WO2016051179A1

WO2016051179A1 - Composition comprising lactoferrine and methods of treatment

Info

Publication number: WO2016051179A1
Application number: PCT/GB2015/052866
Authority: WO
Inventors: John Ridden; Mike Davies; David Cook; Ian Kimber; Christine RIDDEN
Original assignee: Blueberry Therapeutics Limited
Priority date: 2014-09-30
Filing date: 2015-09-30
Publication date: 2016-04-07
Also published as: GB201417248D0

Abstract

The present invention relates to a composition for use in the treatment or prevention of a dermatological condition or in the formulation of a cosmetics product, the composition comprising a polymer capable of forming nanoparticles and lactoferrin and/or derivatives thereof. The composition is particularly suited to the treatment or prevention of acne and/or atopic dermatitis. The invention also relates to novel uses of polyhexamethylene biguanide (PHMB).

Description

COMPOSITION COMPRISING LACTOFERRINE AND METHODS OF TREATMENT

Technical Field of the Invention

The present invention relates to a composition for the treatment of acne, atopic dermatitis and other related skin conditions. Background to the Invention

Acne is a disease of the sebaceous hair follicles, often called pores. At the base of each hair follicle is a gland called the sebaceous gland, which produces sebum. Sebum is an oily substance that keeps the skin moist and pliable, which under normal circumstances travels along the hair follicle to the surface of the skin. A blemish begins approximately 2-3 weeks before it appears on the skin's surface. As the skin renews itself, the old cells die and slough off. When cells are shed unevenly and clump together with the sebum it forms a plug. Sebum which normally drains to the surface gets blocked and bacteria begin to grow. The rapid growth of the bacteria in combination with the accumulated sebum cause the follicle to enlarge and result in a mild form of acne called comedones, which are non-inflammatory. Both whiteheads and blackheads start out as a "microcomedone" and then become skin blemishes called comedones, either a whitehead or a blackhead. Acne is trapped sebum and bacteria (propionibacterium acnes) growing in a plugged follicle. Sebaceous glands are most numerous on the face, chest, back, neck and scalp; consequently, these are the most common sites of acne. The most common factors that cause acne are hormones, increased sebum production, bacteria (Propionibacterium Acnes), and changes inside of the hair follicle. Acne may progress to an inflammatory type of acne lesions that are red in color called papules, pustules and nodules.

There are many types of acne, ranging in severity from mild to severely disfiguring. Acne vulgaris is the most common form of acne which includes several types of pimples. These acne lesions include blackheads, whiteheads, papules, pustules, nodules and cysts.

Mild to moderate acne vulgaris is characterized by whiteheads, blackheads, papules, and pustules. A whitehead is formed when a pore is completely blocked, trapping sebum, bacteria, and dead skin cells below the skin surface causing a white appearance on the surface. Whiteheads are normally quicker in life cycle than blackheads. A blackhead is formed when a pore is only partially blocked, allowing some of the trapped sebum, bacteria, and dead skin cells to drain to the surface slowly. The black colour is due to a reaction of the skin's own pigment, melanin, reacting with the oxygen in the air. A blackhead tends to be a stable structure. Blackheads can often take a long time to clear because the contents very slowly drain to the surface. Papules are small, red, tender bumps with no head. Papules are the earliest stage in the development of what are normally considered the typical "pimple". Papules are an intermediate in the progression of acne between the non-inflammatory and inflammatory stages. Pustules are similar to whiteheads, but are inflamed, and appear as a red circle with a white or yellow center.

Severe acne vulgaris is characterized by nodules and cysts. Nodular acne consists of acne spots which are much larger, can be quite painful, and can sometimes last for months. Nodules are large, hard bumps under the skin's surface. Scarring is common with nodules. An acne cyst can appear similar to a nodule, but is pus-filled, and can been described as having a diameter of 5 mm or more across. They can be painful and scarring is common with cystic acne.

Acne rosacea can look similar to the aforementioned acne vulgaris, and the two types of acne are sometimes confused for one another. Rosacea affects millions of people, most of whom are over the age of 30. It appears as a red rash which is normally confined to the cheeks, nose, forehead and chin. The redness is often accompanied by bumps, pimples, and skin blemishes. Blood vessels may also become more visible on the skin. Blackheads are not a part of rosacea. It is more prevalent in women, but often more severe when found in men. Left untreated, it can cause swelling of the nose and the growth of excess tissue, a condition called rhinophyma.

Acne conglobata is the most severe form of acne vulgaris and is more common in males. It is characterized by numerous large lesions, which are sometimes interconnected, along with widespread blackheads. It can cause severe, irrevocable damage to the skin, and disfiguring scarring. It is found on the face, chest, back, buttocks, upper arms, and thighs. The age of onset for acne conglobata is usually between 18 and 30 years, and the condition can stay active for many years.

Acne fulminans is an abrupt onset of acne conglobata which normally afflicts young men. Symptoms of severe nodulocystic, often ulcerating acne are apparent. As with acne conglobata, extreme, disfiguring scarring is common. Acne fulminans is unique in that it also includes a fever and aching of the joints.

Gram-negative folliculitis is a bacterial infection characterized by pustules and cysts, possibly occurring as a complication resulting from a long term antibiotic treatment of acne vulgaris. It is a rare condition, and prevalence in males versus females is unknown.

Pyoderma Faciale is severe facial acne affects only females, usually between the ages of 20 to 40 years old, and is characterized by painful large nodules, pustules and sores which may leave scarring. It begins abruptly, and may occur on the skin of a woman who has never had acne before. It is confined to the face, and usually does not last longer than one year, but can wreak havoc in a very short time.

