WO2019057899A1

WO2019057899A1 - Aqueous composition comprising at least one phosholipid and further at least one terpene with acaricidal activity against demodex

Info

Publication number: WO2019057899A1
Application number: PCT/EP2018/075613
Authority: WO
Inventors: Tomasz Borowik; Marek Langner; Magdalena Przybylo
Original assignee: Lipid Systems Sp. Z.O.O.
Priority date: 2017-09-22
Filing date: 2018-09-21
Publication date: 2019-03-28

Abstract

The present invention relates to an aqueous composition comprising at least one phospholipid and further at least one terpene with acaricidal activity against Demodex. The present invention further relates to the aqueous composition for use as a medicament. The present invention also relates to the aqueous composition for use in the treatment of Demodex-induced inflammations in a human or a non-human mammal.

Description

AQUEOUS COMPOSITION COMPRISING AT LEAST ONE PHOSHOLIPID AND FURTHER AT LEAST ONE TERPENE WITH ACARICIDAL ACTIVITY AGAINST

DEMODEX

The present invention relates to an aqueous composition comprising at least one phospholipid and further at least one terpene with acaricidal activity against Demodex. The present invention further relates to the aqueous composition for use as a medicament. The present invention also relates to the aqueous composition for use in the treatment of Demodex- induced inflammations in a human or a non-human mammal.

Background Art

Demodex mites (of the class Arachnid and order Acarina) are microscopic ectoparasites that commonly infest the pilosebaceous unit of the skin of humans. Among a wide range of reported species, two {Demodex folliculorum, Demodex brevis) are typically found on the human body surface. Demodex prevalence increases with age and is observed in 84% of the population at age 60 and 100% of the population over the age of 70.

Both Demodex species preferentially gather at the same skin area of the face, cheeks, forehead, nose, and external ear tract. Active sebum excretion favours habitat and breeding in the sebaceous glands and hair follicles. This involves the Meibomian glands, which are sebaceous glands at the rim of the upper and lower eyelids, and the hair follicles of the eyelashes. Debris and waste generated by mites accumulate at the root of lashes forming cylindrical dandruff. Mites also mechanically block sebaceous ducts.

Uncontrolled skin Demodex infestation (demodicosis) is usually related to a suppressed or compromised immune system. Demodicosis is most often seen in folliculitis, an inflammation of the hair follicles, which starts with the introduction of a skin pathogen (e.g. Demodex) to the hair follicle. Depending on the location, an uncontrolled skin Demodex infestation may be the cause of blepharitis, chalazia, rosacea, or acne in humans.

Blepharitis is a common eye condition characterized by inflammation of the eyelid, resulting in inflamed, irritated, itchy, and redden eyelid. Depending on the anatomic location, blepharitis is classified in anterior blepharitis, an infestation of the lid margin, and posterior blepharitis, an infestation of the Meibomian glands, also known as Meibomian gland disease. Chalazia are non-painful cysts on the eyelid resulting from chronic inflammation of a Meibomian gland. The inflammation may be caused by the blockage in one of the Meibomian glands of the upper and lower eyelids following a Demodex entry.

Rosacea typically affects the face and results in redness, pimples, swelling and small and superficial dilated blood vessels. Acne is a long-term skin disease that occurs when hair follicles are clogged with dead skin cells and oil from the skin. It is characterized by blackheads or whiteheads, pimples, oily skin, and possible scarring. It primarily affects areas of the skin with a relatively high number of oil glands, including the face, upper part of the chest, and back.

A Demodex infestation as the cause for blepharitis or chalazia in humans may be diagnosed by slit-lamp examination to detect cylindrical dandruff at the root of lashes. Lash sampling and microscopic examination provide a diagnosis by identifying the Demodex mites in lashes with cylindrical dandruff. In vivo confocal microscopy (IVCM) has been used as a noninvasive method to diagnose Demodex infestation and allows a complete examination of the follicle with detecting mites burrowed deep into sebaceous glands. A Demodex infestation as the cause of rosacea or acne in humans may be e.g. diagnosed by skin surface biopsies using quick hardening polymers such as cyano aery late or by skin scrapings.

