Field of the invention

The present invention relates to nicotinamide, or a precursor or derivative thereof, for use in the inhibition of hair growth. The invention also relates to methods of inhibiting hair growth by providing a subject with an effective amount of nicotinamide, or a precursor or derivative thereof. Furthermore, the invention relates to pharmaceutical compositions comprising nicotinamide, or a precursor or derivative thereof. Suitably the uses, methods and pharmaceutic compositions of the invention utilise nicotinamide.

Introduction

Hair is a characteristic feature of all mammals, including humans. It is primarily composed of proteins, in particular keratin, and is produced in hair follicles, epidermal appendages within the skin.

Hair production occurs in three phases, anagen (during which extension of the hair shaft occurs), telogen (in which extension is paused), and catagen (the cessation of growth).

There are many reasons, including cultural, cosmetic, or therapeutic reasons, why individuals wish to remove some or all hair from sites of the body. Examples of such applications include inhibition of hair growth in individuals who suffer from excess hair growth as a result of disorders such as hirsuitism, or hypertrichosis. Individuals who have hair bearing naevi, or pseudofolliculitis barbae may also wish achieve partial or total inhibition of hair growth.

Typically hair removal may be achieved by depilation or epilation. Such hair removal may be supplemented by the use of compounds able to inhibit hair growth. Merely by way of example, these may be provided by topical application prior to and/or post the hair removal procedure.

At present the existing treatments for hair growth inhibition include eflornithine, Wortmannin, maize propanodiol. Unfortunately, these treatment are typically of limited efficacy and associated with various side effects such as acne (in 7-14% of cases), in-growing hair, hair loss, burning, stinging or tingling sensations, dry skin, itching, redness or rash. Summary of the invention

In a first aspect, the invention provides nicotinamide, or a precursor or derivative thereof, for use in the inhibition of hair growth.

In a second aspect, the invention provides a method of inhibiting hair growth in a subject requiring such inhibition, the method comprising providing to the subject an amount of nicotinamide, or a precursor or derivative thereof, effective to inhibit hair growth.

In a third aspect, the invention provides pharmaceutical composition comprising nicotinamide, or a precursor or derivative thereof, in a therapeutically effective amount to inhibit hair growth, and a pharmaceutically acceptable excipient.

It will be appreciated that, in a suitable embodiment, nicotinamide, or a precursor or derivative thereof, for use in accordance with the first aspect of the invention may be formulated in a pharmaceutical composition of the third aspect of the invention. Similarly, it will also be appreciated that, in a suitable embodiment, a pharmaceutical composition of the third aspect of the invention may be used to provide the effective amount of nicotinamide, or a precursor or derivative thereof, when practicing a method of the second aspect of the invention.

For the sake of brevity, nicotinamide, or a precursor or derivative thereof, for use in accordance with the first aspect of the invention may be referred to in the present disclosure as "an agent of the invention", a method in accordance with the second aspect of the invention may be referred to as "a method of the invention", and a pharmaceutical composition in accordance with the third aspect of the invention may be referred to as "a composition of the invention".

Detailed description of the invention

As set out above, the present invention provides agents, methods, and compositions that are of use in the inhibition of hair growth.

The invention is based upon the inventors' finding that nicotinamide is able to inhibit the growth of hair. This finding is surprising, since a number of previously published reports have indicated that nicotinic acid derivatives act to stimulate hair follicles and thereby promote hair growth.

Without wishing to be bound by any hypothesis, the inventors believe that nicotinamide, or a precursor or derivative thereof, achieves its biological effect through a number of mechanisms. A first such mechanism is an increase in catagen among hair follicles to which such agents have been provided. A second such mechanism is revealed by the finding that provision of nicotinamide decreases proliferation of cells within the hair follicle, in particular matrix keratinocytes, progenitor cells and hair follicle keratinocytes that are known to contribute to the formation of hair shafts. Furthermore, the inventors have found that provision of nicotinamide serves to increase apoptosis among hair follicle cells.

It will be appreciated that these newly identified biological activities of nicotinamide, or precursors or derivatives thereof, contribute to their utility in the inhibition of hair growth, whether for therapeutic or cosmetic purposes. Such uses of these agents may make use of some or all of the biological actions that have been newly identified.

