WO2004087107A1

WO2004087107A1 - Cationic liposome targeting sebaceous glands

Info

Publication number: WO2004087107A1
Application number: PCT/KR2004/000367
Authority: WO
Inventors: Jung-Chul Kim; Mee-Sook Jun; In-Sook Han
Original assignee: Trichogene Inc.
Priority date: 2003-02-24
Filing date: 2004-02-23
Publication date: 2004-10-14

Abstract

A cationic liposome using multi-layered positive ion molecules, which targets sebaceous glands, is disclosed. Cationic medicinal agents to be used for medical treatment of sebaceous glands-related diseases are prepared as the cationic liposome. Cationic intensity is controlled constantly by simply measuring electric conductivity, thereby reducing its own toxicity with keeping the ionic character and function of the cationic medicinal agents. In particular, medicine absorption and adsorption of sebaceous gland cells which are most hydrophobic among the pilosebaceous units are increased, so the cationic liposome has specificity to sebaceous glands.

Description

CATIONIC LIPOSOME TARGETING SEBACEOUS GLANDS

TECHNICAL FIELD

Photodynamic therapy for sebaceous glands involves two major parts: (1) delivering a photosensitizer targeting sebaceous glands to a target site by an applying

method; and (2) exposing the target site with light to activate the delivered photosensitizer for medical treatment of skin disorder associated with the sebaceous glands. The present invention relates to a cationic liposome formulation as a photosensitizer to be applied for delivery of the photosensitizer targeting sebaceous glands. The cationic liposome formulation of the present invention keeps its ionic characteristic as a cationic medicinal agent without changing its stability and intrinsic function, and offers an improved targeting efficiency to sebaceous glands composed of

lipid vesiculae, which are highly capable of absorbing and adsorbing the cationic

liposome. Further, the cationic liposome formulation according to the present

invention is especially effectively used in combination with iontophoresis useful for fast,

high-dose medicinal agent administration.

BACKGROUND ART

A cationic liposome is a liposome which is positively charged, that is, acidic when present in physiological pH. The cationic liposome is produced by mixing cationic lipids or molecules with neutral liposomes. The charge ratio and acidity relative

to the ionic intensity of a cationic medicinal agent to be applied are controlled by

adjusting the quantity of the positive ion molecules. The ionic intensity is controlled to be kept at a constant level by monitoring electric conductivity. Since the cationic

liposome has a very high tendency of absorbing and adsorbing cells, it allows a medicinal agent to be effectively administered into the cells, which is particularly apparent for the sebaceous gland, which is the lipid-richest tissue in the pilosebaceous

unit and skin structure. Meanwhile, although non-ionic or negatively charged

photosensitizers are also used in the cationic liposome, the positive charged medicinal agent is preferably used in the cationic liposome for a synergic effect in delivery efficiency and stability of medicinal agent.

Examples of the cationic medicinal agent to be applied to sebaceous glands

include photosensitizers such as porphyrins, e.g., 5-aminolevulinic acid (hereinafter, to be abbreviated as "ALA"), chlorines, or phtalocyanines, and dyes such as methylene blue, toluidine blue, crystal violet, or rhodamine. These materials are photosensitizers

that are photochemically or photothermally activatable by red light (600-800 nm), and capable of causing damages to cells. The energy source to be irradiated is, for example,

light in the visible to red wavelength regions (600~800 nm) with a fluence of approximately 5~50 J/cm .

Cationic photosensitizers or medicinal agents to be applied to sebaceous glands

are prepared from cationic liposomes with positively charged molecules added thereto, thereby keeping the medicinal agent stable and functionally effective and enabling

cationic liposome formulations to offer sebaceous gland specific absorbance. That is, the cationic liposome formulation can maintain its ionic intensity at a constant level, at

which skin penetration and cell absorbance are maximally exhibited, by adjusting electric conductivity. Acne results from pilosebaceous inflammation and is a very frequently presented disease of facial skin during adolescence or puberty in both

sexes. Endogenous hormones, mainly androgens, which are secreted in unusually high concentrations in the blood during adolescence and puberty, stimulate sebaceous

glands adjacent to hair follicles, giving rise to enlargement of the sebaceous glands and

producing large quantities of sebum. Simultaneously, the horny layer of the infundibulum of the follicle, that is, the follicular opening, becomes thicker and highly adhesive to cause pluggage or stenosis of the follicular opening, resulting in accumulation of sebum and proliferation of the bacteria residing in the skin, such as

Propionibacterium acnes, which in turn leads to inflammation, called acne. Physical or mental stress due to chronic stress, tension or lack of sleep can aggravate the acne lesion.

