HK40081351B - Lipid vesicle compositions with penetration enhancing agents - Google Patents

Description

Lipid vesicle composition with penetration enhancer

Related applications

This application claims the priority of jointly pending U.S. Provisional Patent Application No. 62/904,606, filed September 23, 2019, and U.S. Provisional Patent Application No. 62/904,584, filed September 23, 2019, the contents of which are incorporated herein by reference in their entirety.

Technical Field

This technology typically relates to lipid vesicle formulations for the local delivery of therapeutic compounds, wherein the lipid vesicle formulation contains one or more penetration enhancers, such as one or more surfactants having 10 or less HLB.

Background Technology

The skin's barrier function prevents most external substances from penetrating the body. Most drugs are outside the optimal permeability range, thus requiring some type of facilitator to be therapeutically useful. The primary barrier controlling protein delivery through the skin is the outermost layer, the stratum corneum (SC). In mammalian skin, the SC (10–20 μm thick) consists of dead keratinized cells composed of cross-linked keratin and organized intercellular lipids within the bilayer. Below the SC is the active epidermis (50–100 μm), and deeper still is the dermis (1–2 mm), below which lies a rich capillary bed for drug absorption. The generally accepted size limit for molecules passively delivered through the skin is below 500 Da. Molecules larger than this have extremely low unassisted permeability through intact skin.

Various delivery methods have been developed to facilitate drug diffusion into or through the skin. Enhanced penetration through the skin can be achieved through physical methods (e.g., microneedles, thermal ablation), electrical methods (e.g., electroporation, iontophoresis), or chemical methods (e.g., chemical promoters). While the use of physical and electrical methods to enhance drug penetration through the skin has demonstrated some success in enhancing the delivery of both small and large molecules, significant hurdles remain to be overcome before approval. Several non-invasive delivery solvents for protein delivery have been developed, primarily lipid-based, such as liposomes, delivery bodies, lipid vesicles, and solid lipid nanoparticles. However, compared to other invasive techniques, these delivery systems can only deliver a limited number of proteins to different skin layers.

U.S. Patent Nos. 5,853,755 and 5,993,851 describe biphasic lipid vesicle compositions and methods for their preparation. U.S. Patent No. 5,993,852 describes a biphasic lipid vesicle composition for transdermal administration of immunogens.

Summary of the Invention

This disclosure includes a biphasic lipid vesicle composition comprising:

a) Lipid vesicles, each containing a lipid bilayer, the lipid bilayer containing vesicle-forming lipids.

b) An oil-in-water emulsion, which is embedded in two-phase lipid vesicles and stabilized by one or more surfactants;

c) One or more compounds, which are embedded in a lipid bilayer and/or an oil-in-water emulsion;

d) One or more penetration enhancers embedded in a lipid bilayer and/or an oil-in-water emulsion; wherein the one or more penetration enhancers are one or more nonionic surfactants with a hydrophilic-lipophilic balance (HLB) of about 10 or less.

This application also includes a biphasic lipid vesicle composition comprising:

a) Lipid vesicles, which consist of a lipid bilayer containing vesicle-forming lipids.

b) An oil-in-water emulsion, embedded in two-phase lipid vesicles and containing one or more polycationic surfactants; and

c) One or more compounds embedded in a lipid bilayer and/or an oil-in-water emulsion.

This application further includes a method for preparing the biphasic lipid vesicles of this disclosure, comprising:

a) Prepare an oil-in-water emulsion containing one or more surfactants by mixing the oil component of the oil-in-water emulsion with the aqueous component of the oil-in-water emulsion, wherein the oil component and/or aqueous component of the oil-in-water emulsion contain one or more surfactants.

b) Dissolve vesicles in an acceptable solvent other than water to form lipids;

c) Add one or more compounds and one or more penetration enhancers to the oil component and/or aqueous component of step a), and/or the dissolved vesicle-forming lipids of step b);

d) Adding an oil-in-water emulsion to dissolved vesicle-forming lipids; and

e) Mixing an oil-in-water emulsion and dissolved vesicle-forming lipids under mixed conditions that effectively form two-phase lipid vesicles, wherein the two-phase lipid vesicles comprise a lipid bilayer containing vesicle-forming lipids and an oil-in-water emulsion embedded in the two-phase lipid vesicles.

This application further includes a method of delivering one or more compounds by topically applying the biphasic lipid vesicle composition of this disclosure to the skin or mucous membrane of an object.

This application also includes a method for improving the local delivery of one or more compounds, comprising applying an effective amount of the biphasic lipid vesicle composition of this disclosure to the skin or mucous membrane of a subject in need.

This application further includes a method for treating or preventing skin conditions associated with excessive or deficient collagen production in a subject, comprising applying an effective amount of the lipid vesicle cosmetic composition of this disclosure to a subject in need.

This application further includes a method of treating a disease, condition, or illness treatable by delivering one or more therapeutic compounds by topically applying a therapeutically effective amount of the biphasic lipovesicular pharmaceutical composition of this disclosure to the skin or mucous membranes of a subject in need.

Other features and advantages of this application will become apparent from the following detailed description. However, it should be understood that while indicating embodiments of this disclosure, the detailed description and specific examples are given by way of illustration only, and the scope of the claims should not be limited to these embodiments, but should be given the broadest interpretation consistent with the description as a whole.

Attached Figure Description

Figures 1A and 1B show confocal microscopy images of human skin treated with Figure 1A), which shows exemplary peptide-lipovesicle formulations 1-4 containing rhodamine red-labeled 12-dimeric peptides (peptide molecular weight approximately 1200), FITC insulin (insulin molecular weight approximately 6,000), and FITC-IgG (IgG molecular weight approximately 150,000); and Figure 1B shows a separate control study incorporating Alexa647-labeled IgG (red fluorescence) into biphasic vesicles (contrast formulations); skin sections showing minimal fluorescence in the red channel across the entire epidermis and dermis, i.e., the first small image (three small images: first small image: red channel for Alexa IgG; second small image: general tissue staining (blue nuclear staining Syto 60); third small image: merged image); and the last small image: placebo-treated skin (merged image of red channel and general tissue staining), with no fluorescent background shown in the settings used for protein delivery analysis.

Figure 2 shows confocal microscopy images of mouse skin treated with the formulation of nucleic acid lipid vesicles F-TOM-1-5. For each formulation, three sub-images are shown: First sub-image: red channel for RFP expression (considered as light-colored areas in the epidermis and dermis); Second sub-image: general tissue staining (blue nuclear staining with Syto 60); Third sub-image: merged image.

Detailed Implementation

I. Definition

Unless otherwise stated, the definitions and embodiments described in this and other sections are intended to apply to all embodiments and aspects of this application, as will be understood by those skilled in the art.

The embodiments illustrated herein may be suitably implemented without any one or more elements, limitations, or restrictions not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing,” etc., should be read expansively and without limitation. Furthermore, the terms and expressions used herein have been used as descriptive terms rather than limitations, and are not intended to exclude any equivalents or portions thereof of the features shown and described, but it is recognized that various modifications may be made within the scope of the claimed technology. Additionally, the phrase “consistently composed of…” will be understood to include those specifically listed elements as well as additional elements that do not materially affect the essential and novel features of the claimed technology. The phrase “consisting of…” does not include any unspecified elements.

For example, as used in the application and claims, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”), or “containing” (and any form of containing, such as “contain” and “contains”) are inclusive or open-ended and do not exclude additional unlisted elements or process steps.

As used herein, the term "composition" and its derivatives are intended to be closed terms that specify the presence of the specified feature, element, component, group, integer and/or step, and exclude the presence of other unspecified features, elements, components, groups, integers and/or steps.

The phrase "consistently composed of..." will be understood to include those specifically listed elements as well as additional elements that do not materially affect the essential and novel features of the claimed technology. The phrase "consisting of..." does not include any unspecified elements.

As used herein, the term "and/or" means that the listed items are present or used individually or in combination. In practice, the term means the use or presence of "at least one" or "one or more" of the listed items. With respect to enantiomers, prodrugs, salts, and/or solvates thereof, the term "and/or" means that the compounds of this disclosure are present in combination as individual enantiomers, prodrugs, salts, and hydrates, as well as salts of solvates of, for example, the compounds of this disclosure.

In embodiments that include an “additional” or “second” component or effect (e.g., an additional or second compound), as used herein, the second compound is different from the other compounds or the first compound. A “third” compound is different from the other, first and second compounds, and the further listed or “additional” compounds are also different.

As used herein, “about” will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which it is used. If one of ordinary skill in the art is unaware of the use of the term, “about” will, given the context in which it is used, mean plus or minus 10% of the particular term.

In the context of describing elements (particularly in the context of the following claims), the terms “a,” “an,” and “the,” and similar references, will be interpreted to cover both singular and plural forms, unless otherwise stated herein or clearly contradicted by the context. Unless otherwise indicated herein, the enumeration of numerical ranges herein is intended only as a method of abbreviating each individual value falling within that range, and each individual value is incorporated into the specification as if it were separately stated herein. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. Unless otherwise indicated, the use of any and all instances or exemplary language (e.g., “such as”) provided herein is intended only to better elucidate the embodiments and does not constitute a limitation on the scope of the claims. No language in the specification should be construed as indicating that any unclaimed element is essential.

As used herein, the term "hydrophilic" refers to a compound or additive that is substantially water-soluble, water-dispersible, or generally capable of absorbing and/or transferring water.

The term "hydrophobic" as used refers to compounds or additives that are substantially insoluble in water or can be dispersed in water.

As used herein, the term "nucleic acid" or "oligonucleotide" refers to two or more covalently linked nucleotides. Unless the context clearly indicates otherwise, the term generally includes, but is not limited to, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which can be single-stranded (ss) or double-stranded (ds). For example, the nucleic acid molecules or polynucleotides of this disclosure may consist of: single-stranded DNA and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded RNA and double-stranded RNA, RNA as a mixture of single-stranded and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. Furthermore, nucleic acid molecules may consist of triple-stranded regions comprising RNA or DNA, or both RNA and DNA. As used herein, the term "oligonucleotide" generally refers to a nucleic acid of no more than 200 base pairs in length and may be single-stranded or double-stranded. The sequences provided herein may be DNA sequences, RNA sequences, or hybrid sequences; however, it should be understood that the sequences provided include both DNA and RNA, as well as complementary RNA and DNA sequences, unless the context clearly indicates otherwise. For example, the sequence 5’-GAATCC-3’ is understood to include 5’-GAAUCC-3’, 5’-GGATTC-3’, and 5’GGAUUC-3’. Nucleic acids or oligonucleotides may include naturally occurring bases, including adenine, guanine, cytosine, thymine, and uracil. These sequences may also contain modified bases. Examples of such modified bases include aza- and deazo-adenine, guanine, cytosine, thymine, and uracil; as well as xanthine and hypoxanthine, and others. As used herein, the term “isolated nucleic acid sequence” refers to a nucleic acid that is substantially free of cellular material or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Isolated nucleic acids are also substantially free of sequences with nucleic acid flanking from which the nucleic acid is derived (i.e., sequences located at the 5’ and 3’ ends of the nucleic acid). Nucleic acids can be, for example, plasmid DNA, viral vectors, naked DNA, RNA, DNA/RNA hybrids, and synthetic nucleic acids.

As used herein, the terms “peptide,” “polypeptide,” and “protein” refer to any chain of two or more natural or non-natural amino acid residues, regardless of post-translational modifications (e.g., glycosylation or phosphorylation). Polypeptides incorporated into biphasic vesicles of this disclosure may include, for example, 3 to 3,500 natural or non-natural amino acid residues. Proteins include both single-peptide chains and multi-subunit proteins (e.g., composed of two or more polypeptides).

The term "amino acid" includes all naturally occurring amino acids as well as modified L-amino acids. The atoms of an amino acid can include, for example, different isotopes. For instance, an amino acid can include deuterium replacing hydrogen, nitrogen-15 replacing nitrogen-14, carbon-13 replacing carbon-12, and other similar variations.

As used in this article, "immunogen" refers to a substance that, when administered to a subject, triggers an immune response and leads to the production of antibodies, activation of lymphocytes or other reactive immune cells targeting the antigenic portion of the immunogen.

As used herein, the term "antibody" is intended to include monoclonal antibodies, polyclonal antibodies, single-chain, humanized, and other chimeric antibodies and their binding fragments. Antibodies may be derived from recombinant sources and/or produced in transgenic animals. Human antibodies that can be produced using biochemical techniques or isolated from libraries are also included. Humanized or chimeric antibodies may include sequences derived from one or more homotypes or classes.

As used herein, the term "binding fragment" refers to a portion or fraction of an antibody or antibody chain containing fewer amino acid residues than a complete or whole antibody or antibody chain, and which binds to an antigen or competes with a complete antibody. Exemplary binding fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, nanobodies, small bodies, dimers, and their multimers. Fragments can be obtained by chemical or enzymatic treatment of a complete or whole antibody or antibody chain. Fragments can also be obtained by recombinant methods. For example, an F(ab')2 fragment can be generated by treating an antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bonds, producing a Fab' fragment. Papain digestion can lead to the formation of Fab fragments. Fab, Fab', and F(ab')2, scFv, dsFv, ds-scFv, dimers, small bodies, dimers, bispecific antibody fragments, and other fragments can also be constructed using recombinant expression techniques.

Furthermore, the definitions and embodiments described in particular sections are intended to apply to other embodiments described herein, as will be understood by those skilled in the art. For example, in the following paragraphs, different aspects are defined in more detail. Each aspect thus defined may be combined with any other one or more aspects unless the contrary is explicitly stated. In particular, any feature indicated as preferred or advantageous may be combined with any other feature or feature indicated as preferred or advantageous.

As used herein, the term "compositions of the present disclosure (multiple)" refers to compositions comprising the biphasic lipid vesicles described herein.

As used herein, the term "penetration enhancer" refers to one or more nonionic surfactants or polycationic surfactants with a hydrophilic-lipophilic balance (HLB) of about 10 or less. In one embodiment, one or more penetration enhancers are one or more nonionic surfactants with an HLB of about 10 or less, which are combined with one or more penetration enhancers selected from one or more terpenes, alkaloids, salicylic acid derivatives and polycationic surfactants and combinations thereof.

As used herein, the term "embedded" refers to the non-covalent association of the reagent with one or more lipid bilayers of a biphasic lipid vesicle, the central core of a biphasic lipid vesicle, and/or one or more spaces between adjacent bilayers of a biphasic lipid vesicle.

As used herein, the term "biphase lipid vesicle" refers to a vesicle whose central core compartment is occupied by an oil-in-water emulsion, which consists of an aqueous continuous phase and a dispersed hydrophobic, hydrophilic, or oily phase. In one embodiment, the space between adjacent bilayers of the biphase lipid vesicle may also be occupied by an emulsion.

As used in this article, the term "emulsion" refers to a mixture of two immiscible substances.