Demodex mites also infest mammalian quadrupeds, in particular mammalian domestic animals such as cattle (Demodex bovis), goats (Demodex caprae), guinea pigs (Demodex caviae), horses (Demodex equi), sheep (Demodeex ovis), pigs (Demodex phyloides), dogs (Demodex canis, Demodex cornei, Demodex injai) and cats (Demodex cati, Demodex catoi). Under normal conditions, the infestation does not produce any clinical signs or disease.

Increased stress or a compromised immune system in domestic animals increase the sensitivity to and an overpopulation of Demodex. The mites burrowed into hair follicles and sebaceous glands of the respective animal result in a demodicosis, also called demodectic mange. Demodicosis or demodectic mange may cause pustules, redness, scaling, hair loss, and severe infections of the skin. The demodectic mange presents a difficult clinical problem for veterinarians, as it can involve the face and the entire body of the animal in some cases.

A diagnosis of a Demodex infestation in mammalian quadrupeds may be obtained by the inspection of skin biopsies or skin scrapings. Inflammations caused by Demodex may be treated systemically or topically. Topical administration comprises the application of the active agent to body surfaces such as the skin or mucous membranes to treat conditions via a large range of base formulations such as creams, foams, gels, lotions, and ointments.

Terpenes are a class of organic compounds produced by a variety plants and some insects. Terpenes are the primary constituents of essential oils of many types of plants. The basic molecular formula of terpenes are multiples of isoprene units. Hemiterpenes consist of a single isoprene unit, monoterpenes consist of two isoprene units, sesquiterpenes consist of three isoprene units, diterpenes consist of four isoprene units, and so on.

Monoterpenes classify in acyclic, monocyclic, and bicyclic monoterpenes. Sesquiterpenes classify in acyclic, monocyclic, bicyclic, and tricyclic sesquiterpenes. A broad range of the biological properties of terpenes has been described, including cancer chemopreventive effects, antihyperglycemic and anti- inflammatory activities, antiviral, antimicrobial, antifungal, and acaricidal activities. Acaricides are active agents that kill members of the arachnid subclass Acari, which includes ticks and mites.

It has been previously described that essential oils or specific components are capable of treating and ameliorating Demodex-caused inflammations. Essential oils form tea trea, sage, or peppermint were found to decrease the in vitro survival of Demodex mites (Sedzikowska A. et al, J Bacteriol Parasitol, 2015, 6:3). Isolates of essential oils such as terpinen-4-ol or carvone have been found to decrease the survival time of Demodex in vivo (WO 2006/119174).

Essential oils for topical administration have been disclosed in combination with Vaseline (WO 2009/032773), ethanol, mineral oil, baby shampoo (WO 2006/119174), triglycerides, vegetable oils, or mineral oils (WO 2014/167552, US 2012/0121694). Components of essential oils such as terpinen-4-ol for topical administration have been disclosed in combination with mineral oil (Tighe S et al.,Tansl Vis Sci Technol, 2013, 3(7), Art. 2).

Terpenes are very little or entirely insoluble in water. Nevertheless, water-based compositions may be considered as preferred formulations when easily prepared and low priced, while ensuring an efficient delivery of the active agent at the treated skin areas.

Starting from the prior art, the problems solved by the present invention may be described as the: (i) provision of an improved composition of an acaricidal terpene, (ii) an improved composition of an acaricidal terpene for use as a medicament, or (iii) an improved composition of an acaricidal terpene for use in the treatment of Demodex-mduced inflammations. Detailed Description of the Present Invention

In a first aspect, the present invention provides an aqueous composition comprising at least one phospholipid and further at least one terpene with acaricidal activity against Demodex.

Favourably, the aqueous compositions of the present invention comprise high concentrations of terpenes and display a good skin adhesion. The good skin adhesion ensures an efficient delivery of the terpenes into the skin areas affected by Demodex. Further, when the aqueous compositions of the present inventions are administered on the skin areas of the eye, the good adhesion prevents their flow into the eye chamber. In addition, lipids in the composition will integrate with the stratum corneum supporting the reconstruction of the skin damaged by Demodex.