Teenage (adolescent) acne: Most cases of acne that require treatment occur in individuals 9 to 19 years of age. Boys and girls are equally affected but the condition is usually more severe in boys. No ethnic groups are predisposed to acne, but certain cosmetic practices, such as the use of oily grooming agents, can lead to a specific pattern of lesions. Internal factors that may cause adolescents acne include endogenous hormones

(androgens, progesterone) and specific drugs (oral contraceptives, isoniazid, phenytoin, corticosteroids, lithium-containing compounds). External factors include skin bacteria, especially Propionibacterium acnes; industrial chemicals (petroleum, animal and vegetable oils); oil- or wax-containing cosmetics; greasy sunscreen or suntan preparations; and local pressure from objects such as headbands, shoulder pads, or helmets. Excessive

perspiration and emotional stress can also aggravate acne. Androgens (e.g., testosterone) will increase the size of sebaceous glands and, in people prone to acne, increase the production of sebum. In women, fluctuations in estrogen during the menstrual cycle change the sensitivity of sebaceous glands to androgens. During puberty, the skin cells lining the follicle shed more quickly, mix with the increased levels of sebum and increase the likelihood of the pores becoming clogged.

Adult acne: Twenty percent (20%) of the adult population is afflicted by adult acne, most of them women. Modern-day job related stress, pollution, poor nutrition and bad cosmetics are among the major contributing factors. A common case of adult acne consists of blackheads and whiteheads, while others developed inflamed acne papules or pustules.

Approximately 85% of people worldwide suffer from acne at some point in their lives, leading to more than 103 million affected by acne at any given moment. Approximately 17 million people in the U.S. have acne resulting in approximately 5.5 million visits to the physicians each year.

Acne vulgaris occurs in up to 95% of the population in westernized societies; acne vulgaris is a nearly universal skin disease afflicting 79% to 95% of the adolescent population. In men and women older than 25 years, 40% to 54% have some degree of facial acne, and clinical facial acne persists into middle age in 12% of women and 3% of men. (Cordain L, Lindeberg S, Hurtado M, Hill K, Eaton S B, Brand-Miller J. Acne vulgaris: a disease of Western civilization. Arch Dermatol 2002 December; 138(12): 1584-90).

Current medications include a variety of topical and systemic medications such as antibiotics, anti-infectives, anti-inflammatories, hormone therapies, keratolytics, and retinoids. The over the counter medication include benzoyl peroxide, salicylic acid, sulfur, and resorcinol.

Benzoyl peroxide medication is very effective for killing acne-causing bacteria. Benzoyl peroxide first saw use in the 1930's, and remains a mainstay of acne treatment because it has proven itself to work well. To this day, benzoyl peroxide actually kills propionibacterium acnes (P. Acnes) better than any other medication on the market, prescription or otherwise. Benzoyl peroxide is available in non-prescription concentrations of 2.5%, 5% and 10%.

Many anti-acne agents, including benzoyl peroxide, have a high flux into the skin. While this penetration is advantageous during the initial application, the rapid subsequent diffusion in the skin means that the anti-acne agent will diffuse away from the acne lesion to be treated (a phenomenon known as "outflow"). In turn, this means that the efficacy of the anti-acne agent is lessened, because of the relatively short dwell time of the anti-acne agent in the area of the acne lesion. Thus, it would be advantageous to have a method and a kit that would decrease the outflow of the anti-acne agent from the area of the acne lesion.

Currently Acne is treated in a number of ways. From simple antibacterial skin wash through to oral antibiotics and retinoids. In addition, to these 2 strong treatment regimes acne can also be treated with topical antibiotics, Salicylic acid based medicines, topical retinoids, in woman oral contraceptives and azelaic acid. Of these treatments the most effective are GP prescribed oral antibiotics. Although antibiotic resistance is a growing concern. Retinoids have serious safety concerns in the youth especially with regard to depression, headaches, dry skin etc.

An object of the present invention is to address one or more of the above problems associated with acne and/or atopic dermatitis treatments. It is also an object of the present invention to provide an acne and/or atopic dermatitis treatment. It is additionally an object of the present invention to provide a treatment which allows for better penetration or delivery of existing and/or future acne and/or atopic dermatitis treatment compositions.

Summary of the Invention

In accordance with a first aspect of the present invention, there is provided a composition for use in the treatment or prevention of a dermatological condition, the composition comprising a polymer capable of forming nanoparticles and lactoferrin and/or derivatives thereof.

Related to the first aspect of the present invention, there is provided a composition for the treatment or prevention of a dermatological condition, the composition comprising a polymer capable of forming nanoparticles and lactoferrin and/or derivatives thereof. Further related to the first aspect of the present invention, there is provided the use of a composition comprising a polymer capable of forming nanoparticles and lactoferrin and/or derivatives thereof, in the manufacture or preparation of a medicament. Such a medicament may be for the treatment of a dermatological condition. In another embodiment of the present invention, there is provided a composition for use in the formulation of a cosmetics product, the composition comprising a polymer capable of forming nanoparticles and lactoferrin and/or derivatives thereof.

The polymer may comprise a linear and/or branched or cyclic

polymonoguanide/polyguanidine, polybiguanide, analogue or derivative thereof. By forming nanoparticles from polymers and lactoferrin, it is believed that

advantageously that, the lactoferrin will be taken up by the cells much more efficiently and is able to travel down the hair follicle to effect sebaceous treatment.

