Microsomal triglyceride transfer protein

Abstract

Nucleic acid sequences, particularly DNA sequences, coding for all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, expression vectors containing the DNA sequences, host cells containing the expression vectors, and methods utilizing these materials. The invention also concerns polypeptide molecules comprising all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, and methods for producing these polypeptide molecules. The invention additionally concerns novel methods for preventing, stabilizing or causing regression of atherosclerosis and therapeutic agents having such activity. The invention concerns further novel methods for lowering serum liquid levels and therapeutic agents having such activity.

What is claimed is:  
     
         1 . An isolated nucleic acid molecule comprising a nucleic acid sequence coding for all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, wherein the nucleic acid sequence coding for part of the high molecular weight subunit of microsomal triglyceride transfer protein is at least about 15 sequential nucleotides in length.  
     
     
         2 . The nucleic acid molecule according to  claim 1  which is a DNA molecule and wherein the nucleic acid sequence is a DNA sequence.  
     
     
         3 . The DNA molecule according to  claim 2  wherein the DNA sequence has the nucleotide sequence shown in SEQ. ID. NOS. 1, 2, 5, 7, 8, 1 together with 5, 2 together with 7, the first 108 bases of 2 together with 8, the first 108 bases of 2 together with 7 and 8, or 8 together with 31 and 32.  
     
     
         4 . The DNA molecule according to  claim 2  wherein the DNA sequence has part of the nucleotide sequence shown in SEQ. ID. NOS. 1, 2, 5, 7, 8, 1 together with 5, 2 together with 7, the first 108 bases of 2 together with 8, or the first 108 bases of 2 together with 7 and 8, or 8 together with 31 and 32, wherein the DNA sequence coding for part of the high molecular weight subunit of microsomal triglyceride transfer protein is at least about 15 sequential nucleotides in length.  
     
     
         5 . A DNA molecule having a DNA sequence which is complementary to the DNA sequence according to  claim 2 .  
     
     
         6 . A DNA molecule having a DNA sequence which is complementary to the DNA sequence according to  claim 3 .    
     
     
         13 . A prokaryotic or eukaryotic host cell comprising the expression vector according to claim  10 .  
     
     
         14 . A method for producing a polypeptide molecule having all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, which comprises culturing a host cell according to claim  11  under conditions permitting expression of the polypeptide.  
     
     
         15 . A method for producing a polypeptide molecule having all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, which comprises culturing a host cell according to claim  12  under conditions permitting expression of the polypeptide.  
     
     
         16 . A method for producing a polypeptide molecule having all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, which comprises culturing a host cell according to  claim 13  under conditions permitting expression of the polypeptide.  
     
     
         17 . A method for detecting a nucleic acid sequence coding for all or part of the high molecular weight subunit of microsomal triglyceride transfer protein or a related nucleic acid sequence, which comprises: 
 (a) contacting the nucleic acid sequence with a detectable marker which binds specifically to at least part of the nucleic acid sequence, and    (b) detecting the marker so bound; wherein the presence of bound marker indicates the presence of the nucleic acid sequence.    
     
     
         18 . The method according to  claim 17  wherein the DNA sequence has the nucleotide sequence as shown in SEQ. ID. NOS. 1, 2, 5, 7, 8, 1 together with 5, 2 together with 7, the first 108 bases of 2 together with 8, the first 108 bases of 2 together with 7 and 8, or 8 together with 31 and 32, or a part of any thereof of at least 15 sequential nucleotides in length.  
     
     
         19 . The method according to  claim 17  wherein the detectable marker is a nucleotide sequence of at least about 15 nucleotides in length complementary to at least a portion of the nucleic acid sequence coding for the high molecular weight subunit of microsomal triglyceride transfer protein.  
     
     
         20 . The method according to  claim 19  wherein the nucleotide sequence is selected from the group consisting of a genomic DNA sequence, a cDNA sequence, an RNA sequence, a sense RNA sequence or an antisense RNA sequence.  
     
     
         21 . The method according to  claim 17  wherein the detectable marker is labelled with a radioisotope and the detecting step is carried out by autoradiography.  
     
     
         22 . An isolated polypeptide molecule comprising at least five sequential amino acids of the high molecular weight subunit of microsomal triglyceride transfer protein.  
     
     
         23 . The isolated polypeptide molecule of  claim 22  encoded by all or part of SEQ. ID. NO. 2.  
     
     
         24 . The isolated polypeptide molecule of  claim 22  encoded by part of SEQ. ID. NO. 1.  
     
     
         25 . The polypeptide molecule according to  claim 22  having the amino acid sequence as shown in SEQ. ID. NOS. 3, 4, or 3 together with 6.  
     
     
         26 . The polypeptide molecule according to  claim 22  having part of the amino acid sequence as shown in SEQ. ID. NOS. 3, 4, or 3 together with 6.  
     