The acaricidal activity of the compositions of the present invention may be measured by determining the reduced Demodex survival time in the respective composition relative to a control. The control may be e.g. terpene alone or water. All lipids that contain phosphorus are called phospholipids. Phospholipids are known for their use in pharmaceutical formulation. Natural or synthetic phospholipids are used in oral, dermal, and parenteral products. Phospholipids are surface-active, amphiphilic molecules, which comprise a polar head group and a lipophilic tail. Because of this amphiphilic character, they are used as emulsifier, wetting agent, solubilizer, and liposome former. Liposomes itself are used as drug delivery systems, solvents and permeability enhancers.

Without being bound to theory, it is believed that the phospholipids of the present composition essentially form liposomes. Hydrophobic active agents may be inserted into the bilayer membrane and hydrophilic molecules may be entrapped in the aqueous centre. It is preferred that in the present aqueous composition the phospholipids are present as liposomes. It is preferred that the present aqueous composition comprises at least one natural phospholipid. Preferably, the present aqueous composition comprises at least one natural zwitterionic phospholipid. Preferably, the present aqueous composition comprises at least one natural zwitterionic phospholipid and at least one natural anionic phospholipid.

Preferably, the natural zwitterionic phospholipid is selected from the group consisting of phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), sphingomyelin (SM), cholesterol (CH), and combinations thereof. Even more preferably, the natural zwitterionic phospholipid is PC. Preferably, the natural anionic phospholipid selected form the group consisting of phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatic acid (PA), cardiolipin, and combinations thereof. More preferably, the natural anionic phospholipid is selected from the group consisting of PS, PG, and combinations thereof. Even more preferably, the natural anionic phospholipid is PS.

In a more preferred embodiment, the present aqueous composition comprises natural PC and at least one natural anionic phospholipid selected from the group of phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), phopshatic acid (PA), cardiolipin, and combinations thereof. Preferably, the present aqueous composition comprises natural PC and natural PS.

It is preferred that the present invention comprises at least one synthetic phospholipid. Preferably, the present aqueous composition comprises at least one synthetic zwitterionic phospholipid. Preferably, the present aqueous composition comprises at least one synthetic zwitterionic phospholipid and optionally at least one synthetic anionic phospholipid and optionally at least one synthetic cationic phospholipid.

It is preferred that the synthetic zwitterionic phospholipid is selected from the group consisting of saturated or unsaturated synthetic PC or PE. In a more preferred embodiment, the synthetic zwitterionic phospholipid is selected from the group consisting of 12:0, 13:0, 14:0, 15:0, 16:0, 17:0, 18:0, 19:0, 20:0, 14: 1, 16: 1, 18: 1, 18:2, 20:1 PC or PE, and combinations thereof. More preferably, the synthetic zwitterionic phospholipid is selected from 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, 16:0 PC), l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC, 18:0 PC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 18: 1 PC), and combinations thereof.

It is further preferred that the synthetic anionic phospholipid is selected from the group consisting of saturated or unsaturated synthetic PS or PG. In a more preferred embodiment, the synthetic phospholipid is selected from the group consisting of 12:0, 14:0, 16:0, 18:0, 18: 1 PS or PG, and combinations thereof. More preferably, the synthetic anionic phospholipid is selected from l,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS, 16:0 PS), 1 ,2-distearoyl- sn-glycero-3-phospho-L-serine (DSPS, 18:0 PS), l ,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS, 18: 1 PS), and combinations thereof.

It is further preferred that the synthetic cationic phospholipid selected from the group consisting of dimethyldioctadecylammonium (DDAB) and l,2-stearoyl-3- trimethylammonium-propane (TAP). Preferably the synthetic cationic phospholipid is selected from 18:0 DDAB, l,2-dioleoyl-3-trimethylammonium-propane (DOTAP, 18: 1 TAP), and combinations thereof.

Preferably, the aqueous composition of the present invention comprises at least one synthetic zwitterionic phospholipid and at least one synthetic anionic phospholipid. In a more preferred embodiment, the present aqueous composition comprises DOPC and DOPS.

