HK1096851A - Use of sphingomyelinase to increase the levels of skin and mucosal ceramides - Google Patents

Description

Use of sphingomyelinase for increasing the content of skin and mucosal ceramides

The present application is a divisional application of the application with the filing date of 14/11/1997 and the filing number of 97180647.0 entitled sphingomyelinase composition and use thereof.

Technical Field

The present invention relates to the use of sphingomyelinase derived from gram-positive bacteria, gram-negative bacteria and lactic acid bacteria or mixtures thereof for increasing the level of skin and mucosal ceramides, and dermatological and cosmetic compositions suitable for topical application containing sphingomyelinase.

Background

Ceramide (ceramide) is a lipid metabolite recently proposed as an important intracellular messenger, which is released intracellularly under the stimulation of various mediators over several hours or is produced as a result of serum loss, and is associated with cell arrest in G0/G1 phase and apoptosis. Ceramides are now considered to be second messengers in the context of the sphingomyelin signal transduction pathway. It is released from sphingomyelin under the influence of sphingomyelinase, a form of sphingomyelin-specific phospholipase C. In cells, ceramides can affect growth and differentiation, regulate protein secretion, induce DNA fragmentation and apoptosis, increase cytokine synthesis and secretion. The molecular mechanisms underlying these various actions are not yet fully understood. More so known are extracellular stimulants that cause ceramide release. Exposing cells to exogenous sphingomyelinase or activationSphingomyelin is hydrolyzed rapidly after the stimulant of the source sphingomyelinase. Such agonists include TNF-alpha, Fas ligand, interleukin 1-beta, IFN-gamma, 1 alpha, 25-dihydroxyvitamin D₃And NGF.

Cosmetic compositions containing sphingomyelinase are known. Japanese patent publication 63216813 discloses such a composition: the sphingomyelinase obtained therein has the purpose of counteracting the physiological reduction of this enzyme in aging skin, thus promoting the conversion of sphingomyelin to ceramide, which brings about a favorable moisturizing effect on the skin and limbs.

However, the Japanese publication does not disclose or at least suggest that these compositions can be used therapeutically to treat skin disorders. Moreover, sphingomyelinase is obtained from tissues of higher animals (such as brain and liver) through a complicated and complicated extraction process.

The importance of ceramides in the metabolism of the skin can be clearly understood from the following.

The main cellular components of the epidermis are: keratinocytes, melanocytes, langerhans cells, fibroblasts, endothelial cells and macrophages. During inflammation and swelling, monocytes and polymorphonuclear leukocytes infiltrate the skin. On the other hand, the intracellular space is mainly composed of neutral lipids, glycoproteins, protein degradation products, desmosomes, active enzymes, sebaceous gland products and ceramides. As long as the structure of such "bricks and mortar" is intact, the skin is endowed with the functional role of both the protective layer and the selective permeation filter.

During the differentiation process of the epidermis (cell division starting at the basal layer, ending at the death of keratinocytes and the formation of lipid barriers), the cells change their lipid synthesis capacity. The result is that the basal layer of the epidermis is characterized by phospholipids and cholesterol, while the outermost layer is characterized by cholesterol, free fatty acids and, in particular, ceramides. The lipids of the stratum corneum, the main component of which consists of sphingolipids, play a critical role in maintaining the epidermal permeability barrier to water. The sphingolipids are extruded from lamellar bodies of epidermal granular cells. Ceramides (sphingolipids) which account for 43-46% of the stratum corneum are the main polar lipids of the stratum corneum, and they play a fundamental role in the barrier function of the skin against water leakage during cell adhesion and differentiation of the epidermis. Literature data indicate that ceramides are synthesized de novo in the epidermis, undergoing phospholipid-like intermediates. They are present in the stratum corneum in fairly high concentrations (up to 40% of the total lipids).

Like the appearance of the skin surface, its functional characteristics change with aging. Aged skin is characterized by a reduction in the water content of the stratum corneum and the associated reduction in the transdermal leakage of water. It has been shown that ceramide 2: the proportion of sphingolipids decreases with age. The age-related reduction of ceramides can cause dehydration of the skin, which has been observed during aging.

In addition, abnormal ceramide content (deficiency) has been detected in atopic eczema, dermatosis and dermatitis, particularly atopic dermatitis and psoriasis. Recently, an innate deficiency in ceramide 1 has been found in autosomal recessive sphingolipid deposition diseases. Also, the usual forms of sphingolipidosis are known, such as fabry disease, gaucher disease and tay-sachs disease. The sjogren's syndrome is associated with a deficiency of ceramides 1 and 6, with a concomitant breakdown of the normal skin barrier.