As used herein, the term "bilayer" refers to a structure consisting of two molecular layers of amphiphilic lipid molecules arranged in a manner that includes a hydrophobic tail on the inside and a polar head group on the outside.

As used herein, the terms “topical application” or “topical delivery” refer to the intradermal, transdermal, and/or transmucosal delivery of a compound by applying a composition comprising one or more compounds to the skin and/or mucous membranes.

As used herein, the term "gemini surfactant" refers to a surfactant molecule comprising more than one hydrophobic tail, each hydrophobic tail having a hydrophilic head, wherein the hydrophobic or hydrophilic tails are connected together by spacer groups. The hydrophobic tails may be the same or different. Similarly, the hydrophilic heads may be the same or different. The hydrophilic heads may be anionic, cationic, or neutral.

The term "HLB" or "hydrophilic-lipophilic balance" value refers to the HLB standard according to Griffin, J. Soc. Cosm. Chem., vol. 5, 249 (1954), which indicates the degree of hydrophilicity and lipophilicity of a surfactant.

As used herein, the term "object" includes all members of the animal kingdom, including mammals, and appropriately refers to humans. Therefore, the methods and uses of this application are suitable for human therapeutic and cosmetic applications as well as veterinary applications.

As used herein, the terms “treating” or “treatment,” and as is well known in the art, refer to a method of obtaining a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, alleviating or improving one or more symptoms or conditions, reducing the severity of disease, stabilizing (i.e., not worsening) a disease state, preventing the spread of disease, delaying or slowing disease progression, improving or alleviating a disease state, reducing disease relapses, and achieving remission (whether partial or complete), whether detectable or undetectable. “Treating” and “treatment” also mean extended survival compared to expected survival without treatment. As used herein, “treating” and “treatment” also include preventative treatment. For example, a subject with a skin disease, condition, or ailment may be treated to prevent progression. Treatment methods include administering a therapeutically effective amount of one or more compounds of this disclosure to a subject, and optionally consist of a single administration, or alternatively include a series of administrations.

As used herein, the term "effective dose" or "therapeutic effective dose" refers to the effective dose and time period required to achieve the desired outcome. As used herein, the terms "to treat," "treating," and "treatment," and as is well known in the art, refer to a method for obtaining a beneficial or desired outcome, including clinical outcomes. "To treat," "treating," and "treatment" can also mean extended survival compared to expected survival without treatment. As used herein, "to treat," "treating," and "treatment" also include prophylactic treatment.

When the features or aspects of this disclosure are described in accordance with the Markush Group, those skilled in the art will recognize that this disclosure is also described in accordance with any individual member of the Markush Group or a subgroup of its members.

Furthermore, the definitions and embodiments described in particular sections are intended to apply to other embodiments described herein, as will be understood by those skilled in the art. For example, in the following paragraphs, different aspects are defined in more detail. Each aspect so defined may be combined with any other one or more aspects unless the contrary is explicitly stated. For example, any combination of members of any group may be combined, and optionally, may be combined with a subgroup of any other member. In particular, any feature indicated as preferred or advantageous may be combined with any other feature or feature indicated as preferred or advantageous.

As those skilled in the art will understand, for any and all purposes, particularly for the purpose of providing a written description, all scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. Any listed scope can be readily identified as sufficiently descriptive, and the same scope can be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can be readily decomposed into a lower third, a middle third, and an upper third. As those skilled in the art will also understand, all language, such as “up to,” “at least,” “greater than,” “less than,” etc., includes the listed numbers and refers to a scope that can subsequently be decomposed into subscopes as discussed above. Finally, as those skilled in the art will understand, a scope includes each individual member.

II. The disclosed composition

The applicant has demonstrated that biphasic phospholipid vesicles enhance the skin penetration of compounds having a phospholipid bilayer that isolates a stable oil-in-water emulsion and the compound, the compound comprising one or more penetration enhancers (e.g., compositions and/or products comprising the biphasic vesicles described herein) added to both parts of the phospholipid bilayer or the stable oil-in-water emulsion or delivery system.

The applicant has demonstrated that, in contrast to other combinations, certain penetration enhancers and combinations of penetration enhancers and compounds can be used to more effectively deliver higher amounts of compounds (e.g., in milligrams) to a given amount of skin (e.g., in grams).

The penetration enhancer compound can be selected from a variety of compounds commonly referred to as penetration enhancers. In one embodiment, the applicant has shown that a penetration enhancer, such as a nonionic surfactant with a hydrophilic-lipophilic balance (“HLB”) of 10 or less, or alone or in combination with one or more penetration enhancers (e.g., terpenes, alkaloids, salicylic acid derivatives, polycationic (e.g., dicationic, tricationic, etc.) surfactants such as gemini cationic surfactants or polycationic amino acids or combinations thereof), enhances skin penetration compared to other compositions that are identical or similar in aspects (except for the absence of one or more penetration enhancers).

In another embodiment, the applicant has shown that polycationic surfactants, such as, for example, gemini dicationic surfactants or polycationic amino acids, enhance the skin penetration of compounds relative to other similar or identical compositions (except for the use of monocationic surfactants instead of polycationic surfactants).

Therefore, this application includes a biphasic lipid vesicle composition comprising:

a) Lipid vesicles, each containing a lipid bilayer, the lipid bilayer containing vesicle-forming lipids.

b) An oil-in-water emulsion, embedded in two-phase lipid vesicles and stabilized by one or more surfactants;

c) One or more compounds, embedded in a lipid bilayer or oil-in-water emulsion of two-phase vesicles; and

d) One or more permeation enhancers embedded in a lipid bilayer or oil-in-water emulsion of two-phase vesicles.

in liquid,

The one or more penetration enhancers are one or more nonionic surfactants with a hydrophilic-lipophilic balance (HLB) of about 10 or less.

In one embodiment, the biphasic lipovesicle composition is a cosmetic composition. In another embodiment, the biphasic lipovesicle composition is a pharmaceutical composition.

In one embodiment, a pharmaceutical composition (described herein as a lipovesicle composition) is provided for topical application of a therapeutic compound to achieve local delivery, the composition comprising: lipovesicles; an oil-in-water emulsion; a therapeutic compound; and one or more penetration enhancers; wherein the lipoves comprise an outer lipid bilayer; the oil-in-water emulsion is coated by the outer lipid bilayer; the therapeutic compound is, for example, a small molecule peptide or protein; and the one or more penetration enhancers increase the amount of the therapeutic compound absorbed into a given amount of skin, relative to the composition in the absence of the one or more penetration enhancers.

The applicant has demonstrated that lipid vesicles can be formulated by selectively incorporating compounds and/or penetration enhancers into lipid bilayers and/or oil-in-water emulsions at different stages of biphase lipid vesicle production. For example, during the production of biphase lipid vesicles, the compound may be added only to the oil-in-water emulsion, only to the lipid bilayer component, or to both the oil-in-water emulsion and the lipid bilayer. Similarly, during the production of biphase lipid vesicles, one or more penetration enhancers may be added only to the oil-in-water emulsion, only to the lipid bilayer, or to both the oil-in-water emulsion and the lipid bilayer.

In one embodiment, the biphasic lipid vesicle composition is used for the local delivery of one or more compounds. In one embodiment, local delivery is for intradermal delivery, transdermal delivery, mucosal delivery, or transmucosal delivery.

In one embodiment, the biphase lipid vesicle composition comprises a suspension of biphase lipid vesicles.

In one embodiment, one or more permeation enhancers are embedded in an oil-in-water emulsion of biphasic lipid vesicles. In one embodiment, the oil-in-water emulsion of biphasic lipid vesicles contains about 0.01 wt% to about 20 wt% of one or more permeation enhancers. In one embodiment, the oil-in-water emulsion of biphasic lipid vesicles contains about 0.1 wt% to about 10 wt% of one or more permeation enhancers. In one embodiment, the oil-in-water emulsion of biphasic lipid vesicles contains about 0.5 wt% to about 9 wt%, about 0.5 wt% to about 8 wt%, about 0.5 wt% to about 7 wt%, about 1 wt% to about 6 wt%, about 1 wt% to about 5 wt%, about 1 wt% to about 4 wt%, about 1 wt% to about 3 wt%, or about 1 wt% to about 2 wt% of one or more permeation enhancers.

In one embodiment, one or more penetration enhancers are embedded in the lipid bilayer of a lipovesicle. In one embodiment, the lipid bilayer of the lipovesicle composition comprises 0.1 wt% to 20 wt% of one or more penetration enhancers. In one embodiment, the lipid bilayer comprises 0.1 wt% to 10 wt% of one or more skin penetration enhancers. In one embodiment, the lipid bilayer of a biphasic lipoves comprises about 7 wt% of one or more skin penetration enhancers. In one embodiment, the lipid bilayer of a lipoves comprises about 10 wt%, about 9 wt%, about 8 wt%, about 7 wt%, about 6 wt%, about 5 wt%, about 4 wt%, about 3 wt%, about 2 wt%, about 1 wt%, about 0.5 wt%, or about 0.1 wt% of one or more skin penetration enhancers.

In one embodiment, one or more permeation enhancers are embedded in both the lipid bilayer and the oil-in-water emulsion of the two-phase lipid vesicles.

In one embodiment, the penetration enhancer is one or more nonionic surfactants with a hydrophilic-lipophilic balance (HLB) of about 10 or less, selected from one or more of polyethylene glycol ethers of fatty alcohols, sorbitan esters, polysorbates, sorbitan esters, and polyethylene glycol fatty acid esters and combinations thereof.

In one embodiment, the polyethylene glycol ether of the fatty alcohol is selected from Oleth and combinations thereof. In one embodiment, the polyethylene glycol ether of the fatty alcohol is selected from Oleth, and in another embodiment, the polyethylene glycol ether of the fatty alcohol is Oleth.

In one embodiment, the sorbitan ester is selected from sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trioleate, sorbitan sesquioleate, and sorbitan isostearate, and combinations thereof. In one embodiment, the sorbitan ester is selected from sorbitan monolaurate, sorbitan monopalmitate, and sorbitan monostearate, and combinations thereof. In one embodiment, the sorbitan ester is sorbitan monopalmitate.

In one embodiment, the polyethylene glycol fatty acid ester is selected from one or more PEG-8 dilaurate, PEG-4 dilaurate, PEG-4 laurate, PEG-8 dioleate, PEG-8 distearate, PEG-8 distearate, PEG-7 glyceryl cocoate, and PEG-20 almond glyceride, and combinations thereof. In one embodiment, the polyethylene glycol fatty acid ester is selected from PEG-4 dilaurate, PEG-4 laurate, and combinations thereof. In one embodiment, the polyethylene glycol fatty acid ester is PEG-4 dilaurate.

In one embodiment, the hydrophilic-lipophilic balance (HLB) is one or more nonionic surfactants of about 10 or less, further selected from propylene glycol isostearate, ethylene stearate, glyceryl stearate, glyceryl stearate SE, glyceryl laurate, glyceryl caprylate, PEG-30 dihydroxystearate, ethylene distearate, and combinations thereof.

In one embodiment, the hydrophilic-lipophilic balance (HLB) is one or more nonionic surfactants selected from the surfactants in Table 1:

Table 1

and their combinations.

In one embodiment, one or more penetration enhancers are one or more nonionic surfactants with an HLB of about 10 or less, which are combined with one or more penetration enhancers selected from one or more terpenes, alkaloids, salicylic acid derivatives and dicationic or polycationic surfactants and combinations thereof.

In one embodiment, HLB is about 10 or less of one or more nonionic surfactants as described above.

In one embodiment, one or more terpenes are selected from one or more eugenol, d-limonene, menthol, menthone, farnesol, neridol, camphor, nerol, and thymol, and combinations thereof. In another embodiment, one or more terpenes are selected from one or more menthol, camphor, nerol, and thymol, and combinations thereof.

In one embodiment, one or more salicylic acid derivatives are selected from ethyl salicylate, salicylic acid, acetylsalicylic acid, and triethanolamine salicylate. In one embodiment, the salicylic acid derivative is methyl salicylate.

In one embodiment, one or more alkaloids are selected from piperidine derivatives (e.g., piperine and lobeline), purine derivatives (e.g., caffeine, theobromine, and theophylline), pyridine derivatives (e.g., nicotine), colchicine, pyrrolidine derivatives (e.g., N-methylpyrrolidone and salicylic acid), benzylamine (e.g., capsaicin), isoquinoline derivatives (e.g., berberine and sanguisorbine), or imidazole derivatives (e.g., histamine and pilocarpine). In one embodiment, one or more alkaloids are piperidine derivatives. In one embodiment, one or more alkaloids are piperine or lobeline, or combinations thereof. In one embodiment, one or more alkaloids are piperine.

In one embodiment, the polycationic surfactant is one or more gemini surfactants.

Gemini surfactants are surfactant molecules containing more than one hydrophobic tail. Each hydrophobic tail has a hydrophilic head (Menger and Keiper, 2000; Kirby et al., 2003). The hydrophobic or hydrophilic tails are linked together by spacer groups. The hydrophobic tails can be the same or different. Similarly, the hydrophilic heads can be the same or different. Furthermore, the hydrophilic heads can be anionic (e.g., phosphate, sulfate, or carboxylate type), cationic (e.g., quaternary ammonium type), or neutral (e.g., polyether, peptide, or sugar type) (Menger and Keiper, 2000). In aqueous solutions, gemini surfactants spontaneously aggregate into micelles, the shape and size of which are particularly sensitive to the length of the spacer groups and their hydrophobic or hydrophilic properties. The spacer group can be variable, i.e., short (e.g., 2 methylene groups) or long (e.g., more than 12 methylene groups); rigid (e.g., stilbene) or flexible (e.g., methylene chain); and polar (e.g., polyether, ethoxy, or polyethoxy) or nonpolar (e.g., aliphatic, aromatic) (Menger and Keiper, 2000). Because the hydrophobic tail, hydrophilic head, and spacer group can differ in these respects, countless different molecules can be designed.

In one embodiment, the hydrophobic tail is of the _C3 - _C30 alkyl type, linear or branched, saturated or unsaturated. In one embodiment, the hydrophilic head can be anionic, cationic or neutral. In one embodiment, the hydrophilic head is cationic.

In one embodiment, the gemini surfactant is anionic, cationic, or neutral. In one embodiment, the polycationic surfactant is one or more gemini dicationic surfactants.

In one embodiment, the gemini surfactant comprises a linear hydrocarbon tail group and a quaternary ammonium head group. A typical structure of a type of gemini cationic surfactant includes a head group consisting of two positively charged nitrogen atoms separated by spacer groups (n) of 3, 4, 6, 8, 10, 12, or 16 carbon atoms, each spacer group containing two methyl groups, and a tail consisting of two saturated chains of 12 or 16 carbon atoms (m = 10 or 14).