Hair removal by plucking, waxing, shaving (whether using razors, electrical shavers, or clippers or the like) or using chemical methods such as those making use of agents (such as calcium thioglycolate or potassium thioglycolate) is associated with various side effects. For example plucking and waxing is painful and can cause irritation, as can chemical methods of hair removal. Shaving will often result in cuts. Hair removal may also result in in-grown hairs which can form during hair re-growth, and which can also cause irritation.

In certain embodiments, the uses, methods, and compositions of the invention may be of utility in reducing such side effects, either indirectly (by increasing the delay of hair growth, thus reducing the frequency of hair removal procedures), or directly by reducing irritation. In certain embodiments, the uses, methods, and compositions of the invention may make use of further agents that do not have a direct effect upon hair growth, but serve to reduce irritation. The inventors have surprisingly found that certain compounds that do not exhibit biological activity that would suggest a role in the reduction of irritation when administered on their own, do show biological activity that is indicative of the ability to reduce irritation when provided in combination with nicotinamide, or a precursor or derivative thereof. Examples of such embodiments are described further elsewhere in this document.

Except where the context requires otherwise, the uses, methods, and compositions of the invention may be used in the inhibition of hair growth in both human and non-human subjects. For example it the invention may be used in animals which suffer from in-growing hairs or hypertrichosis, especially domestic animals such as cats and dogs.

Certain terms will now be further defined in order to aid the clear understanding of the invention.

Nicotinamide, and "precursors or derivatives" thereof

Nicotinamide is a water-soluble amide form of nicotinic acid (which is also known as niacin, vitamin B3, or pyridine-3-carboxylic acid). Nicotinamide has previously been disclosed as a nutritional supplement for the promotion of hair and nail growth as well as a topical treatment for stimulate hair growth. It will be recognised that these prior reported uses make the inventors' findings in the present application, in which nicotinamide, or precursors or derivatives thereof, are used in the inhibition of hair growth, especially surprising.

Nicotinamide itself represents a particularly suitable example of an agent suitable for use in accordance with the first aspect of the invention, in methods in accordance with the second aspect of the invention, and used as an active ingredient in pharmaceutical compositions in accordance with the third aspect of the invention.

Nicotinamide possesses a number of properties that make it well suited to use in the agents, methods, or compositions of the invention. It is water soluble, which facilitates its incorporation in a number of different formulations. Apart from its ability to inhibit hair growth, it also demonstrates favourable biological properties in vivo, in that it does not act as a vasodilator (unlike nicotinic acid), and therefore does not give rise to undesirable effects such as "flushing", which may cause reddening of the skin at sites where nicotinic acid is applied. In a suitable embodiment, a derivative or precursor of nicotinamide is a compound other than nicotinic acid.

The nicotinamide precursors or derivatives mentioned herein may, in principle, comprise any agent or compound in the synthesis pathway or metabolism pathway of nicotinamide or any compound that is structurally related to nicotinamide. In a suitable embodiment, a precursor or derivative of nicotinic acid for the purposes of the present invention may be selected from the group consisting of: nipecotic acid hydrochloride, N-nicotinyl-4-amino-phenol, N- benzylnicotinamide, N-nicotinyl-3-amino-phenol, N-nicotinyl dicyandiamide, N-nicotinyl-2- amino-phenol, N-nicotinyl4-aminosalicylic acid, N(2-pyrimidyl)-nicotinamide, N-nicotinyl-p- aminobenzoic acid, 2(N-nicotinyl)-amino-5-carbethoxy-thiazole, N(2-thiazolyl)-nicotinamide, N(2-pyridyl)-nicotinamide, 2-Amino-nicotinamide, N(piperidyl propyl)-nicotinamide, and N- dodecyl-nicotinamide.

In a suitable embodiment a use, method, or composition of the invention may employ more than one agent selected from the group consisting of nicotinamide, or a precursor or derivative thereof. The effective amount required to inhibit hair growth may thus be provided by a combination of these agents.