As mentioned above, it is clearly very advantageous to maintain a high level of ceramides in the skin. We will further understand that the use of sphingomyelinase is also advantageous at the mucosal level.

A large number of ceramide-containing products, obtained by extraction or synthetic methods, for dermatological and cosmetic applications are now available on the market. Topical ceramides have been proposed for remodeling the lipid barrier of the skin, which is altered by aging, drugs, detergents, physical agents, etc. Such external administration is unlikely to result in ceramide variations in nature and/or quantity due to age, anatomical location, seasonal factors, and disease. It is clear that external administration of ceramide only plays an additive role (endogenous ceramide + exogenous ceramide) and not a regulatory role (ceramide changes due to season, anatomical parts, possible disease processes in development, etc.).

Disclosure of Invention

We have now surprisingly found that sphingomyelinase is present in high levels in bacterial cells which is neutral rather than acidic.

It is therefore an object of the present invention to provide the use of sphingomyelinase derived from bacteria for the production of dermatological or cosmetic compositions suitable for topical application to increase the ceramide content of the skin and mucous membranes.

According to the invention, the sphingomyelinase is preferably extracted from gram-positive bacteria, gram-negative bacteria, lactic acid bacteria or a mixture thereof. The lactic acid bacteria should preferably be selected from the group consisting of: lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus buchneri (Lactobacillus buchneri), Lactobacillus casei (Lactobacillus casei), Lactobacillus catenulatum (Lactobacillus catenafare), Lactobacillus cellobiosus (Lactobacillus celliosus), Lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus curvatus (Lactobacillus curvatus), Lactobacillus delbrueckii (Lactobacillus delbrueckii), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus jensenii (Lactobacillus jensenii), Lactobacillus delbrueckii (Lactobacillus leiomyelii), Lactobacillus plantarum (Lactobacillus brevis), Lactobacillus plantarum (Bifidobacterium), Bifidobacterium longum (Bifidobacterium), Bifidobacterium lactis (Bifidobacterium), Bifidobacterium longum (Bifidobacterium longum), Lactobacillus plantarum (Bifidobacterium longum), Bifidobacterium longum (Bifidobacterium) and Bifidobacterium longum (Bifidobacterium) including Bacillus bifidum, Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium plantarum (Bifidobacterium plantarum), Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum), Bifidobacterium pseudolongum subspecies pseudolongum (Bifidobacterium pseudomonologum), lactococcus lactis (Steptococcus lactis), Streptococcus raffinosus (Streptococcus raffinosus) and Streptococcus salivarius (Streptococcus thermophilus).

According to a preferred embodiment of the invention, the cells are applied in the form of lyophilized cells or sonicated cells.

Sphingomyelinase can also be used as a skin penetration or absorption enhancer according to the invention, alone or in combination with other enhancers, for the preparation of pharmaceutical or cosmetic compositions suitable for transdermal administration.

It is a further object of the present invention to provide dermatological or cosmetic compositions characterized in that they comprise an amount of sphingomyelinase effective to increase the ceramide content in the skin or mucous membranes.

According to the invention, the sphingomyelinase comprised in such a composition is preferably extracted from gram-positive bacteria, gram-negative bacteria, lactic acid bacteria or mixtures thereof. The lactic acid bacteria should preferably be selected from the group consisting of: lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus catenulatum, Lactobacillus cellobiosus, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus jensenii, Lactobacillus delbrueckii subsp.

According to a preferred embodiment of the invention, the cells contained in the composition are in the form of lyophilized cells or sonicated cells.

The dermatological or cosmetic composition of the present invention preferably comprises 1 × 10²～1×10¹⁵CFU lactobacillus/gram composition.