     
         27 . A process for detecting an inhibitor of microsomal triglyceride transfer protein comprising: 
 (a) incubating a sample thought to contain an inhibitor of microsomal triglyceride transfer with detectably labeled lipids in donor particles, acceptor particles and microsomal triglyceride transfer protein; and    (b) measuring the transfer of the detectably labeled lipid from the donor particles to the acceptor particles promoted by the microsomal triglyceride transfer protein;    wherein the inhibitor will decrease the rate of transfer of detectably labeled lipid from donor particles to acceptor particles.    
     
     
         28 . A method for preventing, stabilizing or causing regression of atherosclerosis in a mammalian species comprising administration of a therapeutically effective amount of an agent which decreases the amount or activity of microsomal triglyceride transfer protein.  
     
     
         29 . A method for decreasing serum lipid levels in a mammalian species, which comprises administration of a therapeutically effective amount of an agent which decreases the amount or activity of microsomal triglyceride transfer protein.  
     
     
         30 . A method for preventing or treating pancreatitis in a mammalian species comprising administration of a therapeutically effective amount of an agent which decreases the amount or activity of microsomal triglyceride transfer protein.  
     
     
         31 . A method for preventing or treating obesity in a mammalian species comprising administration of a therapeutically effective amount of an agent which decreases the amount or activity of microsomal triglyceride transfer protein.  
     
     
         32 . The method of  claim 28  or  29 , wherein the agent is a compound of the formula  
       
         
           
           
               
               
           
         
       
       or a compound of the formula  
       
         
           
           
               
               
           
         
       
       or a compound of the formula  
       
         
           
           
               
               
           
         
       
       wherein: 
 R 1  is alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl (all optionally substituted through available carbon atoms with 1, 2, or 3 groups selected from halo, alkyl, alkenyl, alkoxy, aryloxy, aryl, arylalkyl, alkylmercapto, arylmercapto, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl);  
 R 2 , R 3 , R 4  are independently hydrogen, halo, alkyl, alkenyl, alkoxy, aryloxy, aryl, arylalkyl, alkylmercapto, arylmercapto, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl;  
 R 5  and R 6  are independently hydrogen, alkyl, alkenyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl (all optionally substituted through available carbon atoms with 1, 2, or 3 groups selected from halo, alkyl, alkenyl, alkoxy, aryloxy, aryl, arylalkyl, alkylmercapto, arylmercapto, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl; with the proviso that when R 5  is CH 3 , R 6  is not hydrogen.  
 R 7  is alkyl (optionally substituted with oxo), aryl, or arylalkyl (wherein the alkyl portion is optionally substituted with oxo).  
 
     
     
         33 . The method of  claim 32 , wherein the agent is a compound of the formula  
       
         
           
           
               
               
           
         
       
       or a compound of the formula  
       
         
           
           
               
               
           
         
       
       wherein: 
 R 1  is —R v —R w  or  
                     
 R v  and R x  are each independently alkylene or cis-alkenylene of up to 6 carbon atoms;  
 R w  is aryl or heteroaryl; and  
 R y  and R z  are each independently alkyl, alkenyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, or cycloalkylalkyl.  
 
     
     
         34 . The method of  claim 33 , wherein R y  and R z  are each independently aryl, arylalkyl, heteroaryl, or heteroarylalkyl.  
     
     
         35 . A compound of the formula  
       
         
           
           
               
               
           
         
       
       wherein: 
 R 1  is alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl (wherein the alkyl comprises at least two carbon atoms), heteroarylalkyl (wherein the alkyl comprises at least two carbon atoms), cycloalkyl, or cycloalkylalkyl, all optionally substituted through available carbon atoms with 1, 2, or 3 groups selected from halo, alkyl, alkenyl, alkoxy, aryloxy, aryl, arylalkyl, alkylmercapto, arylmercapto, cycloalkyl, cycloalkylalkyl, heteroaryl, and heteroarylalkyl; and  
 R 2 , R 3 , R 4  are each independently hydrogen, halo, alkyl, alkenyl, alkoxy, aryloxy, aryl, arylalkyl, alkylmercapto, arylmercapto, cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl.  
 
     
     
         36 . A compound of the formula  
       
         
           
           
               
               
           
         
       
       wherein: 
 R 1  is arylalkyl or heteroarylalkyl wherein the alkyl portion of each comprises at least two carbon atoms and wherein each is optionally substituted through available carbon atoms with 1, 2, or 3 groups selected from halo, alkyl, alkenyl, alkoxy, aryloxy, aryl, arylalkyl, alkylmercapto, arylmercapto, cycloalkyl, cycloalkylalkyl, heteroaryl, and heteroarylalkyl; and  
 R 5  and R 6  are independently hydrogen, alkyl, alkenyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl (all optionally substituted-through available carbon atoms with 1, 2, or 3 groups selected from halo, alkyl, alkenyl, alkoxy, aryloxy, aryl, arylalkyl, alkylmercapto, arylmercapto, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl; with the proviso that when R 5  is CH 3 , R 6  is not hydrogen.  
 
