Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1993-11-04
1999-03-02
Carlson, Karen Cochrane
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
514 2, 530350, C12P 2106, C07K 1400
Patent
active
058769684
DESCRIPTION:
BRIEF SUMMARY
The present invention refers to the substantially pure dimer of apolipoprotein Al-Milano (Apo Al-M/ Apo Al-M) and pharmaceutical compositions containing this dimer. It also relates to the process for the preparation by recombinant technique as well as by isolation from plasma. The product may be used for the treatment of atherosclerosis and cardiovascular diseases and as a retard formulation of the Apo Al-M
BACKGROUND
The clear correlation between elevated levels of serum cholesterol and the development of coronary heart disease (CHD) has been repeatedly confirmed, based on epidemiological and longitudinal studies. The definition, however, of complex mechanisms of cholesterol transport in plasma, has allowed the recognition of a selective function of circulating lipoproteins in determining the risk for CHD.
There are, in fact, four major circulating lipoproteins: chylomicrons (CM), very low density (VLDL), low density (LDL) and high density (HDL) lipoproteins. While CM constitute a short-lived product of intestinal fat absorption, VLDL and, particularly, LDL are responsible for cholesterol transport into tissues, including for example, the arterial walls. In contrast, HDL are directly involved in the removal of cholesterol from peripheral tissues, carrying it back either to the liver or to other lipoproteins, by a mechanism known as "reverse cholesterol transport" (RCT).
The "protective" role of HDL has been confirmed in a number of studies (e.g. Miller et al. Lancet, 1977:965-968 and Whayne et aL Atherosclerosis 1981;39:411-419). In these, the elevated levels of LDL, less so of VLDL, seem to be clearly associated with an increased cardiovascular risk, whereas high HDL levels seem to confer cardiovascular protection. The protective role of HDL has been further strongly supported by the in vivo studies, showing that HDL infusions into rabbits may hinder the development of cholesterol induced arterial. lesions (Badimon et al, Lab. Invest. 60, 455-61, 1989)) and/or induce regression of same (Badimon et al, J Clin Invest. 85, 1234-41, 1990).
Recent interest in the study of the protective mechanism/s of HDL has been focussed on apolipoprotein Al (Apo Al), the major component of HDL. High plasma levels of Apo Al are associated with reduced risk of CHD and presence of coronary lesions (Maciejko et al,. N Engl J Med 1983;309:385-389, Sedlis et al,. Circulation 1986;73:978-984).
Plasma Apo Al is a single polypeptide chain of 243 amino acids, whose primary sequence is known (Brewer et al, Biochem Biophys Res Commun 1978;80:623-630). Apo Al is synthesized as a 267 amino acid precursor in the cell. This pre-pro-apoliprotein is processed by N-terminal cleavage first intracellularly where 18 amino acids are lost and then with a further cleavage of 6 amino acids in the plasma or the lymph by the activity of specific proteases.
The major structural requirement of the Apo Al molecule is believed to be the presence of repeat units of 11 or 22 amino acids, presumed to exist in amphipathic helical conformation (Segrest et al, FEBS Lett 1974;38:247-253). This structure allows for the main biological activities of Apo Al, i.e. lipid binding and lecithin cholesterol acyl transferase (LCAT) activation.
Another recently described property of Apo Al is its antiviral activity. This has been reported from in vitro studies and is exerted both against Herpes virus strains (Srinivas R V et al,, Virology, 1756, 48-57, 1990) and also against the Human Immunodeficiency virus, HIV, (Owe et al,., J Clin Invest, 86, 1142-50, 1990). This activity seems to be exerted by way of an interaction between amphipatic helical portions of Apo Al and envelope glycoproteins of the viruses.
In vitro studies indicate that complexes of Apo Al and lecithin can promote the efflux of free cholesterol from cultured arterial smooth muscle cells (Stein. et al,. Ciochem Biophys Acta 1975;380:106-118). By this mechanism HDL can also reduce the proliferation of these cells (Yoshida et al, Exp Mol Pathol 1 984;41 :258-266).
More recently, the infusion of Apo Al or
REFERENCES:
Weisgraber et al., Apolipoprotein A-I.sub.Milano Detection of Normal A-1 in Affected Subjects and Evidence for a Cysteine for Arginine Substitution in the Variant A-I, The Journal of Biological Chemistry, vol. 258, No. 4, (1993), pp. 2508-2513.
Franceschini et al., High Density Lipoprotein-3 Heterogeneity in Subjects with the Apo-Ai.sub.Milano Variant, The Journal of Biological Chemistry, vol. 257, No. 17, (1982), pp. 9926-9930.
Franceschini et al., "Apolipoprotein Al.sub.Milano . . . ", Journ. Biol. Chem. vol. 265, No. 21, Jul. 25, 1990, pp. 12224-12231.
Weisgraber et al., "Identification of the Disulfide-linked Homodimer of Apolipoprotein E3 in Plasma," Journ. Biol. Chem. vol. 266, No. 18, Jun. 25, 1991, pp. 12029-12034.
Pigiet et al., "Thioredoxin-catalyzed refolding of disulfide-containing proteins," Proc. Natl. Acad. Sci. USA vol. 83, pp. 7643-7647, Oct. 1986.
Nilsson et al., "Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors," The EMBO Journal vol. 4, pp. 1075-1080, 1985.
Abrahmsen Lars
Chmielewska Joanna
Franceschini Guido
Holmgren Erik
Lake Mats
Carlson Karen Cochrane
Pharmacia & Upjohn Aktiebolag
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