The dermatological or cosmetic compositions of the invention may also comprise exogenous ceramides or products containing: exogenous ceramides, sphingomyelins, fatty acids, cholesterol, ceramidase inhibitors, protease inhibitors, immunomodulators, vitamins, growth factors, surfactants, emulsifiers, stabilizers, lipids, rheological additives, moisturizers, antioxidants, preservatives, colorants, lakes, pigments, auxiliary substances (e.g., acids, bases, propellants) and functional substances (astringents, anti-seborrheic agents, anti-dandruff agents, deodorants, skin cleansers, keratinogenic agents, moisturizers, anti-drying agents, smoothing agents, protectants, sunscreens, colorants, anti-depigmenting agents, emollients, restoratives, nutraceuticals (eutrophic agents), anti-wrinkle agents, anti-free radical agents, sclerosing agents, anti-stretch-markers (anti-stretch-markagens), vascular protectants, anti-rash agents, soothing agents, anti-cellulite agents, tonics, stimulants, anti-eruptive agents, anti-seborrheics, anti-seborrheic agents, anti-dandruff agents, anti-wrinkle agents, anti-stretch markers, anti-cellulite agents, anti-cellulite, Ultra-eluting agents (hypereluting agents), depilatory agents, nail protectors).

Finally, the dermatological or cosmetic composition of the invention may be formulated in the form of a liquid, semi-solid, solid or powder product, for example in the form of: creams, ointments, lotions, capsules, pearls, pessaries, mascara, eyewashes, toothpaste, mouthwash, lipstick, liposomes, soap, shaving soap, tonics, douches, enema, shampoos, anti-dandruff preparations, impregnated and/or medicated bandages or gauzes, patches, medicated emulsions, transdermal gels or patches.

Detailed Description

To confirm the ability of these bacteria to induce ceramide formation in skin, we performed experiments based on the detection of sphingomyelinase, which produces ceramide in human skin.

Method of producing a composite material

Determination of neutral and acidic sphingomyelinase in lactic acid bacteria

10mg of lyophilized Streptococcus salivarius thermophilus are resuspended in 500. mu.l of a buffer containing: 20mM HEPES (pH7.4), 10mM MgCl₂EDTA 2mM, DTT 5mM, NA 0.1mM₃VO₄0.1mM Na₂MoO₄30mM p-nitrophenyl phosphate, 10mM beta-glycerophosphate, 750mM ATP, 1. mu.M PMSF, 10. mu.M leupeptin, 10. mu.M pepstatin (Sigma Chemical Co.), and 0.2% TritonX-100 (for determining neutral sphingomyelinase activity) or 500. mu.l of 0.2% TritonX-100 (for determining acid sphingomyelinase activity). The samples thus prepared were then sonicated (time 1 min 50 sec, alternating 10 sec sonication and 10 sec intervals) using a Vibracell sonicator (Sonic and Materials inc., Danbury, CT). The sonicated sample was centrifuged at 14,000rpm at 4 ℃ for 30 minutes, the supernatant was removed, and the protein concentration was determined using the Bio-Rad Laboratories kit (Richmond, Calif.).

Mu.g of the sample was incubated at 37 ℃ for 2 hours in a buffer (final volume 50. mu.l) containing 20mM HEPES, 1mM MgCl₂(pH7.4) and 2.25. mu.l of [ N-methyl-¹⁴C sheath phosphorus to (SM) (0.2. mu. Ci/ml, a.s.56.6mCi/mmol, Amersham).

To detect the activity of acid sphingomyelinase, 100. mu.g of the bacterial lysate were placed in a buffer (final volume 50. mu.l) containing 250mM sodium acetate, 1mM EDTA (pH 5.0) and 2.25. mu.l of N-methyl-¹⁴C, SM. The reaction was stopped by the addition of 250. mu.l chloroform: methanol: acetic acid (4: 2: 1). Phospholipids were extracted, plated and analyzed by TLC and hydrolysis of SM by autoradiography and liquid scintillation.Sphingomyelinase present in the sonicated bacteria is expressed in units/mg protein. 1 unit of neutral sphingomyelinase hydrolyses 1 micromole per minute of sphingomyelin at 37 ℃ and a pH of 7.4. 1 unit of acid sphingomyelinase hydrolyses 1 nanomole of SM to N-acetyl sphingosine and phosphorylcholine per hour at 37 ℃ at pH 5.0.