In one embodiment, one or more gemini-cationic surfactants are of the quaternary ammonium type. In one embodiment, one or more gemini-cationic surfactants are selected from 12-7NH-12, _12-7NCH3-12, 16-3-16, 12-4(OH) _2-12 , and 12-EO1-12. In one embodiment, one or more gemini-cationic surfactants are selected from 12-7NH-12, _12-7NCH3-12 , and 16-3-16.

In one embodiment, one or more polycationic surfactants are polycationic amino acids. In one embodiment, the polycationic amino acid is selected from polylysine, polyarginine, and combinations thereof.

In one embodiment, one or more penetration enhancers are one or more nonionic surfactants with an HLB of about 10 or less, which are combined with one or more penetration enhancers selected from one or more terpenes, alkaloids and salicylic acid derivatives.

In one embodiment, the biphasic lipovesicle composition comprises one to six permeation enhancers. In one embodiment, the biphasic lipovesicle composition comprises one to four permeation enhancers. In one embodiment, the biphasic lipovesicle composition comprises one to three permeation enhancers.

In one embodiment, the penetration enhancer is one or more nonionic surfactants with an HLB of about 9 or less, about 8 or less, about 7 or less, or about 6 or less, and optionally with an HLB of 1 or more, 2 or more, 3 or more, or 4 or more, or any combination thereof (e.g., about 7 or less and about 3 or more). In one embodiment, the penetration enhancer is one or more nonionic surfactants with an HLB of about 1 to about 10, about 1 to about 9, about 2 to about 8, about 3 to about 7, or about 4 to about 7. In one embodiment, the penetration enhancer is one or more nonionic surfactants with a hydrophilic-lipophilic balanced (HLB) of about 3 to about 7 or about 4 to about 7. In one embodiment, the penetration enhancer is one or more nonionic surfactants with an HLB of about 4 to about 7.

In one embodiment, the penetration enhancer is (diethylene glycol monooleyl ether). In one embodiment, the penetration enhancer is a combination with one or more terpenes. In one embodiment, the penetration enhancer is a combination with one or more of menthol, camphor, nerol, or thymol, or combinations thereof. In one embodiment, the penetration enhancer is a combination with menthol or camphor, or combinations thereof. In one embodiment, the penetration enhancer is a combination with menthol and camphor. In one embodiment, the penetration enhancer is a combination with nerol. In one embodiment, the penetration enhancer is a combination with thymol. In one embodiment, the penetration enhancer is a combination with nerol. In one embodiment, the penetration enhancer is a combination with methyl salicylate. In one embodiment, the penetration enhancer is a combination with one or more alkaloids. In one embodiment, the penetration enhancer is a combination with piperidine.

In one embodiment, one or more nonionic surfactants with an HLB of about 10 or less are embedded in a lipid bilayer, and one or more terpenes or one or more alkaloids are embedded in the lipid bilayer, oil-in-water emulsion, or both.

In one embodiment, one or more penetration enhancers are PEG-4 dilaurate. In one embodiment, one or more penetration enhancers are a combination of PEG-4 dilaurate and one or more alkaloids. In one embodiment, one or more penetration enhancers are a combination of PEG-4 dilaurate and piperidine. In one embodiment, one or more penetration enhancers are a combination of PEG-4 dilaurate and methyl salicylate.

In one embodiment, PEG-4 dilaurate is embedded in a lipid bilayer, and one or more alkaloids or methyl salicylate are embedded in the lipid bilayer, oil-in-water emulsion, or both.

In one embodiment, one or more penetration enhancers are Oleth-2, PEG-4 dilaurate, or sorbitan monopalmitate, or combinations thereof. In one embodiment, one or more penetration enhancers are a combination of Oleth-2 and sorbitan monopalmitate. In one embodiment, one or more penetration enhancers are a combination of PEG-4 dilaurate and sorbitan monopalmitate.

In one embodiment, PEG-4 dilaurate or sorbitan monopalmitate or a combination thereof is embedded in a lipid bilayer, an oil-in-water emulsion, or both.

In one embodiment, relative to other identical or similar compositions (except for the absence of one or more penetration enhancers), one or more penetration enhancers increase the amount of compound absorbed into a given amount of skin by at least 10%, 20%, 30%, 40%, or 50%. In another embodiment, relative to other identical or similar compositions (except for the absence of one or more penetration enhancers), one or more penetration enhancers increase the amount of compound absorbed into a given amount of skin by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

In one embodiment, the biphasic lipid vesicle contains about 0.1 wt% to about 5 wt% of alkaloids. In one embodiment, the biphasic lipid vesicle contains about 0.1 wt% to about 4 wt% of alkaloids. In one embodiment, the biphasic lipid vesicle contains about 0.1 wt% to about 3 wt% of alkaloids. In one embodiment, the biphasic lipid vesicle contains about 1 wt% to about 3 wt% of alkaloids. In one embodiment, the lipid bilayer of the lipid vesicle contains 1 wt% to 5 wt% of alkaloids. In some embodiments, the alkaloids are embedded in the lipid bilayer of the biphasic lipid vesicle.

Typically, biphasic lipid vesicles are multilayered lipid vesicles, further comprising one or more internal lipid bilayers. Multilayered biphasic lipid vesicles with multiple concentric lipid bilayer shells encapsulate oil-in-water emulsions.

In one embodiment, the oil-in-water emulsion comprises droplets with an average diameter of less than 1 μm. In one embodiment, the average diameter of the oil-in-water emulsion droplets may be less than 0.5 μm, 0.25 μm, 0.1 μm, or 0.01 μm. In another embodiment, the average diameter of the oil-in-water emulsion droplets may be less than about 0.5 μm, less than about 0.25 μm, less than about 0.1 μm, or less than about 0.01 μm. Because the oil-in-water emulsion contains both aqueous and non-aqueous regions, these submicron oil-in-water emulsion droplets can be modified to incorporate hydrophilic and hydrophobic compounds and excipients.

In one embodiment, the oil-in-water emulsion comprises 40 wt% to 99.9 wt% water. In another embodiment, the oil-in-water emulsion comprises 10 wt% to 95 wt% water, for example, 10 wt% to 25 wt%, 25 wt% to 50 wt%, 50 wt% to 75 wt%, or 75 wt% to 95 wt% water. In one embodiment, the oil-in-water emulsion comprises about 10 wt% to about 99.9 wt% water, about 15 wt% to about 99.9 wt% water, about 25 wt% to about 99.9 wt% water, about 25 wt% to about 50 wt% water, about 40 wt% to about 99 wt% water, about 50 wt% to about 95 wt% water, about 50 wt% to about 75 wt% water, or about 75 wt% to about 95 wt% water.

In one embodiment, the oil-in-water emulsion comprises 0.1 wt% to 60 wt% of oil. In another embodiment, the oil-in-water emulsion comprises about 0.1 wt% to about 60 wt% of oil, about 0.5 wt% to about 50 wt% of oil, about 1 wt% to about 40 wt% of oil, or about 1 wt% to about 20 wt% of oil.

In one embodiment, the oil-in-water emulsion may comprise up to about 95 wt% of the biphasic lipovesicles. In other words, in one embodiment, the biphasic lipovesicles comprise about 1 wt% to about 95 wt% of an oil-in-water emulsion. In one embodiment, the lipovesicle composition may comprise 1 wt% to 10 wt%, 20 wt% to 30 wt%, 30 wt% to 40 wt%, or 40 wt% to 95 wt% of an oil-in-water emulsion. In one embodiment, the lipovesicles comprise about 1 wt% to about 10 wt%, about 20 wt% to about 30 wt%, about 30 wt% to about 40 wt%, about 40 wt% to about 95 wt%, about 50 wt% to about 95 wt%, about 60 wt% to about 95 wt%, or about 70 wt% to about 95 wt% of an oil-in-water emulsion.

In one embodiment, the oil in the oil-in-water emulsion is selected from vegetable oils, monoglycerides, diglycerides and triglycerides, silicone oils and mineral oils, and combinations thereof. It should be understood that the oil-in-water emulsion can be adjusted to have different amounts of water and oil to optimize the solubility of any given compound, permeation-enhancing compound, surfactant and/or emulsifier, etc.

The oil-in-water emulsion of biphase lipid vesicles is stabilized by one or more surfactants. In one embodiment, the oil-in-water emulsion of biphase lipid vesicles comprises 0.01 wt% to 40 wt% of one or more surfactants. Without being theoretically constrained, it is contemplated that surfactants can be added to the oil-in-water emulsion to modify its stability. In one embodiment, the water-in-oil emulsion comprises 0.01 wt% to 10 wt%, 10 wt% to 20 wt%, or 20 wt% to 40 wt% of one or more surfactants. In one embodiment, the water-in-oil emulsion comprises about 0.01 wt% to about 40 wt%, about 0.01 wt% to about 10 wt%, about 10 wt% to about 20 wt%, about 20 wt% to about 30 wt%, about 20 wt% to about 40 wt%, or about 30 wt% to about 40 wt% of one or more surfactants.

In one embodiment, the oil-in-water emulsion of the two-phase lipid vesicles is stabilized by one or more surfactants selected from polyethylene glycol ethers of fatty alcohols, polyethylene glycol fatty acid esters, polysorbate esters, and dehydrated sorbate esters. In one embodiment, the average hydrophilic-lipophilic balance (HLB) number of the one or more surfactants is greater than 10 or more. In one embodiment, the one or more surfactants in the oil-in-water emulsion have an HLB of greater than 10 or more, about 11 or more, about 12 or more, about 13 or more, about 14 or more, about 15 or more, about 16 or more, about 17 or more, about 18 or more, about 19 or more, or about 20 or more, or combinations thereof. In one embodiment, the one or more surfactants in the oil-in-water emulsion have an HLB of greater than 10 to about 20, about 10 to about 18, about 10 to about 16, or about 10 to about 15. In one embodiment, the one or more surfactants in the oil-in-water emulsion have an HLB of about 10 to about 16. In one embodiment, one or more surfactants in the oil-in-water emulsion have an HLB of 10-20 or 10-16.

In one embodiment, one or more nonionic surfactants with a hydrophilic-lipophilic balance (HLB) greater than 10 are selected from the surfactants in Table 2:

Table 2

In one embodiment, the oil-in-water emulsion of the two-phase lipid vesicles is stabilized by one or more surfactants selected from Tween (polysorbate 80 (ethylene glycol)/polyoxyethylene 20 dehydrated sorbitan monooleate).

The hydrophilic-lipophilic balance (HLB) of the penetration enhancer is about 10 or less. One or more nonionic surfactants are not used for the stabilization and emulsification of oil-in-water emulsions. Instead, the hydrophilic-lipophilic balance (HLB) of the penetration enhancer is used as an additional surfactant to the stabilizing surfactant to provide a penetration-enhancing effect.

It should also be understood that, in contrast to known biphasic vesicle compositions in which lipid vesicles contain surfactants as stabilizing structural components for creating oil-in-water emulsions, this disclosure uses one or more penetration enhancers that, when incorporated into vesicle structures (lipid bilayers or oil-in-water emulsions), can enhance the delivery capabilities of a range of compounds.

In one embodiment, the oil-in-water emulsion comprises 10 wt% to 99 wt% water, 0.5 wt% to 60 wt% oil, and further comprises 0.01 wt% to 20 wt% of one or more surfactants for stabilizing the oil-in-water emulsion.

In one embodiment, the vesicle-forming lipid is an amphiphilic lipid having a hydrophobic tail and a head group, which can spontaneously form bilayer vesicles in water. In one embodiment, the vesicle-forming lipid comprises two hydrocarbon chains, such as acyl chains, wherein the head group is polar or nonpolar. In one embodiment, the vesicle-forming lipid is selected from phospholipids, glycolipids, lecithin, and ceramides, such as phosphatidylethanolamine, lysophosphatidylethanolamine, lysophosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin, phosphatidic acid, and cerebrosides. These lipids are commercially available or can be prepared according to published methods.

In one embodiment, the vesicle-forming lipid is a phospholipid. In one embodiment, the phospholipid is an ester of glycerol with one or two (identical or different) fatty acid residues and a phosphate group, wherein the phosphate residue is sequentially bound to a hydrophilic group, such as, for example, choline (phosphatidylcholine-PC), serine (phosphatidylserine-PS), glycerol (phosphatidylglycerol-PG), ethanolamine (phosphatidylethanolamine-PE), or inositol (phosphatidylinositol). Esters of phospholipids containing only one fatty acid residue are commonly referred to in the art as the "lysosomal" form of phospholipids or "lysophospholipids." The fatty acid residues present in phospholipids are typically long-chain fatty acids, typically containing 12 to 24 carbon atoms, or 14 to 22 carbon atoms; the aliphatic chain may contain one or more degrees of unsaturation or be fully saturated. Examples of suitable fatty acids included in phospholipids are, for example, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, and linolenic acid. Saturated fatty acids, such as myristic acid, palmitic acid, stearic acid, and arachidic acid, can be used.

In one embodiment, the phospholipid is phosphatidic acid, i.e., a diester of glycerophosphate and fatty acid; sphingolipid, such as sphingomyelin, i.e., those phosphatidylcholine analogs in which the residues of the diglyceride of fatty acid are replaced by a ceramide chain; cardiolipin, i.e., an ester of 1,3-diphosphatidylglycerol and fatty acid; glycolipid, such as ganglioside GM1 (or GM2) or cerebroside; glycolipid; thioglycoside and sphingolipid.

In one embodiment, the phospholipid is a naturally occurring, semi-synthetic, or synthetically prepared product that can be used alone or as a mixture. In one embodiment, the naturally occurring phospholipid is natural lecithin (a phosphatidylcholine (PC) derivative), such as, typically, soybean or egg yolk lecithin.

In one embodiment, the semi-synthetic phospholipid is a partially or fully hydrogenated derivative of naturally occurring lecithin. In one embodiment, the phospholipid includes fatty acid diesters of phosphatidylcholine, ethylphosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, or sphingomyelin. In one embodiment, the phospholipid is, for example, dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), arachidonicyl phosphatidylcholine (DAPC), distearyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), 1,2-distearyl-sn-glycerol-3-ethylphosphatidylcholine (ethyl-DSPC), dipentadecanoyl phosphatidylcholine (DPDPC), 1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl-phosphatidylcholine... Alkali salts (PMPC), 1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), 1-oleoyl-2-palmitoyl-phosphatidylcholine (OPPC), dilauroyl phosphatidylglycerol (DLPG) and their alkali metal salts, arachidoyl phosphatidylglycerol (DAPG) and their alkali metal salts, dimyristoyl phosphatidylglycerol (DMPG) and their alkali metal salts, dipalmitoyl phosphatidylglycerol (DPPG) and their alkali metal salts, distearyloyl phosphatidylglycerol (DSPG) and their alkali metal salts. Alkali metal salts, dioleoylphosphatidylglycerol (DOPG) and its alkali metal salts, dimyristoylphosphatidyl acid (DMPA) and its alkali metal salts, dipalmitoylphosphatidyl acid (DPPA) and its alkali metal salts, distearylphosphatidyl acid (DSPA), arachidoylphosphatidyl acid (DAPA) and its alkali metal salts, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), arachidoylphosphatidylethanolamine (DAPE), dilinoleylphosphatidylethanolamine (DLPE), dimyristoylphosphatidylethanolamine (DLPE), dimyristoylphosphatidylethanolamine (DM ... Myristoyl phosphatidylserine (DMPS), arachidoyl phosphatidylserine (DAPS), dipalmitoyl phosphatidylserine (DPPS), distearyl phosphatidylserine (DSPS), dioleoyl phosphatidylserine (DOPS), dipalmitoyl sphingomyelin (DPSP), distearyl sphingomyelin (DSSP), dilauroyl phosphatidylinositol (DLPI), arachidoyl phosphatidylinositol (DAPI), dimyristoyl phosphatidylinositol (DMPI), dipalmitoyl phosphatidylinositol (DPPI), distearyl phosphatidylinositol (DSPI), dioleoyl phosphatidylinositol (DOPI).