The term "nanoparticle" is intended to mean a structure having an average diameter in the approximate range of 0.5 - 200 nm. Preferably, the nanoparticles will be in the range of 1 to 150 nm, more preferably in the range of 2 to 120nm and most preferably 5 to 120 nm. In some instances, it is preferred that the nanoparticles are in an upper range of around 100 to 120 nm, more preferred in the range of 50 to 175 nm, even more preferred in the range of 75 to 150nm and most preferred in the range of 1 10 to 140nm. In other instances, it is preferred that the nanoparticles are in a lower range of 0.5 to 10 nm, more preferred in the range of 0.5 to 8 nm, even more preferred 1 to 7 nm and most preferred about 7 nm or lower. It is most preferred that the nanoparticles are equal to or less than about 10 nm in diameter

It is preferred that the polymer comprises a linear and/or branched or cyclic polymonoguanide/polyguanidine, polybiguanide, analogue or derivative thereof. The linear and/or branched or cyclic polymonoguanide/polyguanidine, polybiguanide, analogue or derivative thereof may be according to the following formula 1 a or formula 1 b, with examples provided in tables A and B below:

Formula 1 a

Formula 1 b

wherein:

"n", refers to number of repeating units in the polymer, and n can vary from 2 to 1000, for example from 2 or 5 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800 or 900;

Gi and G₂ independently represent a cationic group comprising biguanide or guanidine, wherein l_i and L₂ are directly joined to a Nitrogen atom of the guanide. Thus, the biguanide or guanidine groups are integral to the polymer backbone. The biguanide or guanidine groups are not side chain moieties in formula 1a.

Example of cationic groups:

Biguanide

(as in PHMB)

or

H H guanidine NH (as in PHMG) In the present invention, l_i and L₂ are the linking groups between the Gi and G₂ cationic groups in the polymer. I_i and L₂ can independently represent an aliphatic group containing C1-C140 carbon atoms, for example an alkyl group such as methylene, ethylene, propylene, C₄, C5, C6, C₇, Cs, C9 or C10; C1-C10, -C20, -C30, -C₄₀, -C50 -Ceo, -C70, -Ceo, -C90, - C100, -C110, -C120, -Ci3o or -Ci₄₀, alkyl ; or Li and L₂ can (independently) be C Ci₄₀ (for example Ci , C₂, C3, C₄, C5, C6, C₇, Cs, C9 or Ci₀; C1-C10, -C₂₀, -C30, -C₄₀, -C50 -Ceo, -C70, - Ceo, -C90, -C100, -C110, -C120, -Ci3o or -Ci4o), cycloaliphatic, heterocyclic, aromatic, aryl, alkylaryl, arylalkyl , oxyalkylene radicals, or l_i and L₂ can (independently) be a

polyalkylene radical optionally interrupted by one or more, preferably one, oxygen, nitrogen or sulphur atoms, functional groups as well as saturated or unsaturated cyclic moiety.

Examples of suitable l_i and L₂ are groups are listed in table A.

L₂, Gi and G₂ may have been modified using aliphatic, cycloaliphatic, heterocyclic, aryl, alkaryl, and oxyalkylene radicals.

N and G₃ are preferably end groups. Typically the polymers of use in the invention have terminal amino (N) and cyanoguanidine (G₃) or guanidine (G₃) end groups. Such end groups may be modified (for example with 1 ,6-diaminohexane, 1 ,6

di(cyanoguanidino)hexane, 1 ,6-diguanidinohexane, 4-guanidinobutyric acid) by linkage to aliphatic, cycloaliphatic heterocyclic, heterocyclic, aryl, alkylaryl, arylalkyl, oxyalkylene radicals. In addition, end groups may be modified by linkage to receptor ligands, dextrans, cyclodextrins, fatty acids or fatty acid derivatives, cholesterol or cholesterol derivatives or polyethylene glycol (PEG). Optionally, the polymer can end with guanidine or biguanide or cyanoamine or amine or cyanoguanidine at N and G₃ positions or cyanoamine at N and cyanoguanidine at G₃ position or guanidine at N and Cyanoguanide at G₃ positions or L1 amine at G3 and cyanoguanidine at N. G3 can be L amine, |_₂-cyanoguanidine or L₂- guanidine. Depending on the number of polymerization (n) or polymer chain breakage and side reactions during synthesis, heterogeneous mixture of end groups can arise as described above as an example. Thus, the N and G3 groups can be interchanged/present as a heterogeneous mixture, as noted above. Alternatively N and G₃ may be absent and the polymer may be cyclic, in which case the respective terminal l_i and G₂ groups are linked directly to one another.

In formula 1 b, X can be either present or absent. L₃, L₄ and X are as noted above for "l_i or L₂". In Thus, L₃ and L₄ and X are the linking groups between the G₄ and G₅ cationic groups in the polymer. L₃ and L₄ and X can independently represent an aliphatic group containing C Ci₄₀ carbon atoms, for example an alkyl group such as methylene, ethylene, propylene, C₄, C5, C6, C₇, Cs, C9 or C10; C1-C10, -C20, -C30, -C₄₀, -C50 -C60, -C₇o, -Cso, -C90, - C100, -C110, -C120, -Ci3o or -Ci₄₀, alkyl ; or L₃ and L₄ and X can independently be C Ci₄₀ (for example Ci, C2, C₃, C₄, C5, C6, C₇, Cs, C9 or C10; C1-C10, -C20, -C₃₀, -C₄₀, -C50 -C60, -C₇o, - Cso, -C90, -C100, -C110, -C120, -Ci3o or -Ci4o), cycloaliphatic, heterocyclic, aromatic, aryl, alkylaryl, arylalkyl , oxyalkylene radicals, or L₃ and L₄ and X can independently be a polyalkylene radical optionally interrupted by one or more, preferably one, oxygen, nitrogen or sulphur atoms, functional groups as well as saturated or unsaturated cyclic moiety. Examples of suitable L₃ and L₄ and X are groups are listed in table B.

