Purified Apo A and Apo E compounds and methods for using them

Details Purified Apo A and Apo E compounds and methods for using them Purified Apo A and Apo E compounds and methods for using them

1999-11-22

2003-01-14

Carlson, Karen Cochrane (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

Lipoproteins, e.g., egg yolk proteins, cylomicrons, etc.

C435S069100, C435S071100

Reexamination Certificate

active

06506879

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a substantially endotoxin-free apolipoprotein A (ApoA) or apolipoprotein E (ApoE) and a process for producing the same, by separating the endotoxins from the ApoA or ApoE, or variants or mixtures thereof, by contacting a first aqueous solution containing said ApoA or ApoE with a matrix containing an immobilized compound with an end group comprising two or three nitrogen atoms bonded to a carbon atom, and subsequently treating the matrix containing an immobilized compound with a second aqueous solution containing a surfactant, or by contacting a first aqueous solution containing said ApoA or ApoE with an anion-exchange matrix, and subsequently treating the anion-exchange matrix with a second aqueous solution containing a compound comprising two or three nitrogen atoms bonded to a carbon atom. The invention further relates to use of a matrix containing an immobilized compound comprising two or three nitrogen atoms bonded to a carbon atom and a solution containing a surfactant, or an anion-exchange matrix and a solution containing a compound comprising two or three nitrogen atoms bonded to a carbon atom, for removing endotoxins from aqueous solutions containing ApoA or ApoE, or variants or mixtures thereof. The thus produced ApoA or ApoE can be used for the manufacture of a medicament in the treatment of atherosclerosis and cardiovascular diseases, as well as in a method for treatment of atherosclerosis and cardiovascular diseases when administered in a therapeutically effective amount.
BACKGROUND OF THE INVENTION
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. Of these, HDL is 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. Recent studies directed to the protective mechanism(s) of HDL have been focused on apolipoprotein A-I (ApoA-I), the major component of HDL. High plasma levels of ApoA-I are associated with a reduced risk of CHD and presence of coronary lesions.
Plasma ApoA-I is a single polypeptide chain of 243 amino acids, whose primary sequence is known (Brewer et al. (1978) Biochem. Biophys. Res. Commun. 80: 623-630). ApoA-I is synthesized as a 267 amino acid precursor in the cell. The major structural requirement of the ApoA-I 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 ApoA-I, i.e. lipid binding and lecithin cholesterol acyl transferase (LCAT) activation.
The apolipoprotein A-IMilano (ApoA-IM) is the first described molecular variant of human ApoA-I (Franceschini et al. (1980) J. Clin. Invest. 66: 892-900). It is characterized by the substitution of Arg 173 with Cys 173 (Weisgraber et al. (1983) J. Biol. Chem. 258:2508-2513). The mutant apolipoprotein is transmitted as an autosomal dominant trait and 8 generations of carriers have been identified (Gualandri et al. (1984) Am. J. Hum. Genet. 37: 1083-1097). The status of a ApoA-IM carrier individual is characterized by a remarkable reduction in HDL-cholesterol level. In spite of this, the affected subjects do not apparently show any increased risk of arterial disease. Indeed, by examination of the genealogical tree it appears that these subjects may be “protected” from atherosclerosis.
The mechanism of the possible protective effect of ApoA-IM in the carriers seems to be linked to a modification in the structure of the mutant apolipoprotein, with the loss of one alpha-helix and an increased exposure of hydrophobic residues (Francheschini et al. (1985) J. Biol. Chem. 260: 1632-1635). The loss of the tight structure of the multiple alpha-helices leads to an increased flexibility of the molecule, which associates more readily with lipids, compared to normal ApoA-I.
Another very specific feature of the ApoA-IM, is its capacity to form dimers with itself and complexes with ApoA-II, in both cases because of the presence of the Cys residue.
To make possible production of sufficient quantities of ApoA-I in general, and more specifically ApoA-IM, use is made of recombinant DNA techniques, e.g. in
E. coli
. Thus, recombinant preparation and use of ApoA-IM, monomers as well as dimers, are disclosed in patent specifications WO-A-88/03166 assigned to Farmitalia Carlo Erba (FICE), WO-A-90/12879 assigned to Sirtori et al, as well as WO-A-93/12143 and WO-A-94/13819 both assigned to Pharmacia AB (formerly Kabi Pharmacia AB).
Use of e.g.
E. coli
as medium introduces certain drawbacks. Thus, endotoxins or lipopolysaccharides (LPS) are high molecular complexes associated with the outer membrane (cell wall) of grain-negative bacteria, such as
E. coli
, Proteus and Salmonella. Endotoxins consist of two main parts, a lipid moiety called lipid A which is embedded in the outer membrane and a polysaccharide (O-antigen) which protrudes into the environment. Lipid A is the region which elicit the toxic effect of the endotoxins, a prerequisite being the presence of the entire lipid A moiety. The polysaccharide is made up of a O-specific chain and a core. The O-specific chain projects from the core and is the outermost part of the endotoxin. The core works as a linkage between lipid A and the O-specific chain.
It is known that endotoxins must be released from the bacterial surface to cause toxic effects. This happens when the bacteria multiply, at lysis and during stress. In aqueous solutions, free endotoxins form aggregates, micelles and vesicles, with a molecular weight of about 5 kDa up to>10
3
kDa.
It is known from the literature that several proteins form complexes with endotoxins. Particularly strong complexes are formed with HDL and apolipoproteins (Emancipator et al. (1992) Infect. Immun. 60: 596-601). According to Ulevitch et al. (1981) J. Clin. Invest. 67: 827-837, formation of a complex between HDL and endotoxins involve a two step mechanism, as follows:
Endotoxins(aggregated)→Endotoxins(disaggregated)  (1)
Endotoxins(disaggregated)+HDL→Endotoxins-HDL  (2)
This behavior has been confirmed e.g. by Munford et al (1981) Infect. Immun. 34: 835-843. There are indications suggesting that lipid A is the main factor in the complex and that the interaction involves both ionic and hydrophobic forces (Freudenberg et al. (1979) Nat. Toxins, Proc. Int. Symp. Anim., Plant Microb. Toxins., 6th, 349-354).
As already stated above, strong complexes are formed between endotoxins and HDL in general and particularly with apolipoproteins. This mechanism has been used in U.S. Pat. No. 5,128,318 assigned to the Rogosin Institute. U.S. Pat. No. 5,128,318 thus relates to HDL associated apolipoprotein containing reconstituted particles, and use thereof in removing lipid soluble materials, including endotoxins, from cells, body fluids, and the like. More particularly, U.S. Pat. No. 5,128,318 relates to a method for treating a subject for endotoxin-caused toxicity, by administering to the subject a reconstituted particle containing ApoA-I or ApoA-II, with or without cholesterol. Here, naturally, the aim is to create and maintain indefinitely the strongest possible complex, to avoid release of the endotoxins in the subject.
The complexes, strong in themselves, can be further strengthened e.g. by the presence of certain chemical compounds. Thus, deoxycholate is kn

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