In one embodiment, the phospholipid is dioleoylphosphatidylethanolamine (DOPE), phosphatidylethanolamine (cephalin) (PE), phosphatidic acid (PA), phosphatidylcholine (PC), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine (DSPE), or phosphatidylserine (PS).

In one embodiment, the biphasic lipovesies of the biphasic lipovesies composition typically comprise 0.1 wt% to 30 wt% of phospholipids. In some embodiments, the lipovesies comprise 1 wt% to 10 wt%, 10 wt% to 20 wt%, or 20 wt% to 30 wt% of phospholipids. In some embodiments, the biphasic lipovesies comprise 9 wt% to 13 wt% of phospholipids. In some embodiments, the biphasic lipovesies comprise 10 wt% of phospholipids. In some embodiments, the biphasic lipovesies comprise 12 wt% of phospholipids. In some embodiments, the biphasic lipovesies comprise about 1 wt% to about 10 wt%, about 10 wt% to about 20 wt%, about 20 wt% to about 30 wt%, about 9 wt% to about 13 wt%, about 13 wt%, about 12 wt%, about 11 wt%, or about 10 wt% of phospholipids.

In one embodiment, one or more compounds are encapsulated in an oil-in-water emulsion of two-phase lipid vesicles. In one embodiment, the oil-in-water emulsion comprises 1 ng/g to 1,000 ng/g of the compound/oil-in-water emulsion. In one embodiment, the oil-in-water emulsion comprises 1 ng/g to 10 ng/g, 10 ng/g to 100 ng/g, or 100 ng/g to 1,000 ng/g of the compound/oil-in-water emulsion.

In one embodiment, the oil-in-water emulsion droplets comprise 0.0000001 wt% to 0.0001 wt%, 0.0001 wt% to 0.1 wt%, 0.1 wt% to 1 wt%, or 1 wt% to 10 wt% of a compound. In one embodiment, the oil-in-water emulsion comprises about 0.0000001 wt% to about 0.0001 wt%, about 0.0001 wt% to about 0.1 wt%, about 0.1 wt% to about 1 wt%, or about 1 wt% to about 10 wt% of a compound. In one embodiment, the oil-in-water emulsion comprises 0.0000001 wt% to 10 wt% of a compound.

In one embodiment, one or more compounds are embedded in a lipid bilayer of a biphasic lipid vesicle. In one embodiment, the lipid bilayer of the lipid vesicle composition may be formulated to contain one or more compounds. In one embodiment, the lipid bilayer of the lipid vesicle composition comprises 0.0000001 wt% to 10 wt% of the compound. In one embodiment, the lipid bilayer comprises about 0.0000001 wt% to about 0.0001 wt%, about 0.0001 wt% to about 0.1 wt%, about 0.1 wt% to about 1 wt%, or about 1 wt% to about 10 wt% of the compound. In one embodiment, the lipid bilayer of the lipid vesicle comprises 1 wt% to 3 wt% of the compound.

In one embodiment, one or more compounds are embedded in a lipid bilayer and an oil-in-water emulsion of a two-phase lipid vesicle. In one embodiment, the one or more compounds embedded in the lipid bilayer are the same as the one or more compounds embedded in the oil-in-water emulsion of the two-phase lipid vesicle. In one embodiment, the one or more compounds embedded in the lipid bilayer are different from the one or more compounds embedded in the oil-in-water emulsion of the two-phase lipid vesicle.

For example, it can be understood that the release rate of one or more compounds embedded in an oil-in-water emulsion will be faster than that of the same one or more compounds embedded in a lipid bilayer.

In one embodiment, one or more compounds are selected from, but not limited to, small molecules, proteins, peptides, carbohydrates, nucleic acids, vaccine antigens, and/or plant extracts.

In one embodiment, one or more compounds are therapeutic compounds. Therefore, the compositions of this disclosure are pharmaceutical compositions.

In one embodiment, the small molecule is a prostaglandin, an anesthetic such as ibuprofen and diclofenac, an analgesic or sedative including opioids such as, for example, buprenorphine, fentanyl, sufentanil, alfentanil and remifentanil, a cardioactive drug, androgenic steroids, estrogens, progestins, antihistamines and antiviral agents, vitamins, anti-inflammatory agents, antifungal agents, corticosteroids, vitamins, anti-infective agents, dermatological reagents, drugs for treating nausea and vomiting, amino acids, short peptides (up to 1000 Da), carbohydrates or natural compounds and combinations thereof.

In one embodiment, the cardioactive drug is an organic nitrate, such as nitroglycerin, isosorbide dinitrate and/or isosorbide mononitrate, quinidine sulfate, procainamide, thiazides such as benzylfluorothiazide, chlorothiazide and/or hydrochlorothiazide, nifedipine, nicardipine, adrenergic blockers such as timolol and/or propranolol, verapamil, diltiazem, captopril, clonidine, or prazosin.

In one implementation, the androgenic steroid is testosterone, methyltestosterone, or fluorohydroxymethyltestosterone.

In one embodiment, the estrogen is estradiol valerate, estradiol valerate, ethinylestradiol methyl ether, estrone, estriol, 17β-ethinylestradiol, or diethylstilbestrol.

In one embodiment, the antihistamine is diphenhydramine, dimenhydrinate, perphenazine, triprolidine, pyramine, chlorcyclorhizine, promethazine, carbisamine, tropineamine, brompheniramine, chlorpheniramine, terfenadine and/or chlorpheniramine.

In one embodiment, the anti-infective agent is an antibiotic, including penicillin, tetracycline, chloramphenicol, sulfacetamide, sulfadiazine, sulfadiazine, sulfamethazine, sulfamethoxazole and/or sulfisoxazole; an antiviral agent; an antibacterial agent such as erythromycin and/or clarithromycin, and/or other anti-infective agents including nitrofurazone, etc.

In one implementation, the dermatological agent is vitamin A and/or vitamin E.

In one implementation, the drugs used to treat nausea and/or vomiting are chlorpromazine, granisetron, perphenazine, prochlorperazine, promethazine, thiotetramethrin, trifluprozine, and/or isobutrazine;

In one embodiment, the progestin is progesterone, 19-norprogesterone, norethindrone, norethindrone acetate, chlormedrone, ethinylprogesterone, etogestene, medroxyprogesterone acetate, hydroxyprogesterone caproate, isethindrone, norethindrone, 17α-hydroxyprogesterone, dydrogesterone, dimethynone, ethinylestradiol, norethindrone, dimegestrol, pramegestrol and/or medroxyprogesterone acetate.

In one embodiment, the small molecule is an anti-inflammatory agent selected from the following: acemetacin, acetaminophen, bendazac, benzylprofen, permoprofen, buccolic acid, butebufen, guimetacin, cyclochloroindica, clopidogrel, biphenylacetic acid, biphenylbutanone, fenclofen, fenprofen, flunoprofen, flurbiprofen, ibuprofen, indomethacin, triphenyloxetine, isocolic acid, ketoprofen, lonazol, loxoprofen, mexaazinic acid, mobendazole, naproxen, oxapazine, pyprazole, pirprofen, pranoprofen, propionic acid, sulindac, sulofen, succinate, thiazolinic acid, tometetin, and/or tropesin. Permoprofen, Buclofen, Isoprofen, Ketoprofen, Loxoprofen, Zaltoprofen, Ampicillin, Bucolon, Celecoxib, Bifenpyramide, Mofebuzoline, Nimesulide, Renitoline, Parecoxib, Persamide, Pyrazoprofen, Tanidoxetine, Tenidap, Terofenadate, Vardecoxib, 21-Acetylpregnenolone, Aclomethasone, Betamethasone, Alpha-Bisabolol, Budesonide, Clobetasol, Cyclosporine, Defocole, Dexamethasone, Diflubenzuron, Desonide, Desoxymethasone, Diflubenzuron, Diflubenzuron The following are listed: difluprednisolone, ditazobactam, everolimus, fluzacroxetine, fludrocortisone, flumethasone, fluocinolone acetonide, flucodone, flucodone acetate, flucodone, flucloxacillin, flumethin, halcinonide, halobetasol propionate, halomethasone, haloprednisolone acetate, hydrocortisone, isobenzo, clotiprednisolone, horseprednidone, memetasone, methylprednisolone, mometasone furoate, hydroxybutazine, piperoxazole, pimecrolimus, prednisolone, prednisone, limexolox, sirolimus, triamcinolone and/or tacrolimus.

In one embodiment, the small molecule is ibuprofen and/or diclofenac.

In one embodiment, the small molecule is a wound-healing compound. In one embodiment, the wound-healing compound is bosentan. In one embodiment, the small molecule is an antibiotic. In one embodiment, the antibiotic is vancomycin.

In one embodiment, the protein is a cytokine or a peptide. In one embodiment, the peptide of the pharmaceutical composition has 2-900 amino acids.

In one embodiment, the amino acid, peptide, or protein has a molecular weight of 50 to 300,000 Daltons. In some embodiments, the therapeutic compound is a carbohydrate or nucleic acid molecule with a molecular weight of 50-5M Daltons.

In one embodiment, the peptide is a polypeptide, such as insulin, cytokine, vaccine antigen, growth hormone-releasing factor, or antibody. In one embodiment, the polypeptide has a molecular weight of 1,000 to 300,000 Daltons.

As described above, the pharmaceutical compositions (sometimes referred to as lipovesicles or lipid compositions or formulations) described herein can be used to deliver therapeutic compounds, including but not limited to small molecules, peptides, proteins, carbohydrates, nucleic acids, vaccine antigens, and/or plant extracts. Lipovesicle formulations comprise one or more lipid (e.g., phospholipid) bilayers containing an oil-in-water emulsion. The oil-in-water emulsion comprises droplets typically smaller than 1 μm within the aqueous interior of the lipoves, which are typically multilayered with multiple lipid bilayers. Biphasic lipoves formulations may also include one or more other lipovesicle components, including but not limited to fatty substances such as cholesterol, penetration enhancers, surfactants, solvents, etc., to adapt the lipovesicle formulation to physicochemical properties relevant to the target skin. Therapeutic compounds, penetration enhancers, surfactants, and/or other lipovesicle components may be incorporated into the lipid bilayer and/or the oil-in-water emulsion.

In one embodiment, lipid vesicles can be formulated to contain compounds, penetration enhancers, surfactants, and/or other lipid vesicle components, selectively incorporated into lipid bilayers and/or oil-in-water emulsions at different stages of production. Therefore, a considerable degree of control can be maintained over the placement of the compounds, penetration enhancers, and/or other lipid vesicle components within the lipid vesicles. For example, during lipid vesicle production, the compound may be added only to the components of the oil-in-water emulsion, only to the components of the lipid bilayer, or to both the oil-in-water emulsion and the lipid bilayer.

The structure and composition of these lipovesicle formulations can be modified to allow one or more compounds to penetrate deeply into the skin. The lipid bilayer and oil-in-water emulsion of the lipovesicle formulation isolate one or more compounds and other pharmaceutical excipients to provide enhanced stability and sustained release of the compounds. In one embodiment, the biphase lipovesicle formulation optionally further comprises one or more other lipovesicle components, including but not limited to fatty substances such as cholesterol, penetration enhancers, surfactants, and solvents, and combinations thereof.

In one embodiment, the lipid bilayer of the lipid vesicle further comprises a fatty substance to, for example, enhance the strength of the lipid bilayer. In one embodiment, the fatty substance is cholesterol, cholesterol derivatives, skeletonol, cholesterol, cholesterolane, or long-chain fatty acids or combinations thereof. In one embodiment, the lipid bilayer of the lipid vesicle composition further comprises 0.1 wt% to 10 wt% cholesterol and/or cholesterol derivatives. In some embodiments, the lipid bilayer comprises 1 wt% to 5 wt% cholesterol and/or cholesterol derivatives.

The lipid bilayer of the lipid vesicle composition may contain 0.1 wt% to 5 wt% cholesterol or a derivative thereof. In some embodiments, the lipid bilayer of the lipid vesicle composition contains 0.1 wt% to 3 wt% cholesterol or a derivative thereof. In some embodiments, the lipid bilayer contains 2 wt% cholesterol or a derivative thereof.

In one embodiment, the lipid bilayer of the lipid vesicle composition optionally further comprises one or more permeation enhancers in addition to one or more permeation enhancers. Skin permeation enhancers include any known skin permeation enhancers, excluding one or more permeation enhancers such as those described in Adrian C. Williams and Brian W. Barry Advanced Drug Delivery Reviews 64 (2012) 128-137; or Majella E. Lane Int. J. Pharm. 447 (2013) 12-21.

It should be understood that, in addition to the penetration enhancers described herein, one or more additional penetration enhancers may be added to the formulation.

In some embodiments, the skin penetration enhancer is selected from one or more alcohols, such as ethanol or isopropanol; amides, such as azone; esters, such as ethyl acetate, amyl dimethylaminobenzoate O, ethyl oleate, glyceryl monooleate, glyceryl monodecanoate, glyceryl tricaprylate, isopropyl myristate, isopropyl palmitate, propylene glycol monolaurate, or propylene glycol monocaprylate; ether alcohols, such as (e.g., carbitol P, 2-(2-ethoxyethoxy)ethanol); fatty acids, such as lauric acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, stearic acid, or isostearic acid; ethylene glycol, such as dipropylene glycol, propylene glycol, 1,2-butanediol, or 1,3-butanediol; pyrrolidone, such as N-methyl-2-pyrrolidone or 2-pyrrolidone; and sulfoxide, such as decanmethyl sulfoxide or dimethyl sulfoxide.

In one embodiment, one or more permeation enhancers are fatty acylated amino acids such as monolauroyl lysine and/or dipalmitoyl lysine.