"G₄" and "G₅" are cationic moieties and can be same or different. At least one of them is a biguanidine moiety or carbamoylguanidine, and the other moiety may be as above (biguanidine or carbamoylguanidine) or amine. For the avoidance of doubt, in formula 1 b, cationic moiety G₄ and G₅ do not contain only single guanidine groups. For example, G₄ and G₅ typically do not contain single guanidine groups. Examples of such compounds are polyallylbiguanide, poly(allylbiguanidnio-co-allylamine),

poly(allylcarbamoylguanidino-co-allylamine), polyvinylbiguanide, as listed in table B.

Example of polyallylbiguanide is as shown below:

In case of polyallylbigunidine L₃ and L₄ are identical, G₄ and G5 are similar, thus polyallylbiguanide can be simplified as below.

Example of poly(allylcarbamoylguanidnio-co-allylamine) is as shown below

The polymers for use in the invention will generally have counter ions associated with them. Suitable counter ions include but are not limited to the following: halide (for example chloride), phosphate, lactate, phosphonate, sulfonate, amino carboxylate, carboxylate, hydroxy carboxylate, organophosphate, organophosphonate, organosulfornate and organosuflate.

Polymers for use in the invention can be either heterogeneous mixtures of polymers of different "n" number or homogenous fractions comprising specified "n" numbers purified by standard purification methods. As indicated above the polymers may also be cyclic and in addition may be branched.

Preferred numbers for "n" include 2-250, 2-100, 2-80 and 2-50.

guanide CH₂-CH₂

Table A. Examples of polymer analogues arising from formula 1 a.

CAS numbers for example compounds arising from formula 1a Name L₃ G₄ L₄ G₅ X

Polyallylbiguanide (CH₂-CH) Biguanide (CH₂-CH) Biguanide CH₂ poly(allylbiguanidnio-co- (CH₂-CH) amine (CH₂-CH) biguanide CH₂ allylamine)

poly(allylcarbamoylguanid (CH₂-CH) amine (CH₂-CH) Carbamoyl CH₂ ino-co-allylamine) guanidine

polyvinylbiguanide (CH₂-CH) Biguanide (CH₂-CH) biguanide absent

Table B. Examples of polymer analogues arising from formula 1 b.

The polymer used in the method of the invention may comprise linear, branched or dendrimeric molecules. The polymer may comprise a combination of linear, branched or dendrimeric molecules. The polymer may comprise one or any combination of molecules of Formula 1 a or Formula 1 b, for example as described above.

For example, the polymer can comprise one or more of polyhexamethylene biguanide (PHMB), polyhexamethylene monoguanide (PHMG), polyethylene biguanide (PEB), polytetramethylene biguanide (PTMB) or polyethylene hexamethylene biguanide (PEHMB). Some examples are listed in table A and B.

Thus, the polymer may comprise homogeneous or heterogeneous mixtures of one or more of polyhexamethylene biguanide (PHMB), polyhexamethylene monoguanide (PHMG), polyethylene biguanide (PEB), polytetramethylene biguanide (PTMB), polyethylene hexamethylene biguanide (PEHMB), polymethylene biguanides (PMB), poly(allylbiguanidnio- co-allylamine), poly(N-vinylbiguanide), polyallybiguanide.

Most preferred the polymer comprises polyhexamethylene biguanide (PHMB).

The nanoparticles may be formed with and/or in the presence of lactoferrin. Various methods may be used to form the nanoparticles and it is envisaged that the nanoparticles will be formed as a polymer and lactoferrin complex. However, polymer nanoparticles may be independently formed and then incubated with lactoferrin so that it is absorbed or attached to the nanoparticles.

It will be apparent to the skilled addressee that the composition may further comprises one or more of the following component: buffers, excipients, binders, oils, water, emulsifiers, glycerin, antioxidants, preservatives and fragrances or any additional components usually found in topical creams, ointments sprays or powders. Furthermore, the composition could be in a number of forms such as a paste or a suspension for use with a spraying device. Preferably, the composition is in the form of a topical medicament.

In the embodiments related to the treatment or prevention of a dermatological condition, the dermatological condition will preferably comprise acne and/or atopic dermatitis or related conditions.

Preferably, the polymer to lactoferrin ratio is in the range of about 4:0.5 to about 2:1.5, more preferably, 3.75:0.75 to about 2.5: 1.5. The polymer to lactoferrin ratio is even more preferably in the ratio of 3: 1 ±0.5. Advantageously, the inventors have identified that highest concentration of nanoparticles takes place when the polymer to lactoferrin ratio is in the region of about 3: 1.

Preferably, the composition comprises in the range of about 200μg/ml to about 400μg/ml of the polymer and in the range of about 50μg/ml to about 150μg/ml of lactoferrin. More preferably, the composition comprises about 300 μg/ml of the polymer and about 100 μg/m I of I actof erri n .

The composition will preferably comprise a polyhexamethylene biguanide (PHMB) polymer present in at least 0.03 mg/ml. If targeting P. acnes it is preferred that PHMB is present in at least 0.062mg/ml PHMB, whereas it is preferred that PHMB is present in at least 0.031 mg/ml if targeting P.granulosom. The lactoferrin may be derived from a number of sources and may be in its native form. It is preferred that the lactoferrin is derived from rice. The lactoferrin may be a rice- derived recombinant human lactoferrin.

In accordance with another aspect of the present invention, there is provided use of polyhexamethylene biguanide (PHMB) to form one or more nanoparticles with, or associated with, lactoferrin in the preparation of a medicament.

In accordance with a related aspect of the present invention, there is provided use of polyhexamethylene biguanide (PHMB) to form one or more nanoparticles with, or associated with, lactoferrin in the preparation of a cosmetics product. The nanoparticles may be used as the delivery vehicle for delivering lactoferrin to a dermatological area. An affected dermatological area may comprise sebaceous hair follicles or pores. The dermatological condition may be affected by, or susceptible to, acne and/or atopic dermatitis or related conditions.