Treatment of acne can be accomplished by reducing production of sebum,

normalizing desquamination (loss) of skin cells within the follicular opening, or

diminishing the bacteria responsible for acne, e.g., Propionibacterium acnes. Topical therapy of acne includes directly applying a medicinal agent containing therapeutic

ingredients such as benzoyl peroxide, retinoic acid or azelaic acid, or an antibiotic

ointment, such as clindamycin or erythromycin, to a site afflicted with acne.

Systemic therapy of acne includes oral administration of antibiotics of tetracycline

family for sterilizing bacteria responsible for acne or reducing inflammation by suppressing production of free fatty acids, or retinoic acids for suppressing production

of sebum.

Also, treatment of acne can be accomplished by photodynamic therapy using various photosensitizers. In particular, ALA, which is a precursor of protoporphyrin IX

(to be abbreviated as "PplX"), is applied to the skin, the PpIX is actively synthesized and stored not only in a transdermal route but also in pilosebaceous units including sebaceous glands. A considerable quantity of porphyrins is also synthesized by Propionibacterium acnes. The sebaceous glands expressly emit red fluorescent light, suggesting that administration of ALA causes accumulation of exogenous porphyrins and the bacteria responsible for acne causes accumulation of endogenous porphyrins (see FIG. 1). These porphyrin precursors are allowed to be sufficiently expressed, followed by exposing the same with light having a wavelength between about 600 to

about 700 nm to excite PplX, thereby diminishing and eradiating the sebaceous glands and destructing the bacteria. Therefore, photodynamic therapy of acne using a photosensitizer having a selectivity to and targeting sebaceous glands, e.g., ALA, can offer satisfactory efficacy in treatment of the skin disorder associated with sebaceous

glands and bacteria, while causing substantially no side effects in the skin.

Many reports and research have been carried out to find formulations that can

deliver medicinal agents targeting the sebaceous glands, and ointments, oils, surfactants, liposomes, and so on, have been employed. Liposomes are microscopic vesicles

having a hydrophilic space in its internal compartment and an enclosed lipid bilayer

membrane in its external compartment. The liposome allows water-soluble molecules

or medicinal agent to be entrapped in the internal hydrophilic space. A lipophilic medicinal agent may be attached to the external lipid bilayer membrane. Charged substances, either positively or negatively, may bind to or coated on the liposome. Since the liposome has various properties and a high tendency of extending and contracting, liposome derivates having new physical properties, functions and uses

can be provided by varying its structure and ingredients. In addition, the liposomes that are present in membranes of living cells, have various advantages, including

bio-adaptability, degradability, stability and so on. These advantages have allowed the liposomes to be widely used as carriers of numerous materials or medicinal agents. Examples of the material that can be combined with liposomes to be used as a carrier for medicinal agent delivery include an anticancer agent, e.g., adriamycin, antifungal agent, e.g., amphotericin B, antimicrobial agent, immunomodulator, antigen and antibody, contrast medium, hemoglobin, peptide like growth factor, protein, lipid,

DNA, and so on, and most of them are currently widely used in clinical applications.

Despite clinical utility, they showed only limited availability to non-ionic, neutral

liposomes without consideration given to stability and functions of ionic medicinal agents. Further, cationic liposomes showing specificity to sebaceous glands have never been available.

DISCLOSURE OF INVENTION

To solve the above problems, the present invention provides a cationic liposome

which has specificity to sebaceous glands by increasing selectivity to and adsorption of

the medicinal agent into a target site, that is, the sebaceous gland, while reducing

toxicity of a cationic medicinal agent itself used for treatment of sebaceous gland related

diseases without changing the ionic characteristic of the cationic medicinal agent.

Also, the present invention provides a cationic liposome formulation which can adjust the ionic intensity of a cationic liposome, at which penetration and absorbance of the cationic liposome into the skin cell are maximally exhibited, by simply adjusting

the electric conductivity.

The present invention provides a cationic liposome which has an appropriate

viscosity suitable to be applied to the skin for delivery of a medicinal agent. To accomplish the above objects of the present invention, the present invention provides a cationic liposome targeting sebaceous glands prepared by mixing a lipid

making a neutral liposome, and a cationic additive.

The lipid may be selected from the group consisting of 1,2-diacyl-sn-glycero-phosphocholin (PC), 1,2-diacyl-sn- glycero-phosphoglycerol (PG), 1,2-diacyl-sn-glycero- phosphoethanolamine (PE) and chlosterol (CH).