As referred to above, agents for use in accordance with the invention may also be used in combination with a further active agent that are not nicotinamide, or a precursor or derivative thereof. By the same token, further active agents that are not nicotinamide, or a precursor or derivative thereof, may also be employed in the methods of the invention, or formulated in the pharmaceutical compositions of the invention.

Suitably such further active agents may have favourable therapeutic activities that complement those of nicotinamide, or a precursor or derivative thereof. Merely by way of example, a further active agent may be an agent that reduces irritation. Irritation may arise as a result of the action of nicotinamide, or a precursor or derivative thereof. Alternatively, irritation may arise in a number of contexts associated with hair removal, including as a result of the development of in-grown hairs, or as a consequence of mechanical, chemical, or other techniques for hair removal.

One suitable example of such an additional active agent that may beneficially be combined in the uses, methods, or compositions of the invention is palmitoylethanolamide (PEA). PEA does not have any direct effect on hair growth (as illustrated by the data set out in the Experimental Results section). Furthermore results obtained by the inventors using an assay employing human skin (the details and results of which are described further in the Experimental Results) indicate that PEA alone does not have any significant effect upon mast cell numbers or degranulation. However, the inventors have found that when skin samples containing mast cells are treated with a combination of nicotinamide and PEA, this combination is able to reduce mast cell degranulation below the level that occurs on treatment with nicotinamide alone.

These results, which are surprising given the lack of effect exerted by PEA itself, are of benefit, since mast cell degranulation (which can be induced by agents, such as nicotinamide or a precursor or derivative thereof) may be associated with itching or other symptoms of skin irritation. Thus the inventors' finding that combinations of agents such as nicotinamide, and PEA are able to reduce mast cell degranulation, while providing beneficial inhibition of hair growth, may be beneficial in preventing the occurrence of unwanted side effects.

Optionally, nicotinamide, or a precursor or derivative thereof, may be used with another "Hair growth" and "inhibition of hair growth"

The ability of an agent, method, or composition of the invention to inhibit hair growth may be assessed with reference to a suitable control. A skilled person will be able to determine a suitable control for an agent, method, or composition of the invention without difficulty.

Inhibition of hair growth may be assessed with reference to the elongation of the hair shaft. It will be appreciated that an agent that reduces elongation of a hair shaft as compared to a suitable control demonstrates the ability to inhibit hair growth.

Inhibition of hair growth may be assessed with reference to hair of human or non-human animals. Examples of suitable experimental models (and suitable controls) for the assessment of inhibition of hair growth are described in the Experimental Results found towards the end of this document.

Merely by way of example, the ability of an agent to inhibit hair growth may be assessed using a suitable in vitro model. For instance, ability to inhibit hair growth may be assessed with reference to the ability of an agent to inhibit elongation of hair shafts of isolated hair follicles maintained in culture. Hair follicles may be incubated with the agent in question for a period of time (for example, around six days in a suitable model), and elongation of the hair shaft assessed during and/or at the end of this period. Suitable hair follicles may be obtained from surgical procedures such as hair transplantation.

Another suitable in vitro model may make use of hair follicle-containing skin biopsies from human or non-human animals. Such biopsies, which may, for example, be derived from human scalp, may be treated with a test agent for a period of time (for example, around six days in a suitable model), and elongation of the hair shaft assessed during and/or at the end of this period. Hair follicle-containing skin biopsies may be maintained in organ culture during the test period. Suitably the agents, methods, or compositions of the invention may be able to bring about a 20% inhibition of hair growth. In a suitable embodiment, an agent, method, or composition of the invention may be able to bring about a 25%, 30%, 35%, 40%, 45%, or 50% inhibition of hair growth. Indeed, in suitable embodiments, an agent, method, or composition of the invention may be able to bring about a 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even 100% inhibition of hair growth. The requisite extent of inhibition may be demonstrated in any of the experimental models of hair growth considered above.

In a suitable embodiment, the hair growth to be inhibited is regrowth of hair after hair removal. The uses, methods, and compositions of the invention may be used to prolong the effects of hair removal. This may allow greater time to elapse between incidences of hair removal. In a suitable embodiment, an agent, method, or composition of the invention may be able to increase the time elapsing between incidences of hair removal by 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days or more. The increase in the time between incidences of hair removal achieved may be determined with reference to suitable controls.

