Lipid cleavage enzyme

Abstract

A membranous enzyme not yet described in the state-of-the-art can be extracted from cellular membrane fractions of blood leukocytes or monocytes/macrophages. Also disclosed is the use of substrates of this enzyme to prepare medicaments that contain these substrates as pharmaceutical active substance. These medicaments are useful to direct pharmacologically active substances to target cells and to enrich target cells with said substances. Also disclosed are in-vitro research systems containing this enzyme used to detect other substrates of this enzyme.

1 . Lipid cleavage enzyme complex (LCE) free of phospholipase C activity, wherein it cleaves the conjugate AZT-DMDOPE [(3′-deoxy-3′-azidothymidine)-5′-phosphoric acid-(3-dodecyl-mercapto-2-decyloxy)-propyl ester] into AZT-MP [3′-deoxy-3′-azido-thymidine-monophosphate] and DMDOP [(3-dodecyl-mercapto-2-decyloxy)-propanol] or the conjugate FLT-DMDOPE [(3′-deoxy-3′-fluorothymidine)-5′-phosphoric acid-(3-dodecylmercapto-2-decyloxy)-propyl ester] into FLT-MP [3′-deoxy-3′-fluoro-thyimidine-5′-monophosphate] and DMDOP or the conjugate 5-FU-DMDOPE [5-fluorouridine-5′-phosphoric acid-(3-dodecylmercapto-2-decyloxy)-propyl ester] into 5-FU-MP [5-fluorouridine monophosphate] and DMDOP.  
     
     
         2 . LCE as claimed in  claim 1  wherein it is obtainable from leucocytes, monocytes, tumour cells, kidney cells, lymphocytes, cells of the immuno/lymphatic system or macrophages.  
     
     
         3 . Isolated subcellular systems for carrying out in vitro test procedures wherein the subcellular systems are enriched with the enzyme as claimed in one of the claims  1  or  2 .  
     
     
         4 . Use of the enzyme as claimed in one of the claims  1  or  2  to find or to identify substrates, ligands, inhibitors or activators of the LCE in a suitable in vitro test system.  
     
     
         5 . Use of subcellular systems as claimed in  claim 3  to find or to identify substrates, ligands, inhibitors or activators of the LCE in a suitable in vitro test system.  
     
     
         6 . Method for finding compounds which act as substrates, ligands, inhibitors or activators of the LCE as claimed in one of the claims  1  or  2 , wherein the cleavage products are detected in a suitable test system.  
     
     
         7 . Method for finding LCE analogues in which compounds found in the method as claimed in  claim 6  are used in a screening system.  
     
     
         8 . LCE analogues obtainable by a method as claimed in  claim 7 .  
     
     
         9 . LCE or LCE analogues as claimed in one of the claims  1 ,  2  or  8 , wherein in the case of conjugates of the type L-B-D in which L represents a lipid-like residue, B represents a phosphate bridge or a thiophosphate bridge and D denotes a pharmacologically active substance or B-D represents an active substance phosphonate the enzyme induces a cleavage of the covalent bond between the lipid moiety L and the residue -B-D.  
     
     
         10 . LCE or LCE analogues as claimed in one of the claims  1 ,  2 ,  8  or  9 , wherein the activity of the enzyme in cleaving the conjugate is at least 2-fold higher in activated immune cells, human leucocytes, monocytes, tumour cells, lymphocytes, kidney cells, cells of the immuno/lymphatic system or macrophages compared to non-activated cells.  
     
     
         11 . LCE or LCE analogues as claimed in one of the claims  1 ,  2  or  8 - 10 , wherein the activity of the enzyme is inhibited by Ca 2+ , Zn 2+  or Mn 2+ .  
     
     
         12 . Use of a covalent conjugate of a lipid derivative and a pharmacologically active substance to produce a pharmaceutical agent for the targeted release of the residue -B-D in suitable target cells wherein the effect of the lipid moiety of the covalent conjugate is to target the conjugate to the target cells, tissues or organs and to transport the conjugate or the residue -B-D through the cell membrane of these target cells, the conjugate is cleaved by a membrane-based enzyme as claimed in one of the claims  1 ,  2  or  9 - 11  resulting in a specific intracellular accumulation of the residue -B-D or D.  
     