The cationic additive may be selected from the group consisting of l,2-diacyl-3-dimethylammonium propane (DAP), l,2-diacyl-3-trimethylammonium

propane (TAP), l,3-diacyl-2-carboxyspermyl-propylamide (DOS), spermine (SM), protamine sulfate (PS) and polyethyleneimine (PEI).

Also, the mixing ratio of the lipid to the cationic additive is preferably

80-90:10-20% by weight.

In the cationic liposome targeting sebaceous glands, PC, CH and the cationic additive are preferably mixed in a ratio of 60-70:20-30:10-20% by weight.

When PC, CH and the cationic additive are preferably mixed in a ratio of

60-70:20-30:10-20% by weight and the cationic liposome comprises four ingredients of PC, PG, CH and the cationic additive, or PC, PE, CH and the cationic additive, the four ingredients are mixed in a ratio of 60-70:20-30:10-20% by weight. BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of preferred embodiments of the present invention will be more fully described in the following detailed description,

taken accompanying drawings. In the drawings: FIG. 1 is a view showing ALA is delivered to a sebaceous gland;

FIG. 2A is a photograph illustrating a pilosebaceous unit in a state in which no substance is applied to skin (control group);

FIG. 2B is a photograph illustrating a pilosebaceous unit in a state in which ALA is applied alone to skin;

FIG. 2C is a photograph illustrating a pilosebaceous unit in a state in which a

topical formulation according to the present invention is applied to skin, the topical formulation being prepared by using a neutral liposome (hereinafter, to be abbreviated

as "N") and a cationic additive (TAP) as a cationic liposome, referred to as "N/TAP";

FIG. 2D is a photograph illustrating a pilosebaceous unit in a state in which a

topical formulation according to the present invention, N/SM, is applied to skin;

FIG. 2E is a photograph illustrating a pilosebaceous unit in a state in which a

topical formulation according to the present invention, N/PS, is applied to skin;

FIGS. 3 A and 3B are photographs of sebaceous glands exhibiting cell death or

destruction due to activation of ALA when treated by the processes of the prior art and

the present invention; and

FIGS. 4A through 4C are photographs illustrating the therapeutic efficacy before and after the topical formulation according to the present invention is administered to

patients afflicted with acne. BEST MODES FOR CARRYING OUT THE INVENTION

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

In order to verify that the topical formulation according to the present invention specifically acts on sebaceous glands, 1-20% by weight of ALA as a cationic medicinal agent is mixed with 80-99% by weight of a cationic liposome.

When the topical formulation is synthesized, 0.1-5% by weight of ALA and 10-20% by weight of a cationic liposome are added in an aqueous solution used as a

solvent.

The cationic liposome is preferably prepared by mixing lipids that make neutral

liposomes with a cationic additive.

Suitable lipids making neutral liposomes useful in the present invention include phospholipids exemplified by PC, PG, PE and CH, and suitable cationic additives useful in the present invention include DAP, TAP, DOS, SM, PS, and PEL These materials

are listed in Table 1.

Table 1

In preparing the formulation, when a cationic lipid, such as DAP, TAP or DOS, is used as a cationic additive, the additive is preferably added to ALA and lipid from an

initial stages and mixed together. When a water-soluble additive such as SM, PS or

PEI, is used as a cationic additive, ALA and the lipid are first mixed and the additive is then added to the mixture, thereby completing the formulation.

The cationic liposome is preferably prepared by mixing the lipid making the

neutral liposome and the cationic additive in a ratio of about 80-90 to about 10~20%

w/w.

Preferred examples of the cationic liposome include a formulation comprising

PC, CH and a cationic additive contained in a ratio of 60-70/20-30/10-20% w/w/w,

and a formulation comprising PC, PG or PE, CH and a cationic additive contained in a

ratio of 60-70/5-10/20-30/5-10% w/w/w/w.

In the present invention, ALA formulated with a cationic liposome, is applied to a site afflicted with acne to be absorbed into the sebaceous gland, to cause PplX to be expressed, followed by exposing the sebaceous gland with visible light, thereby caυsing

damage to the sebaceous gland cells.

Since ALA is a cationic medicinal agent for treatment of acne, it has low toxicity

while keeping its ionic characteristic when formulated with a cationic liposome rather

than a non-ionic or neutral liposome.