In another aspect of the present invention, there is provided a skin cosmetics product comprising a polymer capable of forming nanoparticles and lactoferrin and/or derivatives thereof.

The polymer may comprise the polymer as described with reference to the composition for use in the treatment of a dermatological condition. The skin cosmetics product may comprise a number of products such as make-up, creams, ointments, powders, emulsions, skin masks.

In accordance with a further aspect of the present invention, there is provided a method of producing a composition comprising mixing a polymer capable of forming nanoparticles with lactoferrin and/or derivatives thereof under conditions to allow the formation of nanoparticles. The polymer to lactoferrin may be mixed in a ratio of about 3: 1. The composition may comprise about 300 μςΑτιΙ of the polymer and about ΙΟΟμςΛηΙ of lactoferrin. The

nanoparticles formed may be less than about 10nm in diameter. The polymer may comprise polyhexamethylene biguanide (PHMB) present in at least 0.1 mg/ml. It is preferred that the method is used to produce a composition as herein above described.

In accordance with a further aspect of the present invention, there is provided a composition for use in the treatment of dermatological condition, comprising nanoparticles or nanoparticle conjugates formed of PHMB and a dermatological condition treatment agent. In a related aspect of the present invention, there is provided the use nanoparticles or nanoparticle conjugates formed of PHMB and a dermatological condition treatment agent, for the manufacture or preparation of a medicament for the treatment of a dermatological condition.

The approach taken by the inventors was to combine very effective antibacterials in lactoferrin and PHMB with the inherent anti-inflammatory properties of lactoferrin to deliver into the hair shaft and sebaceous duct by killing the bacterium P Canes and dampening the inflammatory response and hence reduce the redness and local tissue damage that's caused. It is also envisaged that the composition could be used as a formulation as a facial wash as a preventative measure thus reducing the hyperkeratinisation and thus sebaceous duct blockage that occurs in acne spots. Thus a combined over the counter face wash and acne cream would be a powerful but gentle approach to manage acne in adolescents and older patients. Furthermore, the composition could also be formulated with skin cosmetics products so as to provide a product which could be used on skin which had acne or was prone to acne.

Lactoferrin (LF) is an 80 kilo Dalton (kD) iron-binding glycoprotein found in high concentrations in milk and in lower concentrations in other secretions and body fluids. It is one of a number of iron binding proteins, referred to as transferrins, involved in iron binding and delivery in mammals. Montreuil and Mullet, 1960, C.R. Acad. Sci. Paris 250: 1736-1737; Montreuil et al., 1960, Biochem. Biophys. Acta 45:413-421 ; Johansson, 1960, Acta. Chem. Scand. 14: 510-512, Blanc and Isliker, 1961 , Bull. Soc. Chim. Biol. 43:929-943; Masson and Heremans, 1967, Protides Biol. Fluids Proc. Colloq. 21 : 115-124; Querinjenn et al., 1971 , Eur. J Biochem. 20:420-425; Leger et al., 1977, Biol. Anim. Biochim. Biophys. 17:737-747.

Lactoferrin (or lactotransferrin) was originally discovered in milk where it can reach levels of 7 grams/liter in colostrum. Since then, however, it has been detected in a number of other body fluids including tears, saliva and mucosal secretions and also in the secondary granules of polymorphonuclear leukocytes. Biserte et al., 1963, Exp. Ann. Biochim. Med. 25:85-120; Masson, 1970, in: La Lactoferrine, pp. 93-165, Arscia, Bruxelles. Thus, the protein is expressed primarily by glandular epithelial cells and neutrophils associated with both local and central immune defense. Lactoferrin has been shown to play important roles in host defense mechanisms due to its well-established antimicrobial activities. The antimicrobial actions of lactoferrin appear at least in part to be the consequence of the iron binding properties of lactoferrin via sequestration of iron necessary for microbial growth. It has been shown that lactoferrin production is induced by lipopolysaccharides (LPS), which are components of bacterial cell walls. Gutteberg et al., 1990, Scan. J. Clin. Lab. Invest. 50:421-427. There is mounting evidence that in addition to antimicrobial activities, lactoferrin may influence innate and adaptive immune processes, including natural killer cell function, participate in the course of inflammation, and complement activation and affect cytokine production. Lash et al., 1983, Blood 61 :885-888; Mansson et al., 1990, Ann. Rheum. Dis. 49:594-597; Van Snick et al., 1974, J. Exp. Med. 140:1068-1084. With respect to the latter it has been demonstrated, both in vivo and in vitro, that lactoferrin may compromise the production of tumor necrosis factor a (TNF-a) systemically (Machnicki et ai, 1993, Int. J. Exp. Path. 74:433-439), a cytokine that plays important roles in inflammation, sepsis and endotoxemic shock (Beutler et ai, 1985, Science 229:869-871 ; Tracey et al., 1987, Curr. Opinion Immunol. 1 :454-461 ; Waage et al., 1989, J. Exp. Med. 169:333-338; Kunkel et al., 1989, Crit. Rev. Immunol. 9:93-1 17). In previous investigations, however, attention focused upon the regulation of TNF-a production provoked by LPS. Machnicki et al., 1993, Int. J. Exp. Path. 74:433-439; Gutteberg et al., 1990, APMIS 98: 1027-1032; and Gutteberg et al., 1991 , APMIS 99:602-608. Since lactoferrin is known to bind directly to LPS, it was uncertain whether the observed reduction in TNF-a was attributable to transcriptional or post- transcriptional regulation of the cytokine itself or to the reduction by lactoferrin in the availability of LPS due to direct binding and inactivation of the endotoxin. Ellison and Giehl, 1991 , J. Clin. Invest. 88:1080-1091 ; Appelmelk et a/., 1994, Infect. Immunity 62:2628-2632. There has not been a previous demonstration of the allergen-induced effects of lactoferrin. For this reason, experiments have now been performed to examine the ability of

homologous recombinant lactoferrin to influence the induction of TNF-a-dependent biological responses where stimulation of TNF-a expression is independent of LPS. A cDNA encoding lactoferrin and methods for recombinantly producing the same have been disclosed by Conneely et al. in U.S. Patent Nos. 5,571 ,896, 5,571 ,697 and 5,57/1 ,691. Production of lactoferrin as a fusion product has been disclosed in U. S. Serial Nos. 08/453,703,