In one embodiment, the lipid bilayer optionally further comprises a hydrophilic solvent to, for example, dissolve vesicles to form lipids. In one embodiment, the hydrophilic solvent includes, but is not limited to, propylene glycol, glycerol, polyethylene glycol with a molecular weight in the range of 300 to 8000, ethanol, and combinations thereof.

In one embodiment, the oil-in-water emulsion comprises an aqueous medium having water and optionally one or more lipophilic additives, such as preservatives (parabens, phenoxyethanol, benzyl alkyl ammonium salts, etc.), antioxidants (ascorbic acid, ascorbyl palmitate, BHA, BHT, α-tocopherol), waxes, and thickeners (long-chain fatty alcohols and their esters, fatty acids, beeswax, olive oil, glyceryl stearate, cetyl alcohol, stearyl alcohol, myristyl myristate, and cetyl palmitate, stearyl heptanoate, and/or stearyl palmitate).

In one embodiment, the oil-in-water emulsion includes 0.1 wt% to 25 wt% of one or more lipophilic additives.

The applicant also demonstrated that penetration enhancers (e.g., polycationic surfactants) enhance skin penetration of compounds relative to other similar or identical compositions (except for the use of monocationic surfactants instead of polycationic surfactants).

Therefore, this application also includes a biphasic lipid vesicle composition comprising:

a) Lipid vesicles, which consist of a lipid bilayer containing vesicle-forming lipids.

b) An oil-in-water emulsion, embedded in two-phase lipid vesicles and containing one or more polycationic surfactants; and

c) One or more compounds, embedded in a lipid bilayer and/or an oil-in-water emulsion.

In one embodiment, the biphasic lipovesicle composition is a cosmetic composition. In another embodiment, the biphasic lipovesicle composition is a pharmaceutical composition.

In one embodiment, the biphasic lipid vesicle composition is used for the local delivery of one or more compounds. In one embodiment, local delivery is for intradermal delivery, transdermal delivery, and/or transmucosal delivery.

In one embodiment, the biphase lipid vesicle composition comprises a suspension of biphase lipid vesicles.

In one embodiment, the polycationic surfactant is one or more gemini surfactants.

Gemini surfactants are surfactant molecules containing more than one hydrophobic tail. Each hydrophobic tail has a hydrophilic head (Menger and Keiper, 2000; Kirby et al., 2003). The hydrophobic or hydrophilic tails are linked together by spacer groups. The hydrophobic tails can be the same or different. Similarly, the hydrophilic heads can be the same or different. Furthermore, the hydrophilic heads can be anionic (e.g., phosphate, sulfate, or carboxylate type), cationic (e.g., quaternary ammonium type), or neutral (e.g., polyether, peptide, or sugar type) (Menger and Keiper, 2000). In aqueous solutions, gemini surfactants spontaneously aggregate into micelles, the shape and size of which are particularly sensitive to the length of the spacer groups and their hydrophobic or hydrophilic properties. The spacer group can be variable, i.e., short (e.g., 2 methylene groups) or long (e.g., more than 12 methylene groups); rigid (e.g., stilbene) or flexible (e.g., methylene chain); and polar (e.g., polyether, ethoxy, or polyethoxy) or nonpolar (e.g., aliphatic, aromatic) (Menger and Keiper, 2000). Because the hydrophobic tail, hydrophilic head, and spacer group can differ in these respects, countless different molecules can be designed.

In one embodiment, the hydrophobic tail is of the _C3 - _C30 alkyl type, linear or branched, saturated or unsaturated. In one embodiment, the hydrophilic head can be anionic, cationic or neutral. In one embodiment, the hydrophilic head is cationic.

In one embodiment, the polycationic surfactant is one or more gemini dicationic surfactants.

In one embodiment, the gemini surfactant comprises a linear hydrocarbon tail group and a quaternary ammonium head group. A typical structure of a type of gemini cationic surfactant includes a head group consisting of two positively charged nitrogen atoms separated by spacer groups (n) of 3, 4, 6, 8, 10, 12, or 16 carbon atoms, each spacer group containing two methyl groups, and a tail consisting of two saturated chains of 12 or 16 carbon atoms (m = 10 or 14).

In one embodiment, one or more gemini-cationic surfactants are of the quaternary ammonium type. In one embodiment, one or more gemini-cationic surfactants are selected from 12-7NH-12, _12-7NCH3-12 , 16-3-16, 12-4(OH) _2-12 , and 12-EO1-12. In one embodiment, one or more gemini-cationic surfactants are selected from 12-7NH-12, 12-7NCH3-12, and 16-3-16.

In one embodiment, one or more polycationic surfactants are polycationic amino acids. In one embodiment, the polycationic amino acid is selected from polylysine, polyarginine, and combinations thereof.

In one embodiment, the oil-in-water emulsion of biphase lipid vesicles comprises about 0.01% to about 5%, 0.05% to about 5%, 0.1% to about 5%, about 1% to about 5%, or about 2% to about 5% of one or more polycationic surfactants. In another embodiment, the oil-in-water emulsion of biphase lipid vesicles comprises about 0.01% to about 5% of one or more polycationic surfactants.

In one embodiment, the oil-in-water emulsion of biphase lipoves optionally comprises one or more additional surfactants (excluding polycationic surfactants). In one embodiment, the one or more additional surfactants are one or more additional stabilizing surfactants as described above. In one embodiment, the oil-in-water emulsion of biphase lipoves comprises 0.1% to about 10% of one or more surfactants. In one embodiment, the oil-in-water emulsion of biphase lipoves comprises about 0.01% to about 10%, 0.05% to about 10%, 0.1% to about 10%, about 1% to about 10%, about 2% to about 10%, 0.01% to about 7%, 0.05% to about 7%, 0.1% to about 7%, about 1% to about 7%, and about 2% to about 7% of one or more surfactants.

When used with one or more additional surfactants, the oil-in-water emulsion of biphase lipoves comprises about 0.1% to about 10% of one or more polycationic surfactants. In one embodiment, the oil-in-water emulsion of biphase lipoves comprises about 0.01% to about 10%, 0.05% to about 10%, 0.1% to about 10%, about 1% to about 10%, about 2% to about 10%, 0.01% to about 7%, 0.05% to about 7%, 0.1% to about 7%, about 1% to about 7%, and about 2% to about 7% of one or more polycationic surfactants.

In one embodiment, the biphase lipid vesicle composition further comprises one or more penetration enhancers, wherein the one or more penetration enhancers are one or more nonionic surfactants with an HLB of about 10 or less, which alone or in combination with one or more penetration enhancers selected from one or more terpenes, alkaloids, salicylic acid derivatives and polycationic surfactants as described above and combinations thereof.

In one embodiment, the wt% of water and oil in the oil-in-water emulsion is as described above.

In one implementation, the vesicle-forming lipids are as described above.

In one embodiment, one or more compounds are embedded in an oil-in-water emulsion of a two-phase lipid vesicle, i.e., a lipid bilayer.

In one embodiment, as described above, one or more compounds are embedded in a lipid bilayer, an oil-in-water emulsion of two-phase lipid vesicles, or both.

In one embodiment, the amounts of one or more compounds in the lipid bilayer and the oil-in-water emulsion are as described above.

In one embodiment, one or more compounds are selected from, but are not limited to, small molecules, including negatively charged small molecules, carbohydrates, nucleic acids such as RNA or DNA or hybrids thereof, plasmid DNA, oligonucleotides including synthetic oligonucleotides, viral DNA, DNA vaccines, proteins, peptides including peptide antigens such as vaccine antigens, immunoglobulins, immunomodulators, hormones, toxins and/or enzymes, as well as plant extracts and/or vitamins.

In one embodiment, one or more compounds are selected from, but not limited to, peptides, carbohydrates, nucleic acids, vaccine antigens, plasmid DNA, DNA vaccines, peptide vaccines, immunoglobulins, immunomodulators, oligonucleotides, hormones, toxins, and enzymes. In one embodiment, one or more compounds are selected from nucleic acids, plasmid DNA, DNA vaccines, and/or oligonucleotides. In one embodiment, one or more compounds are selected from nucleic acids, plasmid DNA, DNA vaccines, and/or oligonucleotides.

In one embodiment, the biphasic lipid vesicle composition optionally further comprises one or more other lipid vesicle components as described above, including but not limited to fatty substances such as cholesterol, permeation enhancers, surfactants and/or solvents and combinations thereof.

In one embodiment, the biphasic lipid vesicle composition of this disclosure is used for the local delivery of one or more compounds. In one embodiment, local delivery is for intradermal delivery, transdermal delivery, or transmucosal delivery.

As described above, in one embodiment, the biphasic lipid vesicle composition of this disclosure described herein can be a cosmetic composition.

In one embodiment, the biphasic lipovesicular cosmetic compositions of this disclosure may suitably and optionally include components commonly used in cosmetics, such as moisturizers, antioxidants, oily components, UV absorbers, emulsifiers, thickeners, alcohols, powder components, colorants, aqueous components, water, and/or various skin nutrients, to the extent that they do not impair the effectiveness of existing compositions and systems. The cosmetic compositions may include conventional adjuvants and carriers, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and/or fragrances.

In one embodiment, the biphasic lipid vesicle composition of the present disclosure described herein can be formulated as a cream, supplement, ointment, paste, emulsion, gel, oil, liquid spray, foundation, or powder.

In one embodiment, ointments or creams can be formulated with an aqueous or oily base by adding suitable thickeners and/or gelling agents. Such bases may include water and/or oils (such as liquid paraffin) or vegetable oils (such as peanut oil or castor oil). An exemplary base is water. Thickeners that may be used, depending on the nature of the base, include aluminum stearate, hydrogenated lanolin, etc. Furthermore, emulsions can be formulated with an aqueous base and typically include one or more of the following: stabilizers, emulsifiers, dispersants, suspending agents, thickeners, colorants, fragrances, etc. Ointments and creams may also contain excipients such as starch, astragalus gum, cellulose derivatives, carboplatin, polyethylene glycol, silicone, bentonite, veegum (magnesium aluminum silicate), silicic acid, and talc, or mixtures thereof. Emulsions can be formulated with an aqueous or oily base and typically also include one or more of the following: stabilizers, emulsifiers, dispersants, suspending agents, thickeners, colorants, fragrances, etc. Foam can be formed by known foaming agents or surfactants.

In one embodiment, the gel can be formed by mixing a delivery system (such as the biphasic vesicles described herein) with a gelling agent (such as collagen, pectin, gelatin, agarose, chitin, chitosan, and alginate). The delivery system can be incorporated into a liquid to formulate a topical solution, aerosol, mist, spray, drop, or intracavitary infusion solution. For example, the delivery system can be applied to mucous membranes via an aerosol (which may be generated by a topical aerosol spray pump or actuator) or by infusion.

A container comprising the composition described herein is also provided. The container is optionally a spray container, or optionally an aerosol spray pump container.

In one embodiment, the biphasic lipid vesicle composition of the present disclosure described herein is contained in a coating substrate, such as a dressing, filler, membrane, or mesh, that can be coated with the biphasic lipid vesicle composition and applied directly to the skin or mucous membrane.

In one embodiment, the biphasic lipid vesicle composition of the present disclosure described herein may be included in a transdermal delivery system employing one of a variety of forms (e.g., patches or sheet masks).

In one implementation, the transdermal delivery system includes

Backing layer; and

A matrix layer, comprising the biphase lipid vesicle composition described herein, is disposed on the backing layer.

The matrix layer is configured to contact the skin.

In one embodiment, the backing layer is or comprises a polymer selected from polyesters (e.g., polyethylene terephthalate (PET)) and polycarbonates, polyolefins (e.g., polyethylene, polypropylene or polybutene, polyethylene oxide, polyurethane, polystyrene, polyamide, polyimide, polyvinyl acetate, polyvinyl chloride, polyvinylidene chloride), and copolymers (e.g., acrylonitrile-butadiene-styrene terpolymer, or ethylene-vinyl acetate copolymer). Preferred materials for the backing layer are selected from polyesters, and particularly preferred from polyethylene terephthalate. For example, this type of backing layer is available from 3M (USA) under the trade name Scotchpak 1109.

In one implementation, the backing layer is a closed backing layer.

The backing layer can be made of, for example, polyester.

In another embodiment, the backing layer includes an overtape extending laterally beyond the edge of the matrix layer, allowing the transdermal delivery system to adhere to, or better yet, the skin. The overtape may include an adhesive layer free of active ingredients and an overtape film. The overtape film may be a polymer selected from the group consisting of polyolefins, olefin copolymers, polyesters, copolyesters, polyamides, copolyamides, polyurethanes, etc. Examples of suitable materials that may be cited are polyesters, particularly polyethylene terephthalate, as well as polycarbonates, polyolefins (e.g., polyethylene, polypropylene, or polybutene), polyethylene oxide, polyurethanes, polystyrene, polyamides, polyimides, polyvinyl acetate, polyvinyl chloride, polyvinylidene chloride, copolymers (e.g., acrylonitrile-butadiene-styrene terpolymers or ethylene-vinyl acetate copolymers).

In one embodiment, the adhesive may be, for example, a polyisobutylene (PIB) adhesive.

In one embodiment, the backing layer has a thickness of at least about 5 μm, at least about 10 μm, at least about 15 μm, at least about 20 μm, at least about 25 μm, at least about 50 μm, at least about 75 μm, at least about 100 μm, at least about 125 μm, or up to about 250 μm, up to about 200 μm, up to about 150 μm, up to about 100 μm, or up to 50 μm, or any combination thereof. The backing layer may, for example, have a thickness including or in any 0.1 μm increment between 5 μm and 200 μm.

When the transdermal delivery system is a patch, the backing layer thickness may be at least about 75 μm or at least about 100 μm and less than, for example, 200 μm or less than, for example, 150 μm.

When the transdermal delivery system is a face mask, the backing layer thickness may be at least 10 μm or at least 20 μm and less than, for example, 100 μm or less than, for example, 75 μm.

The matrix layer has a surface intended to be placed on the skin, which may be referred to as the application side. The application side may be configured to contain a pressure-sensitive adhesive, such as a surface self-adhesive, on its entire surface, or it may be configured to be an adhesive only on a portion of its surface.

In one embodiment, the transdermal delivery system further includes a protective layer, also known as a release liner, which is applied to the composition comprising the matrix layer and is removed prior to application of the transdermal delivery system to facilitate removal of the protective layer. In some embodiments, the protective layer extends beyond the edge of the backing layer, such as the remaining patch.

In one implementation, the transdermal delivery system is a patch.

In one embodiment, one or more compounds are therapeutic compounds. Therefore, the biphasic lipovesicle compositions of this disclosure described herein are pharmaceutical compositions. Thus, the biphasic lipoves of this disclosure are suitably formulated into pharmaceutical compositions comprising a pharmaceutically acceptable carrier for administration to a subject in a biocompatible form suitable for topical application. In one embodiment, one or more compounds are therapeutic compounds selected from the one or more therapeutic compounds described herein.