Suitably hair removal may be achieved by a variety of known methods. For example, hair removal may be achieved by mechanical methods, such as those selected from the group consisting of plucking, waxing, shaving (whether using razors, electrical shavers, or clippers or the like). Alternatively hair removal may be achieved using chemical methods such as those making use of agents (such as calcium thioglycolate or potassium thioglycolate) that weaken constituents of the hair, thus facilitating its removal. Other methods by which hair may be removed include electrology (in which electrical current is applied to hair follicles), and laser hair removal. The uses, methods, and compositions of the invention may be of utility in inhibiting hair growth after any such methods of removal.

The hair growth to be inhibited may be regrowth of hair after depilation or epilation. For the purposes of the present invention, depilation may be considered to be the removal of that portion of the hair shaft that normal extends above the skin. Depilation may typically occur as a result of shaving or the use of clippers. For the purposes of the present invention, epilation may be considered to be the removal of the entire hair shaft. Merely by way of example, epilation may occur as a result of plucking or waxing.

Suitably the inhibition of hair growth may be localised inhibition. For present purposes, localised inhibition may be taken as being inhibition of hair growth that is substantially restricted to certain sites upon a subject's body. Suitably, localised inhibition of hair growth may be achieved by local administration of nicotinamide, or a precursor or derivative thereof, with inhibition substantially restricted to the area to which the agent has been administered. Topical pharmaceutical compositions for the administration nicotinamide or a nicotinamide precursor or derivative thereof may be of use in embodiments of this sort.

Suitably an agent that comprises nicotinamide or a precursor or derivative thereof may be used to inhibit hair growth on any body part where hair growth inhibition may be desired. Merely by way of example a suitable body part can include those selected from the group consisting of: the head, face, legs, arms, back and chest.

However, the various aspects of the invention may also be of use in achieving global inhibition of hair growth. For present purposes global inhibition of hair growth may be taken as being inhibition occurring at a range of sites upon a subject's body. In particular, global inhibition of hair growth may be taken as being inhibition occurring over substantially the entirety of a subject's body. Suitably global inhibition of hair growth may be achieved by provision of nicotinamide, or a precursor or derivative thereof, at all sites where it is desired to inhibit hair growth, whether this provision is by the uses, methods or pharmaceutical compositions of the invention.

It has previously been reported that topical administration of agents such as nicotinamide, or a derivative or precursor thereof, may give rise to some proportion of systemic uptake of the active agent. Approximately 10% of the total amount administered topically may enter the circulation. For the purposes of the present disclosure, local or topical administration and systemic administration should be construed with reference to the site where the majority of the agent administered is to be found. Thus in the case of routes of administration where the majority of the agent has a local distribution at the site of administration, this may be considered to be local administration, even if some proportion of the agent enters the circulation. Similarly, routes of administration in which the majority of the active agent provided enters the circulation should be considered systemic, even if there is some of the agent is retained at the site of administration.

An individual may wish to inhibit hair growth after depilation or epilation, by topical application of an agent of the invention prior and post the hair removal procedure.

In a suitable embodiment, an individual wishing to inhibit hair growth may suffer from excess hair growth, such as a result of disorders such as hirsuitism, or hypertrichosis. In a suitable embodiment an individual wishing to inhibit hair growth may suffer from pseudofolliculitis barbae, or may have hair bearing naevi. "Pharmaceutical compositions "

A pharmaceutical composition of the third aspect of the invention comprises nicotinamide, or a precursor or derivative thereof, and a pharmaceutically acceptable excipient. Since pharmaceutical compositions of the invention are suitable for practicing the uses and methods of the invention, the skilled reader will appreciate that the following suggestions made regarding suitable pharmaceutical compositions of the invention are also applicable to the uses and methods of the invention.

The inventors have determined that inhibition of hair growth may be achieved by the administration of nicotinamide at a concentration of approximately 200μΜ, or by the administration of nicotinamide at a concentration of approximately 10mM. It will be appreciated that these findings are indicative of a broad range of concentrations of nicotinamide, or a precursor or derivative thereof, which may be used successfully in the inhibition of hair growth.