     
     
         37 . The compound of  claim 35  or 36, wherein: 
 R 1  is —R v —R w  or  
                     
 R v  and R x  are each independently alkylene or cis-alkenylene of up to 6 carbon atoms;  
 R w  is aryl or heteroaryl; and  
 R y  and R z  are each independently alkyl, alkenyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, or cycloalkylalkyl.  
 
     
     
         38 . A compound of the formula  
       
         
           
           
               
               
           
         
       
       wherein: 
 R 2 , R 3 , R 4  are independently hydrogen, halo, alkyl, alkenyl, alkoxy, aryloxy, aryl, arylalkyl, alkylmercapto, arylmercapto; and  
 R 7  is alkyl (optionally substituted with oxo), aryl, or arylalkyl (wherein the alkyl portion is optionally substituted with oxo).  
 
     
     
         39 . The compound of  claim 35 , selected from the group consisting of: 
 2-[1-(3,3-Diphenyl-2-propenyl)-4-piperidinyl]-2,3-dihydro-1H-isoindol-1-one;    2-[1-(3,3-Diphenylpropyl)-4-piperidinyl]-2,3-dihydro-1H-isoindol-1-one;    2,3-Dihydro-2-[1-(phenylmethyl)piperidinyl]-1H-isoindol-1-one;    2,3-Dihydro-2-[1-(3-phenylpropyl)-4-piperidinyl]-1H-isoindol-1-one;    2-[1-(5,5-Diphenylpentyl)-4-piperidinyl]-2,3dihydro-1H-isoindol-1-one;    2-[1-(3-Butylheptyl)-4-piperidinyl]-2,3-dihydro-1H-isoindol-1-one;    (E)-2,3-Dihydro-2-[1-[3-(2-phenoxyphenyl)-2-propenyl]-4-piperidinyl]-1H-isoindol-1-one;    2,3-Dihydro-2-[1-[3-(2-methoxyphenyl)propyl]-4-piperidinyl]-1H-isoindol-1-one;    2-[1-(3-Butyl-2-heptenyl)-4-piperidinyl]-2,3-dihydro-1H-isoindol1-one;    2-[1-(5,5-Diphenyl-2-pentenyl)-4-piperidinyl]-2,3-dihydro-1H-isoindol-1-one;    2,3-Dihydro-2-[1-[3-(2-phenoxyphenyl)propyl]-4-piperidinyl]-1H-isoindol-1-one;    2,3-Dihydro-2-[1-(diphenylmethyl)-4-piperidinyl]-1H-isoindol-1-one;    (Z)-2,3-Dihydro-2-[1-(5,5-diphenyl-2-pentenyl)-4-piperidinyl]-1H-isoindol-1-one;    and pharmaceutically acceptable salts thereof.    
     
     
         40 . The compound of  claim 36 , selected from the group consisting of: 
 N-[1-(3,3-Diphenylpropyl)-4-piperidinyl]-2-pyridineamide;    N-[1-(3,3-Diphenylpropyl)-4-piperidinyl]benzamide    N-[1-(3,3-Diphenylpropyl)-4-piperidinyl]cyclohexane-carboxamide;    N-[1-(3,3-Diphenylpropyl)-4-piperidinyl]benzene acetamide;    N-[1-(3,3-Diphenylpropyl)-4-piperidinyl]pentamide;    N-[1-(Phenylmethyl)-4-piperidinyl]-1H-indole-3-acetamide;    4-Methoxy-α-(4-methoxyphenyl)-N-[1-(2-phenylethyl)-4-piperidinyl]benzeneacetamide;    α-Phenyl-N-[1-(phenylethyl)-4-piperidinyl]-benzeneacetamide,,    N-[1-(3,3-Diphenylpropyl)-4-piperidinyl)phenoxy-acetamide;    N-[1-(3,3-Diphenylpropyl)-4-piperidinyl]-2-methoxybenzamide;    N-[1-(3,3-Diphenylpropyl)-4-piperidinyl]-2-methylbenzamide;    N-[1-(3,3-Diphenylpropyl)-4-piperidinyl]N-(phenylmethyl)acetamide;    N-[1-(3,3-Diphenylpropyl)-4-piperidinyl]-N-(phenylmethyl)benzamide;    N-[1-(3,3-Diphenylpropyl)-4-piperidinyl]-N-methyl-benzamide;    and pharmaceutically acceptable salts thereof.    
     
     
         41 . The compound of  claim 38 , selected from the group consisting of: 
 3,4-Dihydro-3-[[4-phenyl-1-piperazinyl]methyl]-1(2H)-naphthalenone;    3,4-Dihydro-3-[[4-(2-methoxyphenyl)-1-piperazinyl]-carbonyl]-1(2H)-naphthalenone;    and pharmaceutically acceptable salts thereof.    
     
     
         42 . A monohydrochloride salt of the compounds of claims  39 ,  40 , or 41.