III. Method for preparing the composition disclosed herein

The compositions of this disclosure as described above are prepared by mixing an oil component of an oil-in-water emulsion with an aqueous component of the same emulsion, wherein the oil or aqueous component of the oil-in-water emulsion contains one or more surfactants for emulsifying the oil and aqueous components of the oil-in-water emulsion. In one embodiment, the surfactant is mixed with the aqueous component and added to the oil to form an emulsion. The oil-in-water emulsion is then mixed with dissolved vesicles to form lipids and, if any, other lipid components are added, under mixing conditions that effectively form two-phase lipid vesicles.

One or more penetration enhancers and one or more compounds may be added to the oil component of an oil-in-water emulsion, the aqueous component of an oil-in-water emulsion, or both. Alternatively, or otherwise, one or more penetration enhancers and one or more compounds may be added to the lipid component.

Therefore, this application includes a method for preparing biphasic lipid vesicles, comprising:

a) Prepare an oil-in-water emulsion containing one or more surfactants by mixing the oil component of the oil-in-water emulsion with the aqueous component of the oil-in-water emulsion, wherein the oil component and/or aqueous component of the oil-in-water emulsion contain one or more surfactants.

b) Dissolve vesicles in an acceptable solvent other than water to form lipids;

c) Add one or more compounds and one or more penetration enhancers to the oil component and/or aqueous component of step a), and/or the dissolved vesicle-forming lipids of step b);

d) Adding an oil-in-water emulsion to dissolved vesicle-forming lipids; and

e) Mixing an oil-in-water emulsion and dissolved vesicle-forming lipids under mixing conditions that effectively form a two-phase lipid vesicle comprising a lipid bilayer containing vesicle-forming lipids and an oil-in-water emulsion embedded in the two-phase lipid vesicle.

In one embodiment, a pharmaceutical composition for topical application of a compound, namely a lipid vesicle composition, is provided, wherein the composition comprises lipid vesicles comprising an outer lipid bilayer, an oil-in-water emulsion, and a therapeutic compound, the composition being formed by: (a) mixing oil with water to form an oil-in-water emulsion; (b) mixing the oil-in-water emulsion of (a) with at least one vesicle-forming lipid such that the oil-in-water emulsion is coated by an outer lipid bilayer; and (c) adding a therapeutic compound and a penetration enhancer during (a) and/or (b); wherein the compound is a molecule with a molecular weight between 50 and 5 M Daltons; and one or more penetration enhancers increase the amount of compound absorbed into a given amount of skin, relative to the same composition in the absence of one or more penetration enhancers.

In one embodiment, step a) of mixing the oil component of the oil-in-water emulsion with the aqueous component of the oil-in-water emulsion vesicles and/or the mixing conditions in step e) include the use of stirring, such as homogenization or emulsification, or the use of microemulsion techniques that do not involve stirring. In one embodiment, mixing includes high-pressure homogenization. High-pressure homogenization can provide relatively precise control over the composition of the lipid vesicles. High-pressure homogenization is suitable for shear-resistant small molecules and peptides or proteins. In one embodiment, the resulting composition is any of the lipid vesicle compositions described herein.

In one embodiment, other lipid components are added to any one of steps a) through e).

In one embodiment, one or more surfactants are selected from one or more stabilized surfactants and/or one or more polycationic surfactants described herein.

In one embodiment, one or more penetration enhancers, one or more compounds, oil-in-water emulsions, vesicle-forming lipids, acceptable solvents, and/or other lipid components are as described above.

The lipid vesicle compositions of this disclosure can also be prepared by methods known in the art, such as those disclosed herein by reference and incorporated herein by U.S. Patent Nos. 5,993,852, 5,853,755 and 5,993,851.

In one embodiment, the biphasic lipid vesicle composition of this disclosure described herein may be included in a transdermal delivery system employing one of a variety of forms (e.g., patches or sheet masks). In one embodiment, the biphasic lipid vesicle composition is a transdermal patch.

In one embodiment, the transdermal patch can be prepared using procedures known in the field of transdermal patches. The preparation process typically involves formulating a matrix layer comprising two phases (i.e., mixing an adhesive and two-phase lipid vesicles and additives, if any), casting the matrix layer onto a backing layer or release liner, and removing solvents from the matrix.

IV. Methods and Uses of this Disclosure

Biphasic lipid vesicles are liposomes, microscopic vesicles composed of a single phospholipid bilayer or multiple concentric phospholipid bilayers surrounding an oil-in-water emulsion. These lipid vesicles serve as compound carriers for the local delivery of compounds that may be hydrophobic or hydrophilic. Lipid vesicles are typically biocompatible, biodegradable, and non-toxic drug delivery carriers.

The compositions disclosed herein can be used for the topical delivery of one or more compounds. Therefore, this application includes a method for delivering one or more compounds by topical application of the biphasic lipid vesicle compositions of this disclosure to the skin or mucous membranes of a subject.

This application also includes the use of the disclosed lipovesicle compositions for the topical delivery of one or more compounds to the skin or mucous membranes, and the use of the disclosed lipovesicle compositions for the preparation of a medicament for the topical delivery of one or more compounds to the skin or mucous membranes. This application also includes the disclosed lipovesicle compositions for the topical delivery of one or more compounds to the skin or mucous membranes.

The biphasic lipovesicle compositions of this disclosure, comprising one or more penetration enhancers as described herein, have been shown to improve skin penetration of one or more compounds relative to other similar or comparable compositions (except for the absence of one or more penetration enhancers). The biphasic lipovesicle compositions of this disclosure and the biphasic lipovesicle cosmetic compositions of this disclosure, comprising one or more polycationic surfactants as described herein, have been shown to improve skin penetration of one or more compounds relative to other similar or comparable compositions (except for the use of a monocationic surfactant instead of a dicationic or polycationic surfactant).

Therefore, this application also includes an improved method for local delivery of one or more compounds, comprising applying an effective amount of the biphasic lipid vesicle composition of this disclosure to the skin or mucous membrane of a subject in need.

This application also includes the use of the lipovesicle compositions or lipovesicle cosmetic compositions of this disclosure for improving the local delivery of one or more compounds to the skin or mucous membranes, and the use of the lipovesicle compositions or lipovesicle cosmetic compositions of this invention for preparing a medicament for improving the local delivery of one or more compounds to the skin or mucous membranes. This application also includes the use of the lipovesicle compositions or lipovesicle cosmetic compositions of this disclosure for improving the local delivery of one or more compounds to the skin or mucous membranes.

In one embodiment, this application includes a method for treating or preventing skin conditions associated with excessive or deficient collagen production in a subject, comprising applying an effective amount of the lipid vesicle cosmetic composition of this disclosure to the subject in need.

This application also includes the use of the lipovesicle cosmetic compositions of this disclosure for the treatment or prevention of skin conditions associated with excess or deficiency of collagen, and the use of the lipovesicle cosmetic compositions of this disclosure for the preparation of medicaments for the treatment or prevention of skin conditions associated with excess or deficiency of collagen. The application also includes the lipovesicle cosmetic compositions of this disclosure for the treatment or prevention of skin conditions associated with excess or deficiency of collagen.

In one implementation, skin conditions associated with excess or deficiency of collagen include skin aging, skin elasticity, stripes, stretch marks, wrinkles, collagen vascular diseases such as scleroderma, lentigines, lupus, rheumatoid arthritis, temporal arteritis, and hereditary collagen diseases such as Ehlers-Danlos syndrome and Marfan syndrome.

In one embodiment, one or more compounds are one or more therapeutic compounds. Therefore, the biphasic lipovesicle composition is a biphasic lipovesicle pharmaceutical composition.

Therefore, this application also includes methods for treating diseases, conditions, or illnesses treatable by delivering one or more therapeutic compounds to the skin or mucous membranes of a subject in need through topical application of a therapeutically effective amount of the biphasic lipovesicular pharmaceutical composition of this disclosure. In one embodiment, the biphasic lipovesicular composition of this disclosure is applied topically to the skin.

This application also includes the use of the lipovesicle compositions of this disclosure for treating diseases, conditions, or illnesses treatable by topical delivery of one or more therapeutic compounds of this disclosure to the skin or mucous membranes, and the use of the lipovesicle compositions of this disclosure for preparing a medicament for treating diseases, conditions, or illnesses treatable by topical delivery of one or more therapeutic compounds to the skin or mucous membranes of a subject in need. The application also includes the use of the lipovesicle compositions for treating diseases, conditions, or illnesses treatable by topical delivery of one or more therapeutic compounds to the skin or mucous membranes.

In one embodiment, the disease, condition, or ailment treatable by topically applying a therapeutically effective amount of the biphasic lipovesicular pharmaceutical composition of this disclosure to the skin or mucous membrane is a skin condition related to excessive or deficient collagen production, inflammation, pain, fungal infection, viral infection, dermatological/dermatological conditions, rheumatic conditions, joint conditions, skin aging, or cancer. In one embodiment, the disease, condition, or ailment is skin aging. In one embodiment, the disease, condition, or ailment is a skin condition related to excessive or deficient collagen production.

In one implementation, the disease, condition, or lesion is a skin condition. In one implementation, the skin condition is scleroderma, atopic dermatitis, psoriasis, a condition characterized by any cytokine deficiency, a condition characterized by IFNγ deficiency, hereditary skin diseases (hereditary skin disorders), including epidermal fragility disorders, keratosis disorders, pilaris disorders, pigmentary disorders, porphyria, multisystem disorders, and cancerous conditions. In one implementation, the disease, condition, or lesion is a form of hereditary epidermolysis bullosa (e.g., borderline EB and dystrophic EB), lamellar ichthyosis and/or X-linked ichthyosis and xeroderma pigmentosum.

In one implementation, the disease, symptom, or condition is an infection. In one implementation, the infection is a viral infection, a bacterial infection, or a fungal infection.

In one implementation, the disease, symptom, or condition is a sexual dysfunction. In another implementation, sexual dysfunction is erectile dysfunction or impotence.

In one implementation, the disease, symptom, or condition is hereditary warts.

In one implementation, the disease, symptom, or condition is pain or inflammation. In one implementation, the pain is acute pain or chronic pain.

In one implementation, the object is a mammal. In another implementation, the object is a human.

The dosage of the compositions disclosed herein can vary depending on many factors, such as the pharmacodynamic properties of the compound, the route of administration, the age, health and weight of the subject, the nature and severity of symptoms, the frequency of treatment and the type of concurrent treatment (if any), and the clearance of the compound in the subject being treated. Those skilled in the art can determine an appropriate dosage based on the above factors. The compositions disclosed herein can initially be administered at an appropriate dose, which may be adjusted as needed based on clinical response. A dose is typically selected to maintain serum levels of the compounds disclosed herein at about 0.01 μg/mL to about 1000 μg/mL or about 0.1 μg/mL to about 100 μg/mL. Representative amounts are about 0.001 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.01 mg/kg to about 1 mg/kg, or about 0.1 mg/kg to about 1 mg/kg. The compounds disclosed herein can be administered as a single daily, weekly, or monthly dose, or the total daily dose can be divided into two, three, or four daily doses.

In one embodiment, the composition of this disclosure is administered at least once a week. However, in another embodiment, the compound is administered to the subject approximately every two weeks, three weeks, or one month. In another embodiment, the compound is administered approximately once a week to once daily. In yet another embodiment, the compound is administered 2, 3, 4, 5, or 6 times daily. The length of treatment depends on a variety of factors, such as the severity of the disease, condition, or illness, the age of the subject, the concentration and/or activity of the compound of this disclosure, and/or combinations thereof. It should also be understood that the effective dose of the compound used for treatment may increase or decrease during a particular treatment regimen. Dosage changes can become apparent through standard diagnostic analyses known in the art. In some cases, prolonged administration is required. For example, the compound is administered to the subject in an amount and for a duration sufficient to treat the subject.

Example

The following non-limiting embodiments illustrate this application.

Example 1: Exemplary lipid vesicle composition

A. Method

Method 1: In vitro diffusion of cells:

Full-thickness human breast skin was obtained from female donors who underwent selective mastectomy at the University of Saskatchewan Royal University Hospital (Saskatoon, SK, Canada). Skin collection was approved by the University of Saskatchewan Human Ethics Committee. Skin was collected within 2 hours post-surgery, trimmed to remove subcutaneous fat, and stored at -20°C until use. A straight-line Bronaugh flow-through diffusion cell with a 9mm orifice diameter ( ^0.63cm² ) was mounted on a water-insulated cell heater (PermeGear, Inc., Hellertown, PA) and set to a constant temperature of 32°C. Pre-cut ^1cm² slices of skin were placed in the diffusion cell with the stratum corneum facing upwards. Infusion buffer (100mM phosphate buffer containing 0.05% sodium azide) at 37°C was circulated in the lower half of the diffusion cell using a peristaltic pump at a rate of 1mL/h. 0.1mL of the preparation was applied to the skin surface. After 24 hours of incubation, skin samples were removed from the pool and their surfaces were washed three times with 10 mL of water each time. Each skin sample was aspirated and then peeled off twice with transparent adhesive tape to remove excess formulation from the surface. Skin samples were analyzed by UPLC of the skin homogenate or by confocal microscopy of frozen sections.

Method 2: Preparation of skin homogenate for UPLC analysis

Skin samples were individually homogenized using a gentleMACS ^™ dissociator (Miltenyi Biotec, Inc., Auburn, CA). Each skin slice was reconstituted in 1 mL of methanol (for diclofenac samples) or 1 mL of acetonitrile (for ibuprofen samples), added to a gentleMACS ^™ M-tube (Miltenyi Biotec, Inc.), and homogenized using a protein extraction program (10 x 55 seconds). Samples were then filtered into 2 mL LC/GC certified clear glass maximum recovery vials (Waters Corp., Milford, MA) using a 0.2 μm GH Polypro membrane syringe filter (Pall Corp., Ville St. Laurent, QC, Canada).

The ACQUITY H-class UPLC chromatography system consists of a bioQuaternary solvent manager, an autosampler (biosample manager - flowing through the needle), a variable wavelength UV detector (photodiode array eλ), and a column manager, controlled by Empower3 software (Waters Corp.), and was used for analysis and method validation in this study.

Analysis was performed on a 1.7 μm BEH300 C18 50 mm x 2.1 mm i.d. column (Waters Corp.), heated to 30 °C (for diclofenac analysis) and 35 °C (for ibuprofen analysis), with an injection volume of 5 μL. Mobile phases (solvent A – 0.65 mL/min for diclofenac analysis: 0.35 mL/min for ibuprofen analysis) were pumped at 0.45 mL/min (for diclofenac analysis) and 0.55 mL/min (for ibuprofen analysis) in isocratic mode. The total running times for diclofenac and ibuprofen analyses were 5 min and 10 min, respectively. Mobile phase, standard solutions, and sample solutions were filtered through a 0.2 μm GH Polypro membrane syringe filter (Pall Corp.) and used at room temperature. The UV detection range for diclofenac was set to 200-260 nm, and the collected data were plotted at 254 nm. For ibuprofen, the UV detection range was 200-250 nm, and the collected data were plotted at 220 nm. Calibration and quantification (total peak area) were both calculated using Empower 3 software.

