporter is determined by steps comprising:
 (a) providing a cell expressing the at least one efflux transporter;   (b) contacting the cell with the neuropharmaceutical agent or conjugate in vitro;   (d) determining that the agent or conjugate is transported by the at least one efflux transporter;   (d) determining the V max  for the efflux transporter of the neuropharmaceutical agent or conjugate; and   (e) determining the ratio of the V max  for the MCT1 transporter to the V max  for the efflux transporter of the neuropharmaceutical agent and of the conjugate; and   (f) determining whether the ratio of the V max  for the MCT1 transporter to the V max  for the efflux transporter of the conjugate is greater than the ratio of the V max  for the MCT1 transporter to the V max  for the efflux transporter of the neuropharmaceutical agent.   
     
     
         50 . The method of  claim 49 , wherein the V max  for the efflux transporter is determined by conducting an assay selected from the group consisting of:
 (a) an efflux transporter ATPase activity assay;   (b) an efflux transporter competition assay; and   (c) a direct efflux transport assay across a polarized monolayer of cells.