Preparation and treatment (duration and mode) of the cream

Preparation of a composition containing sonicated lactic acid bacteria (20 g of each base cream in 2 tubes and sonicated lactic acid bacteria in 1 small tube (1X 10)¹²CFU) in 20ml of water. In 8 volunteers, the effect of daily application of the cream on the ceramide content of the stratum corneum of the forearm epidermis was analyzed, as shown in table 1 below:

TABLE 1

Serial number	Sex	Age (age)
			12345678	Male, female, male, female	4539292733323825

The subjects were instructed to self-administer the drug twice daily (about 1ml, once in the morning and once in the evening) with the control cream and the lactic acid bacteria-containing experimental cream. The control cream was applied daily to the right forearm and the experimental cream was applied to the left forearm. Both creams were rubbed vigorously until they were completely absorbed. Before the start of cream application (T0) and one week after the start of cream application (T1), the forearm was rinsed with 250ml of 99.5% ethanol and the lipids of the stratum corneum of the epidermis were collected. The ethanol extract was concentrated on a rotary evaporator and then evaporated to dryness. This dry sample was dissolved in 2ml of chloroform, dried under nitrogen, and subjected to DAG kinase quantification of ceramide (Amersham, Buckinghamshire, Great Britain). After three extraction procedures, the lipid was again dried under nitrogen, dissolved in 100. mu.l of chloroform, and subjected to Thin Layer Chromatography (TLC) using chloroform: methanol: acetic acid (65: 15: 5, v/v/v) as a working solvent. Phosphorylated ceramides were determined by autoradiography. The membrane corresponding to ceramide-1-phosphate was cut off and the radioactivity present was counted in a beta-counter using scintillation fluid. The amount of ceramide present was determined based on a standard curve obtained from authentic ceramide (type III, bovine brain ceramide; Sigma Chemical co., st.

Results

Sphingomyelinase Activity in lactic acid bacteria

The value of neutral sphingomyelinase activity in the sonicated lactic acid bacteria samples was about 2X 10^-7Unit/mg bacteria. No acid sphingomyelinase activity was detected.

Effect of sonicated Lactobacillus on ceramide content in skin

The ceramide content of the ethanol extract obtained as described in the "methods" section is given in table 2 below, and the corresponding autoradiogram is shown in fig. 1. It may be noted that the use of both creams was analyzed to cause an increase in the ceramide content in the skin of the forearms of all subjects, regardless of the basic contents of the subjects. However, the increase was more pronounced after application of the cream containing the lactic acid bacteria. Furthermore, the effect of the experimental cream on the ceramide content can be detected earlier than that caused by the basic cream, thus indicating a faster action of the experimental cream.

TABLE 2

Serial number	Right forearm		Left forearm
						T0	T1	T0	T1
12345678	0223883004003010	056750034750048028280	014724.2243523618012	1,9412987003001,390340270278

Claims (5)

1. Use of sphingomyelinase for the manufacture of a dermatological and/or cosmetic composition suitable for topical application for increasing the content of mucosal ceramides, characterized in that the sphingomyelinase is derived from gram-positive bacteria, gram-negative bacteria, lactic acid bacteria or mixtures thereof.

2. Use according to claim 1 for the manufacture of a dermatological composition suitable for topical application for the prevention or treatment of atopic eczema, dermatosis and dermatitis, in particular atopic dermatitis, psoriasis, fabry disease, gaucher disease, tay-sachs disease and sjogren's syndrome.

3. Use according to claim 1 or 2, characterized in that the lactic acid bacteria are selected from the group consisting of: lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus buchneri (Lactobacillus buchneri), Lactobacillus casei (Lactobacillus casei), Lactobacillus catenulatum (Lactobacillus catenae), Lactobacillus cellobiosus (Lactobacillus celliosus), Lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus curvatus (Lactobacillus curvatus), Lactobacillus delbrueckii (Lactobacillus delbrueckii), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus jensenii (Lactobacillus jensenii), Lactobacillus delbrueckii (Lactobacillus brevis), Lactobacillus delbrueckii), Bifidobacterium longum (Lactobacillus sublactis), Lactobacillus plantarum (Lactobacillus bifidus), Lactobacillus plantarum (Lactobacillus), Lactobacillus bifidus (Bifidobacterium), Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium longum (Bifidobacterium) and Bifidobacterium (Bifidobacterium longum (Bifidobacterium) bifidobacteria, Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium plantarum (Bifidobacterium plantarum), Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum), Bifidobacterium pseudolongum subspecies pseudolongum (Bifidobacterium pseudomonologum), lactococcus lactis (Steptococcus lactis), Streptococcus raffinosus (Streptococcus raffinosus) and Streptococcus salivarius (Streptococcus thermophilus).

4. Use according to claim 1 or 2, characterized in that the dermatological or cosmetic composition is suitable for transdermal application.

5. Use according to claim 1 or 2, characterized in that sphingomyelinase is used in the form of freeze-dried cells or sonicated cells.