It is further preferred that the aqueous composition of the present invention comprises a mixture of at least one natural phospholipid and at least one synthetic phospholipid. Preferably, the natural and phospholipids and synthetic phospholipids are as mentioned above. Preferably, the present aqueous composition comprises at least one synthetic zwitterionic phospolipid and at least one natural anionic phospholipid. Preferably, the present aqueous composition comprises at least one natural zwitterionic phospholipid and at least one synthetic anionic phospholipid. Preferably, the present aqueous composition comprises natural zwitterionic PC and/or PE, preferably PC, and synthetic anionic DOPS and/or DSPS, preferably DOPS.

It is preferred, that the weight ratio of the at least one zwitterionic phospholipid to the at least one anionic lipid, irrespective whether natural and/or synthetic, is in the range of 5:0.1 to 5: 1, preferably 3: 1 to 5 : 1.

It is preferred that the at least one terpene is selected from the group consisting of monocyclic monoterpenes, bicyclic monoterpenes, and combinations thereof.

It is more preferred the monocyclic monoterpenes are selected from the group consisting of terpinen-4-ol, a-terpinene, γ-terpinene, limonene, carvone, and combinations thereof. It is even more preferred that the monocyclic monoterpenes are selected from the group consisting of terpinen-4-ol, limonene, and combinations thereof. It is particularly preferred that the monocyclic monoterpene is terpinen-4-ol.

It is more preferred that the bicyclic monoterpenes are selected from the group consisting of a-pinene, sabinene, 1,4-cineole, 1,8-cineole, and combinations thereof. It is more preferred that the bicyclic monoterpenes are selected from the group consisting of sabinene, 1,8-cineole, and combinations thereof. In case not otherwise stated, the compounds comprise all possible constitutional and spatial isomers. It is further preferred that the aqueous composition comprises at least one fatty acid. Preferably, the fatty acid is selected from the group consisting of saturated fatty acids, unsaturated fatty acids, and combinations thereof. Preferably, the fatty acid is selected from the group of long-chain fatty acids with an aliphatic chain length in the range of C13-C20. Preferably, the fatty acid is selected from the group consisting of oleic acid, linoleic acid, and combinations thereof.

It is further preferred that the aqueous composition of the present invention comprises an alcohol selected from the group consisting of ethanol, propylene glycol, and combinations thereof. Preferably the alcohol is propylene glycol. It is preferred that the weight ratio of terpene to phospholipid in the aqueous composition of the present invention is in the range of 1 :0.5 to 1 :50, preferably 1 : 1 to 1 :20, preferably 1 :2 to 1 : 10, preferably 1 :2 to 1 :5, preferably 1 :2.5 to 1 :3.5.

It is preferred that the aqueous composition of the present invention comprises:

1 to 15 wt.-% terpene,

10 to 40 wt.-%.-% phospholipid,

45 to 89 wt.-% water, and

0 to 15 wt.-% ethanol or propylene glycol, preferably propylene glycol,

relative to the total weight of the composition.

It is further preferred that the aqueous composition of the present invention comprises 5 to 15 wt.-%, more preferably 5 to 12 wt.-% terpene relative to the total weight of the composition.

It is further preferred that the aqueous composition of the present invention comprises: 5 to 15 wt.-% terpene,

15 to 40 wt.-% phospholipid,

45 to 85 wt.-% water, and

0 to 15 wt.-% ethanol or propylene glycol, preferably propylene glycol,

relative to the total weight of the composition.

It is further preferred that the aqueous composition of the present invention comprises: 5 to 12 wt.-% terpene,

23 to 35 wt.-% phospholipid, wherein the phospholipid is preferably comprised by a zwitterionic phospholipid and optionally an anionic phospholipid,

53 to 72 wt.-% water, and 0 to 15 wt.-% ethanol or propylene glycol, preferably propylene glycol,

relative to the total weight of the composition.

53 to 72 wt.-% water, and

1 to 10 wt.-%, preferably 3 to 7 wt.-%, ethanol or propylene glycol, preferably propylene glycol, relative to the total weight of the composition. Preferably in the present aqueous compositions, the zwitterionic phospholipids and anionic phospholipids are present in a weight ratio in the range of 5:0.1 to 5: 1, preferably 3 : 1 to 5: 1.

