United States Patent:  6,120,794

Assignee:  University of Pittsburgh (Pittsburgh, PA)

Filed:  September 26, 1995

New emulsion and micelle formulations are described as are complexes of these formulations with biologically active substances. The novel formulations are different from cationic lipid vectors such as cationic liposomes in that the complexes formed between biologically active substances and the emulsion and micellar formulations of this invention are physically stable and their transfection activity is resistant to the presence of serum. These novel formulations are disclosed to be useful in areas such as gene therapy or vaccine delivery.

SUMMARY OF INVENTION

The present invention relates to novel emulsion and micellar formulations useful for delivering biologically active substances to cells. The emulsion and micellar formulations of this invention are compatible with blood, retain activity in the presence of serum and are stable in storage. The emulsions of this invention comprise lipid components and an aqueous carrier, where the lipid components comprise an oil component, a cationic amphiphile component, preferably a cationic lipid, and a nonionic surfactant component. The micellar-formulations comprise lipid components and an aqueous carrier, where the lipid components comprise a cationic amphiphile component and a nonionic surfactant component. The lipid components of the emulsion and micellar formulation of the present invention may further comprise a neutral phospholipid component.

"Component" as used throughout the specification and claims is defined as: comprising at least one cationic amphiphile or a mixture of amphiphiles when used in the phrase "amphiphile component"; comprising at least one oil or a mixture of oils when used in the phrase "oil component"; comprising at least one nonionic surfactant or a mixture of nonionic surfactants when used in the phrase "nonionic surfactant component"; comprising at least one neutral phospholipid or a mixture of neutral phospholipids when used in the phrase "neutral phospholipid component".

The invention further relates to complexes formed by combining biologically active substances and the above-identified emulsion and micellar formulations. These biologically active substance:emulsion and biologically active substance:micelle complexes are stable over time and may have therapeutic and/or prophylactic utility in vivo depending on the activity of the biologically active substance contained in the complex.

This invention also provides a method for delivering a biologically active substance to cells by exposing cells to the complexes of this invention. In one embodiment, a method of exposing cells to a biologically active substance is provided, said method comprising culturing said cells in the presence of a biologically active substance:emulsion complex or a biologically active substance:micelle complex thereby facilitating delivery of the biologically active substance to cells.

The invention further provides a method of delivering a biologically active substance to cells in vivo comprising administering to an animal or human the complexes of this invention. It is to be understood that the complexes used for the delivery of biologically active substances to cells in vitro or in vivo may be freshly prepared by admixture or may be prepared earlier and stored prior to their use.

The invention further relates to a kit containing an emulsion or micellar formulation of the present invention. The invention also provides a kit containing a complex formed between a biologically active substance and an emulsion or micellar formulation of the present invention.

Methods for producing emulsion and micellar formulations according to the invention are also provided herein.

In one embodiment, a method for producing an emulsion formulation of this invention comprises

a) combining an organic solvent with an oil component, a cationic amphiphile component and a nonionic surfactant component;

b) removing the organic solvent to leave a lipid film; and

c) suspending the lipid film in an aqueous carrier to produce said emulsion formulation miscible in aqueous solution.

In an alternative embodiment, the oil may serve as the organic solvent in step (a) such that the method for producing an emulsion formulation of this invention comprises

a) combining an oil component, a cationic amphiphile component and a nonionic surfactant component; and

b) adding an aqueous carrier to the combination of step (a).

When a neutral phospholipid component is to be included in the emulsion, the neutral phospholipid component is combined with the above components in step (a).

The method for producing a micellar formulation miscible in aqueous solution comprises:

a) combining an organic solvent with a cationic amphiphile component and a nonionic surfactant component;

b) removing the organic solvent to leave a lipid film; and

c) suspending the lipid film in an aqueous carrier to produce said micellar formulation miscible in aqueous solution.

When a neutral phospholipid component is to be included in the micellar formulation, the neutral phospholipid component is combined with the above components in step (a).

Claim 1 of 21 Claims

1. An oil-in-water emulsion formulation comprising lipid components, a nucleic acid and an aqueous calmer, wherein the lipid components comprise an oil component, a cationic amphiphile component, and a nonionic surfactant component;

wherein the oil component is present in an amount from about 10 to about 80 weight % of the total lipid components in the formulation, the cationic amphiphile component is present in an amount from about 5 to about 80 weight % of the total lipid components and the nonionic surfactant component is present in an amount from about 5 to about 50 weight % of the total lipid components.

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