Title:  Materials and methods for making improved micelle compositions

United States Patent:  6,322,810

Appl. No.:  462819

PCT Filed:  July 9, 1998

371 Date:  May 18, 2000

PCT PUB.NO.:  WO99/03448

Provided are methods for preparing improved biologically active micelle and crystalline products comprising a biologically active amphipathic compound in association with a micelle or crystalline product. Methods for producing the micelle or crystalline products as well as methods of using the micelle or crystalline products in therapeutic, diagnostic, and cosmetic, applications are also provided.

SUMMARY OF THE INVENTION

The present invention provides improved methods of preparing biologically active micelle products comprising biologically active amphipathic compounds in association with a micelle. The micellar formulations of the invention deliver and enhance bioactivity of the biologically active peptides in a manner which provides improvements in the efficacy and duration of the biological effects of the associated peptides. Increased efficacy and duration of the biological effect is believed to result, at least in part, from interaction of the compound with the micelle in such a manner that the compound attains, and is maintained in, an active or more active conformation than the compound in an aqueous environment, The invention thus overcomes the problems associated with previous liposomal formulations, such as, but not limited to, uptake by the reticuloendothelial system, degradation of the compound, or delivery of the compound in an inactive conformation. According to the present invention, polyethylene-glycol (PEG) is covalently conjugated to DSPE to form polymeric micelles which were then passively loaded with VIP. The PEG-PE forms micelles with a hydrophobic core consisting of phosphatidylethanolamine (PE) fatty acid chains which is surrounded by a hydrophilic "shell" formed by the PEG polymer.

According to one aspect of the invention, a method is provided for preparing a biologically active micelle product comprising a biologically active amphipathic compound in association with a micelle; said method comprising the steps of a) mixing a combination of one or more lipids wherein said combination includes at least one lipid component covalently bonded to a water-soluble polymer; b) forming sterically stabilized micelles from said combination of lipids; and c) incubating micelles from step b) with a biologically active amphipathic compound under conditions in which said compound becomes associated with said micelles from step b) in an active conformation. According to a further aspect of the invention, a biologically active micelle product may be produced by the coprecipitation of a biologically active amphipathic compound with lipids to form micelles with incubation not required. Specifically, a method is provided of preparing a biologically active micelle product comprising a biologically active amphipathic compound in association with a micelle; said method comprising the steps of: a) mixing one or more lipids wherein said combination includes at least one lipid component covalently bonded to a water-soluble polymer with a biologically active amphipathic compound; b) forming sterically stabilized micelles from the mixture of step (a) under conditions in which said compound becomes associated with said micelles in an active conformation.

As one aspect of the invention, the micelles are sterically stabilized micelles (SSM) which are produced from a combination of lipids which includes at least one lipid component covalently bonded to a water-soluble polymer. This polymer bound phospholipid is the micelle forming component. Other lipids are actually solubilized in this micelle to form mixed micelles. The water-soluble polymer, which is preferably polyethylene glycol (PEG) increases the lipid solubility to form micelles instead of vesicles in aqueous media. It also acts to sterically stabilize the resulting micelle against uptake by components of the reticuloendothelial system.

The methods of the invention are useful with any biologically active amphipathic compound which can thereby be stably maintained in an active conformation in association with or within the lipid core of the micelle. Preferred amphipathic compounds include those characterized by having one or more .alpha.- or .pi.-helical domains in their biologically active conformation and particularly those in which polar and apolar residues are separated on opposite sides of the helix. Particularly preferred amphipathic compounds useful with the invention include any member of the vasoactive intestinal peptide (VIP)/growth hormone releasing factor (GRF) family of peptides which includes biologically active analogs thereof. The mammalian and non-mammalian VIP/GRF family of peptides includes functional analogs of VIP and GRF, peptide histidine isoleucine (PHI), peptide histidine methionine (PHM), growth hormone releasing factor (GRF), pituitary adenylate cyclase activating peptide (PACAP), secretin, and glucagon. Like VIP, other members of the VIP/GRF family of peptides, and biologically active analogues thereof, can form amphipathic helices wherein hydrophobic and hydrophilic domains of the peptide are segregated and the hydrophobic domain(s) is capable of binding lipid core. The invention also contemplates receptor antagonists having enhanced bioactivity in association with micelles prepared by a method of the invention. A particularly preferred peptide for use according to the invention is VIP. Preferred micelles according to the invention are characterized by an average diameter of less than about 20 nm. According to one aspect of the invention the micelles further comprise calmodulin.

Claim 1 of 24 Claims

What is claimed is:

1. A method of preparing a biologically active micelle product comprising a biologically active amphipathic compound in association with a micelle; said method comprising the steps of:

a) mixing one or more lipids wherein said combination includes at least one lipid component covalently bonded to a water-soluble polymer;

b) forming sterically stabilized micelles from said combination of lipids;

c) incubating micelles from step (b) with a biologically active amphipathic compound under conditions in which said compound becomes associated with said micelles from step (b) in an active conformation.

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