08/456, 106 both filed May 30, 1995 and 08/691 , 123 filed on August 1 , 1996. Moreover, the use of lactoferrin to modulate or neutralize heparin activity has been disclosed in U.S. Serial No. 08/391 ,986 filed on February 21 , 1995. Additionally, lactoferrin mutants and variants thereof have been disclosed in U.S. Serial No. 08/866,544 filed on May 30, 1997. The disclosures of all the foregoing patents and applications are herein incorporated by reference in their entirety.

PHMB (polyhexamethylene biguanide) is known as a safe and effective biocidal agent and is used as a sanitiser and preservative: US7897553, US 4758595,

US2008261841 ; US 20040009144. PHMB and related molecules are also found to be useful entry-promoting agents. It was surprisingly observed that PHMB (for example) itself enters a wide range of cells, including bacteria, fungi and mammalian cells. More

surprisingly, PHMB (for example) is able to form nanoparticles with a wide range of molecules and deliver these molecules into such cells as described in WO2013054123 (PCT/GB2012/052526). Finally the delivered molecules ranging from nucleic acids to small molecules were found to be functional inside cells. Moreover, work carried out with some natural product molecules such as retinoic acid and vitamin C have demonstrated an enhanced stabilizing effect on the natural products so they are less likely to break down when combined with PHMB.

Here we generally describe the invention of a formulation of lactoferrin with PHMB which forms nanoparticles enhancing the stability of lactoferrin and ensuring penetration of skin via the hair shaft

Lactoferrin advantageously brings both anti-inflammatory and antimicrobial qualities to the combination whilst PHMB advantageously brings antimicrobial and enhanced delivery to the combination.

The combination of Lactoferrin with PHMB produces nanoparticles of a size between 50 nm and up to 900nm although at 3:1 ratio of PHMB to lactoferrin we achieve particle sizes of more than 50nm and less than 200nm

Surprisingly, the inventors have demonstrated nanoparticle formation with PHMB and

Lactoferrin expressed in Rice but not with the native form of Lactoferrin. Formation of nanoparticles with PHMB is pre requisite to enhanced delivery and enhanced stability of the active ingredient being prepared for delivery. Although both forms of Lactoferrin appear equally potent in biological assay of activity including anti inflammatory they clearly behaved differently in combination with PHMB.

Studies have also demonstrated enhanced delivery of small molecules down the hair shaft in human skin samples.

Detailed Description of the Invention

Embodiments of the present invention will now be described, by way of example only, with reference to the following experiments and accompanying figures, in which:

Figure 1 is a graph showing PHMB and Lactoferrin in water after 2 hours- particle concentration; Figure 2 is a graph showing Mean particle size with PHMB and Lactoferrin;

Figure 3 is a graph showing the intradermal administration of lactoferrin: impact on dendritic cell accumulation;

Figure 4 are graphs illustrating how lactoferrin acts through TNF-a; Figure 5 are graphs showing lactoferrin stability in formulation;

Figure 6 is a graph showing nanoparticle concentrations of Rice lactoferrin (RLF) and PHMB. For each solution, the first column relates to 2h, the second column relates to O/N at RT and the third column relates to O/N at 4oC;

Figure 7 is a DLS reading of the size distribution by volume of the nanoparticles formed of after 3 hours incubation. From left to right, the first peak relates to Record 2: sample 5 is PHMB; the second peak relates to Record 3: sample 6 is RLF: PHMB combined; and the third peak relates to Record 1 : sample 4 is RLF;

Figure 8 is a photograph showing the MIC of PHMB vs. P. acnes. The photograph is of a P. acnes lawn grown on Nutrient agar plate overlaid with 6mm paper disks soaked in PHMB solutions at the concentrations (from left to right) 10 mg/ml, 1 mg/ml, 0.1 mg/ml, 0.01 mg/ml and 0.001 mg/ml;

Figure 9 is a photograph showing the MIC of PHMB vs. P. granulosum. The photograph is of a P. granulosum lawn grown on Nutrient agar plate overlaid with 6mm paper disks soaked in PHMB solutions at the concentrations (from left to right) 10 mg/ml, 1 mg/ml, 0.1 mg/ml, 0.01 mg/ml and 0.001 mg/ml;

Figure 10 is a photograph showing the MIC of PHMB vs. P. acnes. The photograph is of a P. acnes lawn grown on Nutrient agar plate overlaid with 6mm paper disks soaked in PHMB solutions at the concentrations (from left to right) 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.12 mg/ml, 0.062mg/ml and 0.031 mg/ml; and Figure 1 1 is a photograph showing the MIC of PHMB vs. P. granulosum. The photograph is of a P. granulosum lawn grown on Nutrient agar plate overlaid with 6mm paper disks soaked in PHMB solutions at the concentrations (from left to right) 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.12 mg/ml, 0.062mg/ml and 0.031 mg/ml.

Testing in water

The lactoferrins were combined at a concentration of 10ug/ml and the PHMB (also referred to herein as "Nanocin" in parts of the specification and figure legends) was tested at two different concentrations of either 20 or 30μg/ml.

As shown in Fig.1 , the lactoferrins showed a large number of nanoparticles alone (rice lacto more so than native), suggesting that it has come out of solution in water. On combination with PHMB the number of particles dropped significantly.