Also, since the human skin is mildly acidic, the cationic liposome has a higher penetrating or absorbing capability into the skin than the non-ionic or neutral liposome. Further, as described above, since ALA is positively charged, that is, acidic when present in physiological pH, its activity is considerably reduced in a neutral or acidic

solution. Taking this into consideration, the cationic liposome has advantages of enhancing the function of ALA as well as serving as a carrier. The functionally enhanced ALA definitely increased the level of the expression of PplX in the sebaceous

gland.

ALA formulation as a cationic liposome exhibits excellent penetration capability

and enables its increased absorption into the cell. As a result, ALA is transported to the sebaceous gland within a very shot time, so expression of PplX is explicitly shown.

This phenomenon cannot be observed by simply applying the non-ionic or neutral

liposome, suggesting that the cationic liposome enhances the function of ALA by

keeping the ionic characteristic of ALA, while addressing several prior art problems associated with selectivity, depth of penetration, penetration time and so on.

Further, according to the present invention, when ALA is sufficiently absorbed

into the sebaceous gland cells and the expression of PplX becomes explicit, followed by exposing the sebaceous gland applied with ALA to visible light, the sebaceous gland cells can be damaged. In other words, according to the present invention in which 0.1-5% of ALA as a cationic liposome formulation is applied to a target site afflicted with acne, followed by exposing the target site with visible light, the sebaceous gland cells can be selectively, effectively damaged, thereby greatly improving the therapeutic

effect of acne while minimizing adverse side effects.

The invention will now be more fully described by reference to the following examples, which are provided by way of illustration and are not intended to limit the

invention.

The liposomes and cationic additives used in various examples of the present

invention are presented in Table 1.

Formulation Example: Preparation of Liposomes

The liposomes of the invention were prepared from neutral liposomes and lipids mixed in various formulation ratios and dissolved at about 70°C. The resulting

solution was put into a previously warmed syringe and transferred to an extrusion set

connected to another syringe including ALA-containing physiological saline or 5%

dextrose aqueous solution. The solutions in both containers were well mixed by

pumping the syringes and the lipids were homogenized by repeated mixing steps several times. Thereafter, sonication was carried out at room temperature for about 3 to 5

minutes. Here, the concentration of the liposome in the aqueous solution was adjusted

to be about 100 to about 200 mg/ml, and ALA was contained in an amount of about 0.1 to about 5% by weight, on the basis of the total weight of the aqueous solution. When

a cationic lipid, such as DAP, TAP or DOS, was used as a cationic additive, the additive was added to ALA and the lipid at an initial stage and mixed together. When a

water-soluble additive, such as SM, PS or PEI, was used as a cationic additive, ALA and the lipid were first mixed, giving an ALA-lipid mixture, and the additive was then added to the ALA-lipid mixture, yielding final formulations Several topical formulations having different ratios were prepared using the

foregoing method.

Example 1: Preparation of cationic liposome- ALA (1%)

PC (14:0) 130 mg

CH 40 mg

Cationic additive (TAP) 30 mg

A topical formulation according to the present invention was prepared in the

same manner as in the Formulation Example, except that 1% by weight (10 mg) of ALA,

20% by weight of the cationic liposome containing the above listed ingredients and 1

ιn£ of physiological saline were used.

Example 2: Preparation of cationic liposome-ALA (1%)

PC (14:0) 120 mg

PG 20 mg

CH 40 mg

Cationic additive (TAP) 20 mg

A topical formulation according to the present invention was prepared in the same manner as in the Formulation Example, except that 1% by weight (10 mg) of ALA,

ιn£ of physiological saline were used.

Example 3: Preparation of cationic liposome -ALA (5%)

PC (14:0) 100 mg

PE 40 mg

CH 40 mg

Cationic additive (PS) 20 mg

A topical formulation according to the present invention was prepared in the

same manner as in the Formulation Example, except that 5% by weight (50 mg) of ALA,

mC of physiological saline were used.

Example 4: Preparation of cationic liposome-ALA (0.1%)

PC (14:0) 65 mg

CH 20 mg

Cationic additive (SM) 15 mg

A topical formulation according to the present invention was prepared in the

same manner as in the Formulation Example, except that 0.1% by weight (1 mg) of

ALA, 10% by weight of the cationic liposome containing the above listed ingredients

and 1 ml of 5% dextrose aqueous solution were used. Experimental Example 1: Entrapment Efficiency (EE) and Stability

As the concentration of ALA was decreased, the entrapment efficiency (EE

percent) was reduced. When the concentration was 100 mg/ml, the EE was about 40-60%. In a case of a liposome-medicinal agent for skin administration to skin, since isolation of the medicinal agent from the entrapped complex is not necessary, unlike in a case of a liposome-medicinal agent suitable for intravenous administration, the formulated liposome solution was entirely used for treatment. The cationic liposome-ALA formulation was stored in a refrigerator for about one month, and both the liposome and ALA were kept stable while exhibiting little change over time.