Method 3: In vivo studies

Animal studies were approved by the Animal Care Committee Protocol Review Committee of the University of Waterloo. In vivo delivery was performed using CD1 mice (Charles River). All animals (including controls) were anesthetized with isoflurane and shaved along the root the day before treatment. The shaved area was cleaned with distilled water and dried using sterile gauze. Naked plasmid DNA solution or plasmid DNA preparation (50 μL of plasmid containing 25 μg of tD-Tomato Red Fluorescent Protein (RFP) encoded by each animal) was applied to the shaved area and covered with a paraffin-sealed/Opsite occlusive dressing, which was secured in place with plastic tape for 24 hours. The treated skin area was excised 24 hours after treatment.

Method 4: Confocal microscopy

Mouse or human skin samples were characterized using confocal microscopy with a Zeiss LSM 710 confocal microscope. All samples were embedded in an OCT compound matrix and cryosectioned. Skin samples were cryosectioned to 10 μm sections using a Leica CM1850 cryostat. Confocal microscopy images of skin sections were obtained using a Zeiss LSM 710CLSM with HeNe laser lines (543 and 633 nm) for tdTomato (546/579) and Rhodamine (570/590), a 488 nm laser for FITC insulin and FITC-IgG, and a Plan-Apochromer 20x/0.80 dry objective or a 63x/1.40 oil immersion objective. Optical zoom selection was applied in selected cases. Laser intensity, pinhole, and gain settings were kept consistent across sample sets to allow for comparison of relative fluorescence intensity measurements between different treatments. Images were captured and processed using Zen 2009 software.

"Untreated" samples were used to confirm gain and pinhole settings to exclude noise and autofluorescence background from subsequent treated samples.

B: Exemplary lipid vesicle formulation composition

1. Exemplary ibuprofen lipovesicle formulation

Step 1: Preparation of System A (oil-in-water emulsion):

The following is an example of System A for an ibuprofen lipovesicle formulation IB1-IB-6 (water-in-oil submicron emulsion):

Step 2: Preparation procedure for System A (water-in-oil submicron emulsion) formulation (applicable to all formulations):

1. Weigh the oil phase and aqueous phase components in separate beakers.

2. Heat both beakers to approximately 70°C to completely melt and mix all components.

3. Add the aqueous phase quickly to the oil phase in one go, while stirring vigorously with a spatula to form an o/w original emulsion. This will effectively produce a homogeneous emulsion solution in a 70°C water bath (~2 minutes).

4. The formulation was processed three times in batches at 20,000 psi using an LV1 microfluidic device or a nano DeBee homogenizer with a Z5 module.

Step 3: Vesicle preparation:

Example ibuprofen formulation IB1:

Vesicle formation procedure (applicable to all formulations):

1. Weigh the lipid phase component into a 20 mL glass bottle.

2. Heat the bottle in a water bath to ~70°C to completely melt and mix all components.

3. Quickly add the aqueous phase (System A) to the liquid phase in one go.

4. Vortex the mixture at 5-second intervals and heat for 8-10 cycles until a uniform creamy emulsion is formed.

The following exemplary lipid vesicle formulations were prepared using the process described above for ibuprofen formulation IB1.

b. Ibuprofen formulation IB2

Note: Menthol and camphor were premixed with a spatula in a glass flask without heating to form a eutectic mixture. After the mixture was fully mixed and in a liquid state, system A was added and the mixture was vortexed thoroughly. The mixture was then added to the lipid phase as described above.

c) Exemplary ibuprofen formulation IB3A

lipid phase Phospholipon 90H 10% cholesterol 2% Propylene glycol 7% Piperine 0.1% Oleth-2 1% Ibuprofen 5% Aqueous phase System A Qs to 100

d) Exemplary ibuprofen formulation IB3B

lipid phase Phospholipon 90H 10% cholesterol 2% Propylene glycol 7% Piperine 0.1% Oleth-2 2% Ibuprofen 5% Aqueous phase System A Qs to 100

e) Example Ibuprofen formulation IB4A

lipid phase Phospholipon 90H 10% cholesterol 2% Propylene glycol 7% Methyl salicylate 2.5% Oleth-2 1% Ibuprofen 5% Aqueous phase System A Qs to 100

f) Example ibuprofen formulation IB4B

g) Exemplary formulation IB5A

h) Example ibuprofen formulation IB5B

lipid phase Phospholipon 90H 10% cholesterol 2% Propylene glycol 7% Neroli 1% Oleth-2 2% Ibuprofen 5% Aqueous phase System A Qs to 100

i) Exemplary ibuprofen formulation IB6A

j): Exemplary ibuprofen formulation IB6B

2. Exemplary diclofenac lipid vesicle formulation

Step 1: Preparation of System A (Water-in-Oil Submicron Emulsion)

The following is an example of System A for diclofenac lipid vesicle formulations DF1 and DF2:

System A was prepared using the process described above for ibuprofen formulation IB1.

Step 2: Vesicle preparation

The following exemplary diclofenac lipid vesicle formulations were prepared using the process described above for ibuprofen formulation IB1.

a) Exemplary diclofenac formulation DF1

b) Exemplary diclofenac formulation DF2

3. Exemplary peptide and protein lipid vesicle formulations

The following exemplary peptide and protein lipid vesicle formulations were prepared using the process described above for the exemplary ibuprofen formulation IB1.

a) Exemplary 12-dimeric peptide (mwt 1200), insulin (mwt 6000), and IgG (150,000) lipid vesicle formulation 1 (peptide-liposome formulation 1)

Preparation of System A (oil-in-water submicron emulsion) for formulation:

oil phase Labrafac CC 5% glyceryl monostearate (NE) 1.2% cetyl alcohol 0.6% Synchrowax BB4 (beeswax) 0.3% propylparaben 0.05% Aqueous phase Arlasilk EFA (phospholipid EFA) 5% Methylparaben 0.15% Milli-Q Water Qs to 100

Vesicle preparation:

b) Exemplary 12-dimeric peptide (mwt 1200), insulin (mwt 6000) and IgG (150,000) lipid vesicle formulation 2 (peptide-liposome formulation 2)

Preparation of System A (Water-in-Oil Submicron Emulsion)

Vesicle preparation:

c) Exemplary 12-dimeric peptide (mwt 1200), insulin (mwt 6000), and IgG (150,000) lipid vesicle formulation 3 (peptide-liposome formulation 3)

Preparation of System A (Water-in-Oil Submicron Emulsion)

Vesicle preparation:

d) Examples of 12-dimeric peptide (mwt 1200), insulin (mwt 6000), and IgG (150,000)

Lipid vesicle formulation 4 (peptide lipid vesicle formulation 4)

Preparation of System A (oil-in-water submicron emulsion) for formulation:

oil phase Labrafac CC 5% glyceryl monostearate (NE) 1.2% cetyl alcohol 0.6% Synchrowax BB4 (beeswax) 0.3% propylparaben 0.05% Aqueous phase Arlasilk EFA (phospholipid EFA) 5% Methylparaben 0.15% Milli-Q Water Qs to 100

Vesicle preparation:

4) Nucleic acid lipid vesicle formulations

The following exemplary nucleic acid lipid vesicle formulations were prepared using the process described above for the exemplary ibuprofen formulation IB1.

a) Comparison of the properties of granular lipid vesicle formulation F-TOM-1

Preparation of System A (oil-in-water submicron emulsion) for F-TOM-1

Vesicle preparation:

b) Exemplary plasmid formulation: lipid vesicle F-TOM-2

Preparation of System A (Water-in-Oil Submicron Emulsion) for F-TOM-2

oil phase olive oil 5% Glyceryl monostearate 1.2% cetyl alcohol 0.6% Synchrowax BB4 (beeswax) 0.3% propylparaben 0.05% Aqueous phase Gemini surfactant 16-3-16 0.1% Twain 80 0.5% Methylparaben 0.1% Milli-Q Water Qs to 100  

Vesicle preparation:

lipid phase Phospholipon 90H 10% cholesterol 2% Propylene glycol 7% Aqueous phase System A Qs to 100

c) Exemplary plasmid lipid vesicle formulation F-TOM-3

Preparation of System A (oil-in-water submicron emulsion) for F-TOM-3

oil phase Labrafac CC (medium-chain triglyceride) 3% Phospholipids 2% Aqueous phase Gemini Surfactant 12-3-12 0.1% Milli-Q Water Qs to 100

Vesicle preparation:

lipid phase Phospholipon 90H 10% cholesterol 2% Propylene glycol 7% Aqueous phase System A Qs to 100

d) Exemplary plasmid lipid vesicle formulation F-TOM-4

Preparation of System A (oil-in-water submicron emulsion) for F-TOM-4

oil phase Labrafac CC (medium-chain triglyceride) 3% Phospholipids 2% Aqueous phase Gemini surfactant 12-7NCH3-12 0.1% Milli-Q Water Qs to 100

Vesicle preparation:

lipid phase Phospholipon 90H 10% cholesterol 2% Propylene glycol 7% Aqueous phase System A Qs to 100

e) Exemplary plasmid lipid vesicle formulation F-TOM-5

Preparation of System A (oil-in-water submicron emulsion) for F-TOM-5

Vesicle preparation:

lipid phase Phospholipon 90H 10% Lauroyl-Octanyl Lysine Methyl Ester 2.5% Propylene glycol 7% Aqueous phase System A Qs to 100

C. Results and Discussion

Ibuprofen and diclofenac skin delivery

Results of in vitro cell diffusion and skin homogenate analyses (see Tables 3 and 4 below) indicate that delivery of both IB and DF is improved by incorporating penetration enhancer components into exemplary biphasic lipovesicle formulations. The addition of hydrophobic nonionic surfactants with HLB <10, such as Oleth-2 with HLB 4–7, was found to enhance delivery to the active epidermis. Further enhancements can be achieved by adding additional penetration enhancers such as terpenes (e.g., menthol, camphor, methyl salicylate) or alkaloids (e.g., piperine) (Table 3). The enhanced penetration effect of hydrophobic nonionic surfactants (e.g., Oleth-2) can be further enhanced by increasing their concentration in the formulation (e.g., from 1% to 2%) (Table 3).

Table 3. Ibuprofen skin delivery. IB concentration in skin homogenates was measured using UPLC. Data are presented as mean ± s.d. (N = 4). (Whole skin = surface-bound drug removed by two D-squame tapes. Peeled skin = active skin layer only; skin peeled 2+10 times with D-squame tape)

Table 4. Diclofenac skin delivery: Concentration of DF in skin homogenate was measured using UPLC. Data are presented as mean ± s.d. (N = 4). (Whole skin = surface-bound drug removed by two D-squame tapes. Peeled skin = active skin layer only; skin peeled 2+10 times with D-squame tape)

*This data is relatively volatile.

Skin delivery of peptide and protein therapeutic agents

The presence of fluorescent proteins was assessed by cryosectioning human skin samples treated in vitro in a diffusion cell with topical formulations containing fluorescently labeled peptides and proteins. Three compounds with increased molecular weights showed enhanced delivery of the protein and peptide compounds (Figure 1). Increased delivery of these proteins and peptides was demonstrated by incorporating penetration enhancers (HLB < 10) and hydrophobic nonionic surfactants such as Oleth-2, sorbitan monopalmitate [Span 40], or PEG-4 dilaurate (Figure 1).

Table 5 shows the relative fluorescence intensities measured in the active epidermis. While all these hydrophobic nonionic surfactants with HLB < 10 were effective in enhancing delivery in two-phase vesicles, the enhancement levels were as follows (from highest to lowest): PEFA/Oleth-2 > Tween 80/Span 40/Oleth-2 > Tween 80/Span 40/PEG-4-dilaurate > PEFA/PEG-4-dilaurate. (Italicized surfactants are present in the oil and water emulsion components of the comparative two-phase vesicles used for emulsification; bold surfactants indicate additional penetration enhancers used for penetration enhancer functions).

Table 5. Average relative fluorescence intensity values obtained from confocal microscopy images

preparation Mean relative fluorescence intensity of active epidermal layer F1-TAMRA-13 polymeric peptide (mwt 1440) twenty four F2-TAMRA-13 polymeric peptide (mwt 1440) 40 F3-TAMRA-13 polymeric peptide (mwt 1200) 50 F4-TAMRA-13 polymeric peptide (mwt 1440) 212 F1-FITC-insulin (mwt 6,000) 10 F2-FITC-Insulin (mwt 6,000) 40 F3-FITC-Insulin (mwt 6,000) 15 F4-FITC-Insulin (mwt 6,000) 20 F1-FITC-IgG (mwt 150,000) 15 F2-FITC-IgG (mwt 150,000) 20 F3-FITC-IgG (mwt 150,000) 40 F4-FITC-IgG (mwt 150,000) 50

Skin delivery of nucleic acids

The expression of tdTomato red fluorescent protein was assessed in mouse skin samples treated with a topical formulation containing plasmid DNA encoding the red tdTomato reporter gene. Other formulations containing PEFA alternatives, i.e., bicationic gemini surfactants acting as complexing agents for negatively charged plasmid DNA, increased plasmid DNA delivery and in vivo skin gene expression in mice compared to comparative biphasic vesicles (F-TOM-1 containing the monocationic surfactant PEFA). All the bicationic gemini surfactants used were effective in delivering plasmid DNA when incorporated into the biphasic vesicle structure. The enhancements are as follows (from highest to lowest): F-TOM-5 dicationic gemini surfactant 12-7NH-12/phospholipid emulsifier > F-TOM-4 dicationic gemini surfactant 12-7CH3-12/phospholipid emulsifier > F-TOM-3 dicationic gemini surfactant 12-3-12/phospholipid emulsifier > F-TOM-2*Tween 80/dicationic gemini surfactant 16-3-16 (surfactants in italics are improved functional surfactants for two-phase vesicles to improve the encapsulation of highly negatively charged nucleic acids; surfactants in bold indicate the added synergistic permeation enhancer function of HLB<10) (Table 6). *F-TOM-2 is a variant of the control formulation in which the original two-phase vesicles prepared with Tween 80/PEFA are replaced with Tween 80/gemini surfactant.

All blank samples showed almost no background fluorescence (Fig. 2). Samples treated with naked pDNA intradermally showed abundant tdTomato expression (image not shown). For each formulation, three sub-images are shown: First sub-image: red channel for RFP expression (considered as light-colored areas in the epidermis and dermis); Second sub-image: general tissue staining (blue nuclear staining with Syto60); Third sub-image: merged image.

Table 6. Average relative fluorescence intensity values obtained from confocal microscopy images.