The present aqueous compositions are prepared by mixing the phospholipid and terpene at temperatures in the range of 30 to 50 °C, preferably 35 to 45 °C, and preferably for 1 to 10 h, preferably 2 to 8 h, preferably 2 to 4 h.

The phospholipid/terpene mixture is further mixed with water and optionally an alcohol at temperatures in the range of 30 to 50 °C, preferably 35 to 45 °C, and preferably for 1 to 10 h, preferably 2 to 8 h, preferably 2 to 4 h, so that the aqueous compositions of the present invention are provided. In a second aspect, the present invention provides aqueous compositions comprising at least one phospholipid and further at least one terpene with acaricidal activity against Demodex for use as medicament. Preferably, the aqueous compositions comprising at least one phospholipid and further at least one terpene with acaricidal activity against Demodex are provided for use as medicament in a human or non-human mammal. The water-based compositions of the present inventions may be administered in the form a topical solution, lotion, cream, ointment, gel, foam, paste, or tincture. The compositions of the present invention may be provided in a tube, a jar, a bottle, a bottle squeezer, a dropper, a spray bottle, or as wet wipes impregnated therewith.

Suitable excipients which may be further added to the compositions of the present invention are fillers, humectants, emulsifiers, solubilizers, buffers, thickeners, binders, antioxidants, preservatives, absorption enhancers, and combinations thereof. In a third aspect, the present invention provides an aqueous composition comprising at least one phospholipid and further at least one terpene with acaricidal activity for use in the treatment and prophylaxis of Demodex- induced inflammations in a human.

When used in the treatment of a human, the inflammations are selected from the group consisting of Demodex- induced blepharitis, Demodex- induced rosacea, Demodex- induced acne and Demodex- induced Meibomian gland dysfunction.

In a forth aspect, the present invention provides an aqueous composition comprising at least one phospholipid and further at least one terpene with acaricidal activity for use in the treatment and prophylaxis of Demodex-mduced inflammations in a non-human mammal. When used in the treatment of a non-human mammal, the inflammations are selected from the group consisting of demodectic mange.

Preferably, the non-human mammal is a domestic animal, preferably selected from the group consisting of cattle, goats, guinea pigs, horses, sheep, pigs, dogs, and cats, more preferably selected from the group consisting of dogs and cats.

Examples

Adhesive force measurement

Adhesive force values between skin and terpene compositions of the present inventions were determined with a torsion balance (WTW, Techniprot, Poland) relative to water. For each composition a fresh skin sample was used. Every composition was measured in triplicate with standard deviation value smaller than 5% and each skin sample was used for one composition measurement only. For each individual skin sample the adhesive force value between water and skin was determined first, followed by the determination of the adhesive force between composition and skin. Skin was obtained from Polish Landrace pigs that were sacrificed for non-scientific purposes before the skin was harvested. Samples of whole skin were excised from the shank area. Strips of skin samples were cut at a thickness of approximately 1 mm using a scalpel. Then, the skin samples of 15 x 15 mm were prepared for the adhesive force value measurements. First, the weight of skin samples was determined. A single 15 x 15 mm piece of skin was stapled, with stratum corneum side facing out to give the area of contact with the surface of the examined medium (water) equal to 4 x 15mm (60 mm²). The piece of skin was connected to the torsion balance arm with a 50 mm long silk tread.

4 ml water were transferred into a disposable tissue culture dish, the skin attached to the thread was immersed in the water and the adhesive force value between the water and the skin was determined. The skin was dried with a paper towel and weighted again. 4 ml composition of interest were transferred into a fresh, 35 mm² disposable polystyrene tissue culture dish (VWR, United States), the skin was immersed in the composition and the adhesive force value between the respective composition and the skin was determined.