     
         13 . Use as claimed in  claim 12 , wherein the covalent conjugate is cleaved essentially on or in the membrane or in the respective target cells.  
     
     
         14 . Use as claimed in  claim 12 , wherein the pharmaceutical agent reduces the side effects compared to the administration of the free residue -B-D or D not bound to a lipid.  
     
     
         15 . Use as claimed in  claim 12  or  13 , wherein there is no significant cleavage of the conjugate in the plasma or in the liver.  
     
     
         16 . Use as claimed in  claim 12  or  13 , wherein the target cells are human leucocytes, monocytes, macrophages, immunological cells, tumour cells or cells of the lymphatic system, of the kidney, spleen or brain.  
     
     
         17 . Use as claimed in one of the claims  12 - 16 , wherein the pharmaceutical agent is capable of increasing the intracellular concentration of the residue -B-D or D in the target cells by at least 10% compared to the administration of free -B-D or D not bound to a lipid residue.  
     
     
         18 . Use as claimed in  claim 17 , wherein the concentration of the active substance in the target cells is increased by at least 50%.  
     
     
         19 . Use as claimed in one of the claims  12 - 18 , wherein the pharmaceutical agent is administered in a dosage unit which has a lower equivalent content of the conjugated substance -B-D or D compared to the form of administration containing the free non-conjugated substance.  
     
     
         20 . Use as claimed in  claim 19 , wherein the equivalent content of the conjugated substance -B-D or D is less than 50% compared to the form of administration containing the free non-conjugated substance.  
     
     
         21 . Use as claimed in one of the claims  12 - 19  for the production of pharmaceutical agents for reducing organ toxicities of pharmacologically active substances.  
     
     
         22 . Use as claimed in one of the claims  12 - 20  for the production of pharmaceutical agents for reducing the bone marrow toxicity of pharmacologically active substances.  
     
     
         23 . Use as claimed in one of the claims  12 - 20  for the production of pharmaceutical agents for reducing the cardiotoxicity, nephrotoxicity, haemotoxicity, hepatoxicity or neurotoxicity of pharmacologically active substances.  
     
     
         24 . Use as claimed in one of the claims  12 - 20  for the production of pharmaceutical agents for the targeted transport of pharmacologically active substances through the blood-brain barrier.  
     
     
         25 . Use as claimed in  claim 24 , wherein the pharmacologically active substance accumulates in cells of the brain.  
     
     
         26 . Use as claimed in  claim 24  or  25 , wherein the pharmacologically active substance is a therapeutic agent that can be administered orally.  
     
     
         27 . Use as claimed in claims  12 - 26 , wherein the conjugate is an active substance of formula L-B-D and L is a lipid moiety, B is a valency dash, a phosphate bridge or a thiophosphate bridge and D represents a pharmacologically active substance or B-D is an active substance phosphonate.  
     
     
         28 . Use of a covalent conjugate L-B-D as claimed in  claim 27 , wherein L represents a residue of formula II  
       
         
           
           
               
               
           
         
         in which  
         R 1  is a straight-chain or branched, saturated or unsaturated alkyl chain with 1-30 carbon atoms which can be optionally substituted once or several times by halogen, C 5 -C 7  cycloalkyl, phenyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylmercapto, C 1 -C 6  alkoxycarbonyl, C 1 -C 6  alkylsulfinyl or C 1 -C 6  alkylsulfonyl groups,  
         R 2  is hydrogen, a straight-chain or branched, saturated or unsaturated alkyl chain with 1-20 carbon atoms which can be optionally substituted once or several times by halogen, C 5 -C 7  cycloalkyl, phenyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylmercapto, C 1 -C 6  alkoxycarbonyl or C 1 -C 6  alkylsulfonyl groups  
         X represents a valency dash, oxygen, sulphur, aminocarbonyl, oxycarbonyl, carboxyamino, carbonyloxy, a sulfinyl or sulfonyl group  
         Y is a valency dash, aminocarbonyl, oxycarbonyl, carbonylamino, carbonyloxy, an oxygen or sulphur atom and  
         m represents an integer between 1 and 5.  
       