This disclosure is not limited to the specific embodiments described in this application. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. In addition to those listed herein, functionally equivalent methods and compositions within the scope of this disclosure will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. This disclosure is limited only by the terms of the appended claims and the full scope of their equivalents. It should be understood that this disclosure is not limited to specific methods, reagents, compound compositions, or biological systems, which can, of course, be varied. It should also be understood that, as used herein, terminology is used for the purpose of describing particular embodiments only and is not intended to be limiting.

All publications, patent applications, granted patents and other documents mentioned in this specification are incorporated herein by reference as if each individual publication, patent application, granted patent or other document expressly and individually indicated that it is incorporated by reference in its entirety. Definitions contained in the text incorporated by reference that contradict the definitions in this disclosure are excluded.

While certain embodiments have been shown and described, it should be understood that they may be altered and modified in accordance with common art without departing from the art as defined in the broader aspects of the claims provided below.

Claims (59)

1. A biphasic lipid vesicle composition comprising: a) Lipid vesicles, each of which comprises a lipid bilayer containing vesicle-forming lipids. b) An oil-in-water emulsion, which is embedded in the two-phase lipid vesicles and stabilized by a surfactant; c) A penetration enhancer, which is embedded in the lipid bilayer and/or the oil-in-water emulsion; The penetration enhancer (i) includes a nonionic surfactant having a hydrophilic-lipophilic balance (HLB) of 10 or less, and (ii) is selected from diethylene glycol monooleate, sorbitan monopalmitate and polyoxyethylene (4) dilaurate. 2. The two-phase lipid vesicle composition according to claim 1, wherein the penetration enhancer is embedded in an oil-in-water emulsion. 3. The two-phase lipid vesicle composition according to claim 1, wherein the permeation enhancer is embedded in the lipid bilayer and the oil-in-water emulsion. 4. The biphasic lipid vesicle composition according to claim 1, wherein the biphasic lipid vesicle composition is a cosmetic composition. 5. The biphasic lipid vesicle composition according to claim 1, wherein the biphasic lipid vesicle composition is a pharmaceutical composition. 6. The biphasic lipid vesicle composition according to claim 1, wherein the biphasic lipid vesicle composition is used for local delivery of the one or more compounds. 7. The biphase lipid vesicle composition according to claim 1, wherein the biphase lipid vesicle composition comprises a suspension of the biphase lipid vesicles. 8. The two-phase lipid vesicle composition according to claim 1, wherein the permeation enhancer is embedded in the oil-in-water emulsion of the two-phase lipid vesicles. 9. The two-phase lipid vesicle composition of claim 8, wherein the oil-in-water emulsion comprises 0.5 wt% to 9 wt%, 0.5 wt% to 8 wt%, 0.5 wt% to 7 wt%, 1 wt% to 6 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, or 1 wt% to 2 wt% of one or more of the penetration enhancers. 10. The two-phase lipid vesicle composition according to claim 1, wherein the permeation enhancer is embedded in the lipid bilayer. 11. The biphase lipid vesicle composition of claim 10, wherein the lipid bilayer comprises 10 wt%, 9 wt%, 8 wt%, 7 wt%, 6 wt%, 5 wt%, 4 wt%, 3 wt%, 2 wt%, 1 wt%, 0.5 wt%, or 0.1 wt% of the permeation enhancer. 12. The two-phase lipid vesicle composition according to claim 1, wherein the permeation enhancer is embedded in both the lipid bilayer and the oil-in-water emulsion of the two-phase lipid vesicle. 13. The two-phase lipid vesicle composition according to claim 1, wherein the penetration enhancer is diethylene glycol monooleyl ether. 14. The two-phase lipid vesicle composition according to claim 1, wherein the penetration enhancer is PEG-4 dilaurate. 15. The biphasic lipid vesicle composition of claim 1, wherein the biphasic lipid vesicle composition further comprises an additional permeation enhancer selected from one or more terpenes, alkaloids, salicylic acid derivatives, and polycationic surfactants and combinations thereof. 16. The biphasic lipid vesicle composition according to claim 15, wherein the one or more terpenes are selected from one or more of eugenol, d-limonene, menthol, menthone, farnesol, neridol, camphor, nerol and thymol, and combinations thereof. 17. The biphasic lipid vesicle composition according to claim 16, wherein the one or more terpenes are selected from one or more of menthol, camphor, nerol and thymol and combinations thereof. 18. The biphasic lipid vesicle composition according to claim 15, wherein the one or more salicylic acid derivatives are selected from one or more of ethyl salicylate, salicylic acid, acetylsalicylic acid and triethanolamine salicylate and combinations thereof. 19. The biphasic lipid vesicle composition according to claim 18, wherein the salicylic acid derivative is methyl salicylate. 20. The biphasic lipid vesicle composition according to claim 15, wherein the one or more alkaloids are selected from piperine, lobeline, caffeine, theobromine, theophylline, nicotine, colchicine, N-methylpyrrolidone, guaranaine, capsaicin, berberine, sanguisorbin, histamine, and/or pilocarpine. 21. The biphasic lipid vesicle composition according to claim 20, wherein one or more alkaloids are piperine. 22. The biphase lipid vesicle composition of claim 15, wherein the polycationic surfactant is one or more gemini cationic surfactants and the one or more gemini cationic surfactants are quaternary ammonium surfactants. 23. The two-phase lipid vesicle composition according to claim 15, wherein the polycationic surfactant is a polycationic amino acid. 24. The two-phase lipid vesicle composition according to claim 1, wherein the penetration enhancer is one or more nonionic surfactants with an HLB of 10 to 4. 25. The biphasic lipid vesicle composition of claim 15, wherein the penetration enhancer is a diethylene glycol monooleyl ether, and the additional penetration enhancer is selected from menthol, camphor, nerol, or thymol or a combination thereof. 26. The biphasic lipid vesicle composition of claim 15, wherein the penetration enhancer is diethylene glycol monooleyl ether, and the additional penetration enhancer is methyl salicylate. 27. The two-phase lipid vesicle composition of claim 15, wherein the penetration enhancer is a diethylene glycol monooleyl ether, and the additional penetration enhancer is piperidine. 28. The two-phase lipid vesicle composition of claim 15, wherein the permeation enhancer is embedded in the lipid bilayer, and the one or more terpenes or the one or more alkaloids are embedded in the lipid bilayer, the oil-in-water emulsion, or both. 29. The biphasic lipid vesicle composition of claim 15, wherein the penetration enhancer is PEG-4 dilaurate, and the additional penetration enhancer is piperidine. 30. The biphasic lipid vesicle composition of claim 15, wherein the penetration enhancer is PEG-4 dilaurate, and the additional penetration enhancer is methyl salicylate. 31. The biphase lipid vesicle composition according to claim 1, wherein the oil-in-water emulsion of the biphase lipid vesicle is stabilized by one or more surfactants selected from polyoxyethylene (10) hexadecyl ether and polysorbate 80. 32. The biphasic lipid vesicle composition according to claim 1, wherein the vesicle-forming lipid is selected from phospholipids, glycolipids, lecithin and/or ceramides. 33. The biphasic lipid vesicle composition according to claim 1, wherein the vesicle-forming lipid is selected from phosphatidylethanolamine, lysophosphatidylethanolamine, lysophosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin, phosphatidic acid and cerebroside. 34. The two-phase lipid vesicle composition according to claim 32, wherein the vesicle-forming lipid is a phospholipid. 35. The two-phase lipid vesicle composition according to claim 1, further comprising one or more compounds embedded in the lipid bilayer and/or oil-in-water emulsion. 36. The biphasic lipid vesicle composition of claim 35, wherein one or more compounds are embedded in an oil-in-water emulsion of the biphasic lipid vesicle, a lipid bilayer of the biphasic lipid vesicle, or both the oil-in-water emulsion and the lipid bilayer. 37. The biphasic lipid vesicle composition according to claim 35, wherein one or more compounds are selected from small molecules, proteins, peptides and/or nucleic acids. 38. The biphasic lipid vesicle composition according to claim 35, wherein one or more compounds are carbohydrates. 39. The biphasic lipid vesicle composition according to claim 35, wherein one or more compounds are vaccine antigens. 40. The biphasic lipid vesicle composition of claim 37, wherein the small molecule is selected from the group consisting of prostaglandins; analgesics or sedatives; cardioactive drugs; adrenergic blockers; estrogens; progestins; antihistamines; antiviral agents; vitamins; anti-inflammatory agents; corticosteroids; anti-infective agents; drugs for treating nausea and vomiting; amino acids; short peptides; carbohydrates; and combinations thereof. 41. The biphasic lipid vesicle composition according to claim 37, wherein the small molecule is an anesthetic. 42. The biphasic lipid vesicle composition according to claim 41, wherein the anesthetic agent is selected from ibuprofen and diclofenac. 43. The biphasic lipid vesicle composition according to claim 37, wherein the small molecule is an antifungal agent. 44. The biphasic lipid vesicle composition according to claim 37, wherein the small molecule is an antibacterial agent. 45. The biphasic lipid vesicle composition of claim 40, wherein the analgesic comprises an opioid. 46. The biphasic lipid vesicle composition according to claim 45, wherein the opioid is selected from buprenorphine, fentanyl, sufentanil, alfentanil, and remifentanil. 47. The biphasic lipid vesicle composition according to claim 40, wherein the cardioactive drug is selected from nitroglycerin, isosorbide dinitrate, isosorbide mononitrate, quinidine sulfate, procainamide, benzylfluorothiazide, chlorothiazide, hydrochlorothiazide, nifedipine, and nicardipine. 48. The biphasic lipid vesicle composition according to claim 40, wherein the adrenergic blocker is selected from timolol, propranolol, verapamil, diltiazem, captopril, clonidine, prazosin, testosterone, methyltestosterone, and fluorohydroxymethyltestosterone. 49. The biphasic lipid vesicle composition according to claim 40, wherein the estrogen is selected from estradiol valerate, estradiol valerate, ethinylestradiol methyl ether, estrone, estriol, 17β-ethinylestradiol and diethylstilbestrol. 50. The biphasic lipid vesicle composition according to claim 40, wherein the progestin is selected from progesterone, 19-norprogesterone, norethindrone, norethindrone acetate, chlormedroxyprogesterone, ethinylprogesterone, etogestene, medroxyprogesterone acetate, hydroxyprogesterone caproate, isethindrone, norethindrone, 17α-hydroxyprogesterone, dydrogesterone, dimethynone, ethinylestradiol, norethindrone, dimegestrol, pramegestrol and/or medroxyprogesterone acetate. 51. The biphasic lipid vesicle composition according to claim 40, wherein the antihistamine is selected from diphenhydramine, diphenhydramine, perphenazine, triprolidine, piracetam, chlorcyclorhizine, promethazine, carbisamine, tropineamine, brompheniramine, chlorpheniramine, terfenadine, and chlorpheniramine. 52. The biphasic lipid vesicle composition according to claim 40, wherein the anti-inflammatory agent is selected from acemetidine, acetaminophen, benzalkonium chloride, benzalkonium chloride, phenoxyprofen, permoprofen, buccolic acid, butebufen, guimetidine, cyclochloroindole, clopidogrel, biphenylacetic acid, biphenylbutanolate, fenclopidogrel, fenprofen, flunoprofen, flurbiprofen, ibuprofen, indomethacin, triphenylazole acid, isochoric acid, ketoprofen, chlorpromazine, etc. Nazoline, Loxoprofen, Methamphetamine, Mobendazole, Naproxen, Oxaprazin, Pyprazole, Pyprofen, Pramipexole, Propylazine, Sulindac, Suprofen, Succinate, Thiamethoxazole, Tometrine, Tropesin, Permorofen, Buclofen, Isoprofen, Ketoprofen, Loxoprofen, Zaltoprofen, Ampicillin, Bucolone, Celecoxib, Bifenpyridine, Mofebuzoline, Nimesulide, Renitolin, Parexel Coxib, Parsamid, Piraruprofen, Tanidoxime, Tenidapril, Terofenadate, Vardecoxib, 21-Acetylpregnenolone, Aclomethasone, Betamethasone, Alpha-Bisabolol, Budesonide, Clobetasone, Cyclosporine, Dexamethasone, Diflupine, Desonide, Desoxymethasone, Diflupine, Diflucanone, Difluprednisolone, Ditazazole, Everolimus, Fluzacrolimus, Fludrocortisone, Flumethasone Fluocinolone, fluocinolone acetonide, flucodone, flucortone, fluprednisolone acetate, glucosamine, halcinonide, halobetasol propionate, halometasone, haloprednisolone acetate, hydrocortisone, isobenzo, clotiprednisolone, horseprednisolone, memetasone, methylprednisolone, mometasone furoate, hydroxybutazine, piperoxazole, pimecrolimus, prednisolone, prednisone, limesoloxone, sirolimus, triamcinolone acetonide, and tacrolimus. 53. The biphasic lipid vesicle composition of claim 40, wherein the anti-infective agent comprises an antibiotic. 54. The biphasic lipid vesicle composition according to claim 53, wherein the antibiotic is selected from penicillin, tetracycline, chloramphenicol, sulfacetamide, sulfadiazine, sulfadiazine, sulfamethazine, sulfamethazine, sulfadiazine, and sulfamethoxazole. 55. The biphasic lipid vesicle composition according to claim 44, wherein the antibacterial agent comprises erythromycin, clarithromycin, and furacilin. 56. The biphasic lipid vesicle composition according to claim 40, wherein the drug for treating nausea and vomiting is selected from chlorpromazine, granisetron, perphenazine, prochlorperazine, promethazine, thiotetramethrin, trifluprozine, and isoprazine. 57. The biphasic lipid vesicle composition of claim 1, wherein the biphasic lipid vesicle formulation further comprises one or more other lipid vesicle components, including but not limited to fatty substances, permeation enhancers, surfactants and/or solvents and combinations thereof. 58. A method for preparing a biphasic lipid vesicle composition according to any one of claims 1-57, the method comprising: a) The oil-in-water emulsion is prepared by mixing the oil component of the oil-in-water emulsion with the aqueous component of the oil-in-water emulsion; b) Dissolve vesicles in an acceptable solvent other than water to form lipids; c) Add one or more penetration enhancers to the oil component and/or the aqueous component of step a) or the dissolved vesicle-forming lipid of step b), wherein the one or more penetration enhancers are one or more nonionic surfactants with a hydrophilic-lipophilic balance (HLB) of 10 or less. d) Adding the oil-in-water emulsion to the dissolved vesicle-forming lipids; and e) Mix the oil-in-water emulsion and the dissolved vesicle-forming lipids under mixed conditions that effectively form the two-phase lipid vesicles, wherein the two-phase lipid vesicles comprise a lipid bilayer containing vesicle-forming lipids and an oil-in-water emulsion embedded in the two-phase lipid vesicles. 59. The method of claim 58, further comprising adding one or more compounds in b).