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.
Reexamination Certificate
1999-06-01
2003-12-30
Low, Christopher S. F. (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.
C530S350000, C435S007100, C536S023500
Reexamination Certificate
active
06670452
ABSTRACT:
BACKGROUND
The present invention relates to lipoprotein particles, a process for preparing such particles and their use. In particular, the invention relates to non-naturally occurring low density lipoprotein particles, methods for their manufacture and use thereof.
Low density lipoprotein (LDL) is a natural component of plasma which is involved in the transport of cholesterol in the form of cholesterol esters around the body. Naturally occurring LDL is known to occur as roughly spherical-shaped particles (20-22 nm in diameter) which comprise an internal core of about 1500 cholesterol esters containing small amounts of triglyceride (TG). The internal core is typically surrounded by a solubilising monolayer of about 800 phospholipid molecules and small quantities of free cholesterol (about 500 molecules). Located in the monolayer is a large receptor protein, Apo protein B, of approx 500,000 daltons, (Apo B) which accounts for about twenty percent of the weight of the LDL particle.
Naturally occurring LDL finds use in a number of areas, for example, in studies on atherosclerosis and lipid metabolism. LDL also finds use as a drug-targeting vector in cancer chemotherapy. Certain cancerous cells display high rates of receptor mediated LDL uptake relative to normal cells and as such, LDL has also found use as a targeting vector for anti-cancer drugs.
Currently, naturally occurring LDL needs to be isolated from fresh plasma samples. The isolation procedure is lengthy (e.g. up to 48 hours) and depending on the donor's plasma LDL levels the yield of LDL can be any amount up to about 100 mg ApoB/100 ml plasma in healthy individuals. Thus, yields are generally low.
Isolated naturally occurring LDL is known to be unstable. Attempts have been made to produce LDL-like particles, generally in the form of microemulsions of similar size and lipid composition to naturally occurring LDL, however such particles lack receptor competency. Apo B may be grafted onto such microemulsion particles, however, the grafting process still requires a source of the protein from fresh plasma.
Apo B is difficult to graft onto microemulsion particles partly because of its large size and a tendency for it to aggregate due to its amphipathic character. As such, grafting of Apo B onto microemulsion particles is not satisfactory because of inter alia inherent problems associated with the grafting-on process, and instability of the Apo B component.
It has now been found that a non-naturally occurring LDL can be produced which possesses LDL receptor competency, yet does not require the use of substantially whole Apo B or substantially whole analogues thereof. Furthermore, a process for the production of non-naturally occurring LDL has been developed which does not require the use of plasma derived LDL and/or plasma derived Apo B.
SUMMARY OF INVENTION
An object of the present invention is to provide a non-naturally occurring LDL possessing Apo B receptor competence.
Another object of the present invention is to provide a process for producing non-naturally occurring LDL particles.
These and other objects of the invention will become apparent from the following description and examples.
STATEMENT OF INVENTION
According to a first aspect of the present invention there is provided a non-naturally occurring, receptor competent LDL particle comprising at least one peptide component wherein the said peptide component comprises at least a binding site for an Apo B protein receptor and at least one lipophilic substituent.
A non-naturally occurring LDL particle is one which is not found occurring naturally in vivo. A non-naturally occurring LDL must be receptor competent i.e. capable of binding to Apo B receptors and/or capable of eliciting an Apo B protein-like physiological effect on and/or after binding. Thus, the non-naturally occurring LDL particle comprises at least a sequence of amino acids such as a protein, polypeptide or peptide capable of binding to Apo B receptors, which polypeptide may or may not be identical in respect of its binding region with the amino acid sequence of an Apo-B binding site, for example, an Apo B 100 binding site or physiologically functional peptide analogues thereof. Naturally, the skilled addressee will appreciate that the polypeptide capable of binding to Apo B receptors on target cells, such as cancer cells expressing Apo B receptors, is able to elicit an Apo B protein-like physiological effect on and/or after binding i.e. to be receptor competent.
The LDL particle comprises at least two components, a lipid component (L-component) and a peptide component (P-component). The L-component generally comprises a lipid emulsion comprising a core of lipophilic molecules such as cholesteryl esters, for example, cholesterol oleate, cholesterol linoleate, cholesterol stearate and the like. Other suitable lipophilic core molecules can comprise triglycerides, for example, triolein, plant oils such as soya bean oil and even lipophilic drugs, for example, estramustine, prednimustine and lipophilic modifications of known drugs, such as anti-cancer drugs, for example, cholesteryl esters of methotrexate and the like. The core of the L-component is typically solubilised by a lipid, such as an amphiphilic lipid comprising a charged or hydrophilic group. Such amphiphilic lipids include unesterified cholesterol and suitable non-ionic surfactants as well as phospholipids such as phosphatidyl choline, sphingomyelin and phosphatidyl glycerol. Preferably, the cholesteryl esters are solubilised by a monolayer of phospholipid. The preparation of the L-component is known in the art and may be performed using a variety of methods as described in the art, e.g. Ginsburg, G. S. et al (1982) J. Biol. Chem 257 (14) pp 8216-8227; Owens M. D. and Halbert G. W. (1993) J. Pharm. Pharmacol. 45 (Suppl.) p68P; Owens M. D. and Halbert G. W. (1995) Eur. J. Pharm. Biopharm 41 (2) pp 120-126, herein incorporated by reference.
Preferably, the L-component is made up of at least two biologically acceptable components. A first component can be a biologically acceptable saturated or unsaturated long chain charged polar component such as a phospholipid. Examples of suitable charged polar components include phosphatidyl choline (PC), phosphatidyl serine (PS), phosphatidyl glycerol (PG), sphingomyelin, and unesterified cholesterol and the like. The second component can be a biologically acceptable lipophilic component such as a cholesteryl ester, for example cholesteryl oleate. Biologically acceptable components are ones which may be administered to cells in vitro or in vivo and which have substantially no deleterious effect on cell viability. In a preferment the L-component can comprise three or more components in a defined ratio, such as a molar ratio, for example, phospholipid; triolein; cholesteryl ester (P:T:C) The molar ratio may be in any molar ratio as long as the components are capable of forming an L-component suitable for use in the preparation of non-naturally occurring LDL particles of the present invention. The molar ratio of outer core solubilising lipid such as phospholipid (PL) sphingomyelin (SM), phosphatidyl choline (PC) and unesterified cholesterol (UC) to core lipid such as cholesteryl ester (CE), triolein (TO) cholesteryl oleate (CO) or lipophilic drug can be in the range of from about 0.7:1 up to 5:1, preferably 1:1 to 3:1 depending on design. A preferred ratio of PL:CE is about 2:1. Where a third L-component is not employed the ratio of PL:CE can be in the range of from about 1:1 to about 2:1. A suitable molar ratio for a three component system such as a phosphatidylcholine: triolein: cholesteryl oleate is 3:2:1 respectively.
A suitable molar ratio for a five component system comprising three outer core lipids and two core lipids may lie in the range of from 0.7-6.5:0-2:0-1 (outer core lipid): 0-5:0-2.5 (core lipid). Preferably, the molar ratio lies in the range of from 2.5-4.5:1-2:0.5-1 (outer core lipid): 2-4.5:1-2.5 (core lipid). More preferably the molar ratio lies in the range of from 4-4.5:1.5-2:0.7-0.9 (out
Baillie George
Halbert Gavin William
Owens Moira Doreen
Alston & Bird LLP
Low Christopher S. F.
Robinson Hope A.
University of Strathclyde
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