The adhesive force values between skin and measured samples were calculated as follows:

where:

Fn - determined force value

F - force needed to break contact between skin and sample Q - weight of the skin sample [mN]

P - force needed to break contact between skin and water

In vitro Demodex mites survival time determination

Demodex mites were collected from eyelashes of diagnosed patients. Three eyelashes were collected from each eye, by means of tweezers. Immediately after collection, the eyelashes were placed on a slide and examined for Demodex presence and their motility under 200x magnification. Vital mites were placed in a drop of tested substance and the motility of mites was observed continuously from 0-30 min, then every 10 minutes up to the end of the experiment after 180 min. Demodex survival time was measured starting from the substance application on mites ending with the lack of mite signs of vitality. Movements of legs were used as the survival indicator. As a control, Demodex survival time in saline (140 mM NaCl) was used.

Preparation of composition

Terpinen-4-ol was mixed with phospholipids and optionally propylene glycol at 40 °C, 200 rpm and 0.4 bar for 3 h in a thermostated mixing device (UMC5 electronic, Stephan Machinery GmbH, Germany) to obtain an amphiphilic phase. The amphiphilic phase was hydrated by the addition of water or water + propylene glycol at 40 °C, 200 rpm and 0.4 bar for 12 h (UMC5 electronic, Stephan Machinery GmbH, Germany) to give a high viscosity composition (Ex. 1 to Ex. 5). A sample prepared as described was further mixed with a thickener at 25 °C, 200 rpm and 0.4 bar for 180 min (Ex. 6).

As a control terpinen-4-ol only was used (Control).

Results

Clearly, the compositions according to the present invention are superior to the use of terpinen-4-ol alone. The addition of alcohol or alcohol and negatively charged phospholipid (DOPS Avanti, USA) further increase the adhesion force.

Table 1

Demodex survival time in saline (140 mM NaCl) was found to be longer than 180 min the endpoint of the measurement.

Claims

Aqueous composition comprising at least one phospholipid and further at least one terpene with acaricidal activity against Demodex.

The aqueous composition of claim 1, wherein the at least one phospholipid is a zwitterionic phospholipid.

The aqueous composition of claims 1 or 2, wherein the at least one terpene is selected from the group consisting of monocyclic monoterpenes, bicyclic monoterpenes, and combinations thereof.

The aqueous composition of claim 3, wherein the monocyclic monoterpenes is selected from the group consisting of terpinen-4-ol, a-terpinene, γ-terpinene, limonene, carvone, and combinations thereof.

The aqueous composition of claim 3, wherein the bicyclic monoterpenes is selected from the group consisting of a-pinene, sabinene, 1,4-cineole, 1,8-cineole, and combinations thereof.

The aqueous composition of any of claims 1 to 5, further comprising at least on further compound selected from the group consisting of ethanol, propylene glycol, and combinations thereof.

The aqueous composition of any of claims 1 to 6, wherein the ratio of terpene to phospholipid is in the range of 1 :0.2 to 1 :50, preferably 1 : 1 to 1 :20, preferably 1 :2 to 1 : 10, preferably 1 :2 to 1 :5, preferably 1 :2.5 to 1 :3.5.

The aqueous composition of any of claims 1 to 7, comprising:

1 to 15 wt.-% terpene,

10 to 45 wt.-% phospholipid,

45 to 89 wt.-% water, and

0 to 15 wt.-% ethanol or propylene glycol, preferably propylene glycol,

relative to the total weight of the composition

9. Aqueous composition comprising at least one phospholipid and further at least one terpene with acaricidal activity against Demodex according to any of claim 1 to 9 for use as a medicament in a human or non-human mammal.

10. Aqueous composition comprising at least one phospholipid and further at least one terpene with acaricidal activity against Demodex according to any of claims 1 to 9 for use in the treatment and prophylaxis of Demodex- induced inflammations in a human or a non- human mammal.

11. The aqueous composition of claim 10 for use in the treatment and prophylaxis of Demodex-induced inflammations in humans, wherein the inflammations are selected from the group consisting of Demodex-induced blepharitis, Demodex-induced rosacea, Demodex- induced acne and Demodex- induced Meibomian gland dysfunction.

12. The aqueous composition of claim 10 for use in the treatment and prophylaxis of Demodex-induced inflammations in non-human mammals, wherein the inflammation is demodectic mange.