     
     
         29 . Use of a covalent conjugate L-B-D as claimed in  claim 28 , wherein R 1  denotes a straight-chain or branched C 8 -C 15  alkyl group which can be substituted by a C 1 -C 6  alkoxy or a C 1 -C 6  alkylmercapto group and in particular represents a nonyl, decyl, undecyl, dodecyl, tridecyl or tetradecyl group.  
     
     
         30 . Use of a covalent conjugate L-B-D as claimed in one of the claims  28  or 29, wherein R 2  denotes a straight-chain or branched C 8 -C 15  alkyl group which can be substituted by a C 1 -C 6  alkoxy group or a C 1 -C 6  alkylmercapto group and in particular represents an octyl, nonyl, decyl, undecyl, dodecyl, tridecyl or tetradecyl group.  
     
     
         31 . Use of a covalent conjugate L-B-D as claimed in one of the claims  27 - 30 , wherein X denotes a sulphur atom, a sulfinyl or sulfonyl group.  
     
     
         32 . Use of a covalent conjugate L-B-D as claimed in one of the claims  27 - 31 , wherein Y denotes an oxygen atom.  
     
     
         33 . Use of a covalent conjugate L-B-D as claimed in one of the claims  27 - 32 , wherein m denotes the numbers 1 or 2.  
     
     
         34 . Use of a covalent conjugate L-B-D as claimed in one of the claims  27 - 32 , wherein m equals 1, X is S, Y is O, R 1  is a dodecyl residue and R 2  represents a decyl residue.  
     
     
         35 . Use of a covalent conjugate L-B-D as claimed in  claim 27 , wherein X and Y represent a valency dash, R 2  is hydrogen and R 1  represents a C 1 -C 30  alkyl chain which can be optionally substituted by C 1 -C 6  alkoxy or C 1 -C 6  alkylmercapto.  
     
     
         36 . Use of a covalent conjugate L-B-D as claimed in one of the claims  27 - 35 , wherein B represents a phosphate bridge of formula III  
       —O—[PZ(OH)A]—  (III),  
       in which n denotes the numbers 1, 2 or 3 and Z is O or S, and A denotes either O, S or a valency dash.  
     
     
         37 . Use of a covalent conjugate L-B-D as claimed in one of the claims  27 - 36 , wherein D or -B-D represents a pharmacologically active substance selected from the group of antiviral, antiretroviral, cytotoxic, cytostatic, antitumoral, immunosuppressive or immunostimulatory substances.  
     
     
         38 . Process for the production of pharmaceutical agents for the targeted release of substances -B-D or D in target cells, wherein these target cells contain the lipid cleavage enzyme or LCE analogues as claimed in one of the claims  1 ,  2  or  8 - 11  comprising the steps 
 a) selecting a pharmacologically active substance  
 b) producing a covalent conjugate of the pharmacologically active substance and a lipid-like carrier molecule  
 c) and producing a pharmaceutical form of administration which contains the conjugate produced according to b) as well as further pharmaceutical auxiliary or carrier substances.  
 
     
     
         39 . Lipid conjugate of formula I of type L-B-D (I) selected from the group of the following substances: 
 2-fluoro-9-(b-D-arabinofuranosyl)adenine-5′-phosphoric acid-(3-dodecylmercapto-2-decyloxypropyl) ester, 2-chloro-2′-deoxyadenosine-5′-phosphoric acid-(3-dodecylmercapto-2-decyloxypropyl) ester, 3-(2-deoxy-b-D-erythropentofuranosyl)-3,6,7,8-tetrahydro-imidazo-[4,5-d] [1,3]-diazepin-8-ol-5′-phosphoric acid-(3-dodecylmercapto-2-decyloxy-propyl) ester.    
     
     
         40 . Diagnostic agent containing LCE or LCE analogues as claimed in one of the claims  1 ,  2  or  8 - 11  for carrying out methods of determination for substrates, ligands, activators or inhihibitors of the lipid cleavage enzyme.  
     
     
         41 . Agent for finding substances which act as a substrate or ligand of the enzyme, containing an adequate amount of the LCE or LCE analogues in an isolated form or concentrated in cell preparations as claimed in one of the claims  1 ,  2  or  8 - 11  as well as suitable stabilizers.  
     
     
         42 . Diagnostic agent containing a compound as claimed in  claim 27  for the determination of pathological changes due to increased or reduced enzyme activity or enzyme affinity which are associated with corresponding diseases or disease symptoms.