Experimental Example 2: Animal Experiments

(1) Applying to skin

Various ALA formulations were applied to the backs of white mice. Before application, hair was removed from each mouse. The results are shown in FIG. 2. No

red fluorescent light was detected from the control group without treatment with ALA

(FIG. 2A). When ALA was applied to the skin alone, the expression of PplX was

detected in the sebaceous gland after about 2 hours, and the highest level was shown after about 8 hours. Then, substantially no emission of red fluorescence was detected

after about 24 hours. Comparison of the results observed for the cases where ALA was

applied alone without liposome (FIG. 2B) and where cationic liposome-ALA was applied (FIGS. 2C-2E) indicated that the level of the red fluorescent light of PplX was

about 3 times higher for the latter case (ALA alone). This effect is presumably

attributed to functionally enhanced ALA due to remarkable penetrating and absorbing capability into the skin and increased cationic intensity, as described above.

(2) Exposing to sunlight

The topical formulation prepared in Example 2 was applied to the backs of white mice, followed by exposing to sunlight after 2 hours. At day 7, a change occurring to the sebaceous gland was observed. When compared with the control group (FIG. 3A), the sebaceous gland cell of the site treated with the medicinal agent according to the present invention showed selective death (FIG. 3B).

(3) Applying to patients with acne

The topical formulation prepared in Example 2 was applied to patients afflicted with acne once a day for a period of 5 days. After 2 weeks of observation, the efficacy

of the topical formulation prepared in Example 2 was evaluated. The results showed that the severity of their acne conditions was considerably alleviated. The results observed before the topical formulation was applied to the patients with acne (FIG. 4A)

and after the topical formulation was applied to the patients with acne (FIG. 4C) were

compared. The comparison results indicated that the acne conditions were considerably cured such that secretion of sebum was noticeably reduced after one week

(FIG. 4B) and that skin coloration slightly appeared and crust was removed after 2

weeks (FIG. 4C).

INDUSTRIAL APPLICABILITY

As described above, according to the present invention, use of the topical formulation containing the cationic medicinal agent and cationic liposome can keep the

stability and function of the cationic medicinal agent without changing the ionic

characteristic thereof. Also, the cationic liposome can reduce the toxicity of the

medicinal agent itself and can increase selectivity to and adsorption of the medicinal agent into a target site, that is, the sebaceous gland. Also, the cationic liposome can be penetrated into the target site within a short penetration time to a desired depth of penetration. That is, a large amount of the cationic medicinal agent can be selectively transported to the sebaceous gland, causing damage to the sebaceous gland. Also, the

effect of applying the formulation can be maximized by appropriately adjusting the viscosity.

The present invention has been described in detail. However, it should be

understood that the detailed description and specific examples, while indicating

preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will

become apparent to those skilled in the art from this detailed description.

Claims

What is claimed is:

1. A cationic liposome targeting sebaceous glands prepared by mixing a

lipid making a neutral liposome, and a cationic additive.

2. The cationic liposome targeting sebaceous glands of claim 1, wherein the lipid is selected from the group consisting of 1,2-diacyl-sn-glycero-phosphocholin (PC), 1,2-diacyl-sn- glycero-phosphoglycerol (PG), 1,2-diacyl-s/τ-glycero-

phosphoethanolamine (PE) and chlosterol (CH).

3. The cationic liposome targeting sebaceous glands of claim 1, wherein the cationic additive is selected from the group consisting of

l,2-diacyl-3-dimethylammonium propane (DAP), l,2-diacyl-3-trimethylammonium propane (TAP), l,3-diacyl-2-carboxyspermyl-propylamide (DOS), spermine (SM),

protamine sulfate (PS) and polyethyleneimine (PEI).

4. The cationic liposome targeting sebaceous glands of claim 1, wherein the

mixing ratio of the lipid to the cationic additive is 80-90:10-20% by weight.

5. The cationic liposome targeting sebaceous glands of any one of claims 1

through 3, wherein PC, CH and the cationic additive are mixed in the cationic liposome

in a ratio of 60-70:20-30:10-20% by weight.

6. The cationic liposome targeting sebaceous glands of any one of claims 1 through 3, wherein PC, PG, CH and the cationic additive or PC, PE, CH and the cationic additive are mixed in a ratio of 60-70:20-30:10-20% by weight.