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Methods of prevention and treatment using apolipoprotein analogues

a technology of apolipoprotein and analogues, applied in the field of prevention and treatment using apolipoprotein analogues, can solve the problems of increasing the risk of infarction, and achieve the effects of reducing the rate of filtration through the kidneys, reducing the half-life and increasing the size of the apolipoprotein constru

Inactive Publication Date: 2005-05-05
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0021] The construct comprising apolipoprotein and a heterologous moiety is capable of performing reverse cholesterol transport as well as or even better than native apolipoproteins, despite the modification caused by the addition of a heterologous moiety. The plasma half-life of the construct is preferably increased compared to that of the wild-type apolipoprotein. The increased half-life can be due either to the increased size of the apolipoprotein construct, which may reduce the rate of filtration through the kidneys, it may be due to increased binding to HDL, or it may be due to reduced breakdown of the construct compared to native Apo A.
[0022] Preferably the plasma half-life is at least doubled or tripled, or at least quadrupled, or at least 10 doubled. Similarly, the binding affinity such as the lipid binding affinity, and / or the cholesterol binding affinity of the construct is preferably increased as compared to wild-type apolipoprotein. Preferably, the lipid binding affinity is increased by at least 5%, such as at least 10%, for example at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 40% such as at least 50%, for example at least 75%, such as at least 100%, such as at least 150%, for example at least 200%, such as at least 300%. Even in the cases where the lipid binding affinity of the constructs according to the invention is the same or lower than the lipid binding affinity of native apolipoprotein, the clinical effect may be enhanced due to increased plasma half life of the constructs according to the invention.
[0023] An increased plasma half-time and / or increased lipid binding affinity have profound implications for the use of the apolipoprotein constructs in the treatment of arterosclerosis. It is therefore expected that the clinical effect of the apolipoprotein constructs according to the invention is superior to the effect of wild-type apolipoproteins.

Problems solved by technology

One of the pathogenic factors causing atherosclerosis is the deposition of cholesterol in the vessel walls leading to plaque formation and eventually to arterosclerosis and increased risk of infarction.

Method used

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  • Methods of prevention and treatment using apolipoprotein analogues
  • Methods of prevention and treatment using apolipoprotein analogues
  • Methods of prevention and treatment using apolipoprotein analogues

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning of Apo A-I

[0229] The cDNA encoding Apo A-I was amplified from a human liver cDNA library (Clontech) using standard PCR techniques. For the construction of Ubi-A-I the primers used were: 5′-CAC GGA TCC ATC GAG GGT AGG GGT GGA GAT GAA CCC CCC CAG AGC-3′ and 5′-TCC AAG CTT ATT ACT GGG TGT TGA GCT TCT TAG TG-3′. The product was cloned into the vector pT7H6Ubi, described in (Ellgaard L. et al Eur. J. Biochem. 1997; 244(2):544-51) using the Bam HI and Hind III cloning sites. For the construction of Trip-A-A-I the primers used were 5′-AAG GGA TCC GAT GAA CCC CCC CAG AGC CCC-3′ and 5′-TCC AAG CTT ATT ACT GGG TGT TGA GCT TCT TAG TG-3′. The PCR product was cloned into the pT7H6tripa vector described in WO 98 / 56906 using the Bam HI and Hind III cloning sites. For the construction of Trip-A-I-del43 the primers used were 5′-AGG GGA TCC CTA AAG CTC CTT GAC AAC TGG G-3′ and 5′-TCC AAG CTT ATT ACT GGG TGT TGA GCT TCT TAG TG -3′. The PCR product was cloned into the pT7H6tripa vector describ...

example 2

Expression of Apolipoprotein A-I (Apo A-I) in E. coli

[0230] Ubi-A-I and Trip-A-I as well as the other constructs disclosed in the figures are conveniently expressed in E. coli AV-1 cells (Stratagene Inc.). Other cell lines may be used as well. Culturing of the cells and induction of expression were performed as described for tetranectin in WO 98 / 56906.

example 3

Isolation and Processing of Protein

[0231] Crude protein was isolated by phenol extraction as described for tetranectin in WO 98 / 56906. The re-dissolved pellet from 6 litres of expression culture was centrifuged to remove non-dissolved material and then batch adsorbed to 50 ml Ni2+-NTA-Sepharose, prepared as described in WO 98 / 56906. The column material was packed on a column and then washed with 500 ml 8 M urea, 500 mM NaCl, 50 mM Tris-HCl pH 8.0, then 200 ml of 6 M Guanidinium-HCl, 50 mM Tris-HCl pH 8.0 and finally 300 ml of 500 mM NaCl, 50 mM Tris-HCl pH 8.0. The protein was eluted with 500 mM NaCl, 50 mM Tris-HCl pH 8.0 and 10 mM EDTA. The protein was added 0.5 mg of Factor Xa and digested overnight at room temperature. Thrombin may be used for this purpose as well. The protein was gelfiltrated on a G-25 sephadex (Pharmacia) column in to a 500 mM NaCl, 50 mM Tris-HCl pH 8.0 buffer. Undigested protein was removed by passing the protein solution over a Ni2+-NTA-Sepharose column pr...

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Abstract

The invention relates to a pharmaceutical composition comprising an apolipoprotein construct, to an apolipoprotein construct, a nucleic acid sequence encoding the apolipoprotein construct, a vector comprising the nucleic acid sequence, a method for producing the apolipoprotein construct, and a method of treatment comprising administering the apolipoprotein construct. The presented data document that the constructs according to the invention are capable of binding lipids, are capable of binding cubilin, which is a strong Apo AI receptor, stronger than native Apo A-I and that the plasma half life of the constructs is at least tripled compared to native Apo A-I. Together these data document that the constructs according to the invention are strong candidates for treatment of cardiovascular diseases.

Description

[0001] The invention relates to a pharmaceutical composition comprising an apolipoprotein construct, to an apolipoprotein construct, a nucleic acid sequence encoding the apolipoprotein construct, a vector comprising the nucleic acid sequence, a method for producing the apolipoprotein construct, and a method of treatment comprising administering the apolipoprotein construct. Prior Art [0002] In the following, the term Apo A or apolipoprotein A will be used to designate any of the three apolipoproteins, Apolipoprotein A I, Apolipoprotein A II, or Apolipoprotein A IV. [0003] Cardiovascular diseases caused by atherosclerosis in the vessels is the most frequent cause of death in the industrialised countries of the World. One of the pathogenic factors causing atherosclerosis is the deposition of cholesterol in the vessel walls leading to plaque formation and eventually to arterosclerosis and increased risk of infarction. [0004] Apolipoprotein A-1 (apo-A-1) is the main component of plasma ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/09A61K38/00A61K38/47A61K47/14A61K47/24A61K47/28A61K47/48A61K48/00A61P7/00A61P9/10C07K14/775C07K19/00C12N1/15C12N1/19C12N1/21C12N5/10C12N15/62C12P21/02
CPCA61K38/47C07K14/775C07K2319/00C07K2319/02C12N15/62C07K2319/31C07K2319/50C07K2319/73C07K2319/21A61P7/00A61P9/00A61P9/08A61P9/10A61P39/02
Inventor GRAVERSEN, JONASMOESTRUP, SOREN
Owner F HOFFMANN LA ROCHE & CO AG
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