Testing in PBS

Lactoferrins and PHMB were then combined in PBS at ratios of 10:20 and 10:30 ug/ml (Lactoferrin: PHMB). The lactoferrins alone at this concentration had a much lower background particle count compared to when they were in water.

When combined with PHMB, at a 1 :2 or 1 :3 ratios (lactoferrin: PHMB); the rice lactoferrin showed an increase in particle concentration whereas the native lactoferrin did not.

Mean particle size - lactoferrin with PHMB

Fig. 2 shows the mean particle sizes for a range of lactoferrin:PHMB combinatons.

Lactoferrin impact on dendritic cells in skin

Fig. 3 shows the appearance of dendritic cells in lymph nodes following

administration of either bovine serum albumin or different concentrations of lactoferrin topically to the skin. Lactofer ns anti-inflammatory action is mediated through TNF-ct suppression

Fig. 4 shows that lactoferrin anti-inflammatory action is mediated through TNF-a suppression.

Improved stability of lactoferrin with PHMB Lactoferrin is unstable in current formulations. Fig. 5 shows that in combination with

PHMB stability is greatly enhanced.

Dynamic Light Scattering (DLS) detection of Nanoparticle formation with Rice Lactoferrin and PHMB

PHMB had been combined with rice lactoferrin (RLF) at a 3: 1 ratio and as illustrated in Figure 6, shows good levels of nanoparticle formation after 2 hours, which significantly reduced overnight.

The solutions left overnight overnight (and further) appeared to have particles that were visible under the microscope but not detectable by the Nanosight LM10

instrumentation, and so it was necessary to check the solutions with DLS to identify if the particles had gone to a size beyond the limit of detection using the Nanosight LM10 instrumentation.

A 1 mg/ml solution of RLF in PBS (without calcium and magnesium) was stored at - 20°C; this was thawed to room temperature (RT) and vortexed before use. A 1 mg/ml PHMB was also prepared in PBS (or water) but stored at RT. A concentration of 10C^g/ml RLF and 30C^g/ml PHMB was used at it had previously been shown to be the most effective ratio and concentrations to provide the highest concentrations of nanoparticles.

To combine the lactoferrin with PHMB, the lactoferrin was first added to PBS (or water) and the mix was then vortexed. PHMB was then added to the mixture, which was then sheared up and down 10-times with a 1 ml pipette tip. For an initial reading the mix was then left for 3 hours at RT to form nanoparticles.

The nanoparticles were then left to incubate in water or PBS at room temperature over a time period of a week. For incubation with water, the resultant size distribution by volume is provided in

Table 1 below.

Table 1

Figure 7 shows the DLS reading of the size distribution by volume of the

nanoparticles formed of after 3 hours incubation. From left to right, the first peak relates to Record 2: sample 5 is PHMB; the second peak relates to Record 3: sample 6 is RLF: PHMB combined; and the third peak relates to Record 1 : sample 4 is RLF.

For incubation with PBS, the resultant size distribution by volume is provided in Table 2 below.

Table 2

It appeared that nanoparticles are formed between RLF and PHMB which are below 10nm and hence undetectable by Nanosight LM 10. The nanoparticles appear stable in PBS and water over 1 week. Minimum inhibitory concentrations (MICs) assessment of PHMB against Propionibacterium acnes and Propionibacterium granulosum

The antibacterial effect of PHMB against two species of bacteria (Propionibacterium acnes and Probionibacterium granulosum) associated with acne (Williams et al. The Lancet. 2012, 379 (9813). P361-72, Burkhart et al. Postgrad Med J. 1999. 75, P328-331) was then determined.

A single colony of Propionibacterium acnes (P. acnes) or Propionibacterium granulosum (P. granulosum) was picked from a Nutrient agar plate and grown overnight in 2ml of Nutrient Broth for 16 hours at 37°C under anaerobic conditions in an anaerobic chamber. 100μΙ of each culture was spread over a Nutrient agar plate to create bacterial lawns. A 10mg/ml (1 %(w/v)) solution of PHMB in phosphate buffered saline (PBS) was serially diluted 1 : 10 in PBS from 10mg/ml to 0.001 mg/ml. 20μΙ of each dilution was spotted onto a sterile 6mm paper filter disk. In addition a 6mm paper filter disk was also spotted with 20μΙ of vehicle (PBS) as a negative control. Each of the paper disks were carefully placed onto the bacterial lawns. The Nutrient agar plates were incubated for 48 hours at 37°C under anaerobic conditions in an anaerobic chamber. Zones of clearance around the paper disks indicate antibicrobial activity of PHMB. MICs were determined as lying between the lowest concentrations of PHMB that demonstrated a clear zone of clearance.

Zones of clearance were observed around 6mm paper disks soaked with PHMB solutions for both P. acnes and P.granulosum (as shown in Figures 8 and 9). No zones of clearance were observed for vehicle alone controls. For P. acnes clearance was seen at 1 mg/ml PHMB but not at 0.1 mg/ml PHMB. The zone of clearance at 1 mg/ml PHMB was still pronounced and so the MIC for PHMB against P. acnes is <1 mg/ml and >0.1 mg/ml. For P.granulosom a small zone of clearance could still be observed at 0.1 mg/ml and so the MIC for PHMB against P.granulsosum is approximately 0.1 mg/ml. The experiment was repeated again in order to try and establish a more accurate level of MIC. Again, a single colony of Propionibacterium acnes (P. acnes) or

Propionibacterium granulosum (P. granulosum) was picked from a Nutrient agar plate and grown overnight in 2ml of Nutrient Broth for 16 hours at 37°C under anaerobic conditions in an anaerobic chamber. 10ΟμΙ of each culture was spread over a Nutrient agar plate to create bacterial lawns. A 1 mg/ml (0.1 %(w/v)) solution of PHMB in phosphate buffered saline (PBS) was serially diluted 1 :2 in PBS from 1 mg/ml to 0.031 mg/ml. 20μΙ of each dilution was spotted onto a sterile 6mm paper filter disk. In addition a 6mm paper filter disk was also spotted with 20μΙ of vehicle (PBS) as a negative control. Each of the paper disks were carefully placed onto the bacterial lawns. The Nutrient agar plates were incubated for 72 hours at 37°C under anaerobic conditions in an anaerobic chamber. Zones of clearance around the paper disks indicate antibicrobial activity of PHMB. MICs were determined as the lowest concentrations of PHMB that demonstrated a clear zone of clearance.

Zones of clearance were observed around 6mm paper disks soaked with PHMB solutions for both P. acnes and P.granulosum (as shown in Figures 10 and 11). No zones of clearance were observed for vehicle alone controls. For P. acnes the MIC was determined as 0.062mg/ml PHMB. For P.granulosom the MIC was determined as 0.031 mg/ml.

Example Formulation

The following example formulation is intended to provide an indication as to the actual likely formulation which may be incorporated into a wash, cream or gel using standard excipients. The lactoferrin concentration may be in the range of 2-20μg/ml (based on the in vivo efficacy data) and a PHMB concentration in the 0.1-1 mg/ml (0.01-0.1 %) based on achieving exposure in excess of the MIC vs. P. acnes. The lactoferrin concentration might be higher to enable effective nanoparticle formulation at a concentration of PHMB that would achieve efficacy. Based on the nanoparticle studies this is likely to be in the range of about 30C^g/ml PHMB: about 10C^g/ml lactoferrin down to about 10C^g/ml PHMB: about 3C^g/ml lactoferrin.

The forgoing embodiments are not intended to limit the scope of the protection afforded by the claims, but rather to describe examples of how the invention may be put into practice.

Claims

1. A composition for use in the treatment or prevention of a dermatological condition or in the formulation of a cosmetics product, the composition comprising a polymer capable of forming nanoparticles and lactoferrin and/or derivatives thereof.

2. A composition as claimed in claim 1 , wherein the polymer comprises a linear and/or branched or cyclic polymonoguanide/polyguanidine, polybiguanide, analogue or derivative thereof.

3. A composition as claimed in either claim 1 or 2, wherein the polymer comprises polyhexamethylene biguanide (PHMB).

4. A composition as claimed in any preceding claim, wherein the nanoparticles are formed with and/or in the presence of lactoferrin and/or derivatives thereof.

5. A composition as claimed in any preceding claim, wherein the composition further comprises one or more of the following component: buffers, excipients, binders, oils, water, emulsifiers, glycerin, antioxidants, preservatives and fragrances.

6. A composition as claimed in any preceding claim, wherein the composition is in the form of a topical medicament.

7. A composition as claimed in any preceding claim, wherein the composition is for use in the treatment or prevention of a dermatological condition comprising acne and/or atopic dermatitis or related conditions.

8. A composition as claimed in any preceding claim, wherein the lactoferrin is derived from rice.

9. A composition as claimed in claim 8, wherein the lactoferrin is a rice-derived

recombinant human lactoferrin.

10. A composition as claimed in any preceding claim, wherein the composition is in the form of a cream, ointment, spray or powder.

1 1. A composition as claimed in any preceding claim, wherein the polymer to lactoferrin ratio is in the range of about 4:0.5 to about 2:1.5.

12. A composition as claimed in claim 11 , wherein the polymer to lactoferrin ratio is about 3: 1.

13. A composition as claimed in any preceding claim, wherein the composition comprises in the range of about 200μg/ml to about 400μg/ml of the polymer and in the range of about 50μg/ml to about 150μg/ml of lactoferrin.

14. A composition as claimed in claim 13, wherein the composition comprises about 300 μg/ml of the polymer and about 100μg/ml of lactoferrin.

15. A composition as claimed in any preceding claim, wherein the nanoparticles formed are equal to or less than about 10nm in diameter.

16. A composition as claimed in any preceding claim, wherein the polymer comprises PHMB present in at least 0.03 mg/ml.

17. Use of polyhexamethylene biguanide (PHMB) to form one or more nanoparticles with, or associated with, lactoferrin in the preparation of a medicament or cosmetics product.

18. Use of PHMB as claimed in claim 17, wherein the nanoparticles are used as the

delivery vehicle for delivering lactoferrin to a dermatological area.

19. Use of PHMB as claimed in claim 18, wherein the dermatological area comprises sebaceous hair follicles or pores.

20. Use of PHMB as claimed in claim 19, wherein the dermatological area is affected by, or is susceptible to, acne and/or atopic dermatitis or related conditions.

21. A method of producing a composition comprising mixing a polymer capable of forming nanoparticles with lactoferrin and/or derivatives thereof under conditions to allow the formation of nanoparticles.

22. A method as claimed in claimed in claim 21 , wherein the polymer to lactoferrin is mixed in a ratio in range of about 4: 1 to about 2: 1.

23. A method as claimed in claim 22, wherein the polymer to lactoferrin is mixed in a ratio of about 3: 1.

24. A method as claimed in claim 23, wherein the composition comprises about 300 μg/ml of the polymer and about 100μg/ml of lactoferrin.

25. A method as claimed in any one of claims 21 to 24, wherein the nanoparticles formed are less than about 10nm in diameter.

26. A method as claimed in any one of claims 21 to 25, wherein the polymer comprises polyhexamethylene biguanide (PHMB) present in at least 0.03 mg/ml.

27. A method as claimed in any one of claims 21 to 26, wherein the method is used to produce a composition as claimed in any one of claims 1 to 16.