Suitably, a pharmaceutical composition of the invention may comprise nicotinamide, or a precursor or derivative thereof at a concentration of 100μΜ or above. A pharmaceutical composition of the invention may comprise nicotinamide, or a precursor or derivative thereof at a concentration of 150μΜ or above, 250μΜ or above, 350μΜ or above, 450μΜ, or above, 550μΜ or above, 650μΜ or above, 750μΜ or above, 850μΜ or above, or 950μΜ or above.

A pharmaceutical composition of the invention may comprise nicotinamide, or a precursor or derivative thereof, at a concentration of 1 mM or above, 5mM or above, 10mM or above, 15mM or above, 16mM (approximately equivalent to a 2% composition) or above, 20mM or above, 25mM or above, 30mM or above, 35mM or above, 40mM (approximately equivalent to a 5% composition) or above, 45mM or above, 50mM or above, or more

A pharmaceutical composition of the invention may comprise nicotinamide, or a precursor or derivative thereof, at a concentration of 100mM or above, 250mM or above, 500mM or above, 750mM or above, 1 M (approximately equivalent to a 12% composition) or above, 1.1 M or above, 1.2M or above, 1.3M or above, 1.4M or above, 1.5M or above, 2M (approximately equivalent to a 25% composition) or above, or more.

A pharmaceutical composition of the invention may comprise nicotinamide, or a precursor or derivative thereof, at a concentration of between approximately 100μΜ and approximately 50mM. Suitably a pharmaceutical composition of the invention may comprise nicotinamide, or a precursor or derivative thereof, at a concentration of between approximately 150μΜ and approximately 45mM. Indeed, a pharmaceutical composition of the invention may comprise nicotinamide, or a precursor or derivative thereof, at a concentration of between approximately 200μΜ and approximately 40mM.

The amount of a pharmaceutical composition of the invention to be administered to a subject may be determined with reference to the concentration at which the composition comprises an agent, such a nicotinamide, or a precursor or derivative thereof, and to other factors including, the route of administration, the area to be treated, the age of the subject, the half- life of the agent, and the extent of inhibition required.

A pharmaceutical composition of the invention may be formulated to provide an effective amount of nicotinamide, or a precursor or derivative thereof. Merely by way of example, a pharmaceutical composition of the invention may be formulated so that, in each incidence of treatment, it provides an amount of between approximately 0.0005 and 0.002 gram of nicotinamide per 1cm ² of an area to be treated.

In a suitable embodiment, a pharmaceutical composition of the invention may incorporate nicotinamide as a 16.5% w/v cream (equivalent to a 1.3M solution). In such an embodiment, a volume of 10ml of the cream spread over a leg (taken as having a surface area of approximately 1600cm2) would provide a therapeutically effective dose of 1.65g nicotinamide per leg. It will be appreciated that equivalent calculations may be undertaken to determine concentrations of precursors or variant of nicotinamide able to provide similar therapeutically effective amounts.

Pharmaceutical compositions of the invention may take many different forms, depending upon the way in which it is intended that they are to be used. Merely by way of example, a pharmaceutical composition of the invention may take the form of a composition for topical administration. A pharmaceutical composition of the invention in accordance with this embodiment may be used to provide localised administration of an effective amount nicotinamide, or a precursor or derivative thereof to a site where it is desired to inhibit hair growth.

Suitable examples of forms of pharmaceutical compositions of the invention include those selected from the group consisting of: creams; pastes; lotions; serums; ointments; gels; oils; sprays and foams. A pharmaceutical composition of the invention may comprise the agent, such as nicotinamide, or a precursor or derivative thereof, in combination with a pharmaceutically acceptable carrier such as polyethylene glycol (PEG).

Merely by way of example, the inventors have found that a composition comprising 10mM nicotinamide in a PEG6000 (a form of polyethylene glycol with a mean molecular weight of 6000 Daltons) carrier is effective in inhibiting hair growth. This pharmaceutical composition has been shown to be suitable for topical administration to the skin, in particular human scalp skin, in order to bring about statistically significant inhibition of hair growth.

In another embodiment nicotinamide, or a precursor or derivative thereof can be provided to the individual as a pharmaceutical composition comprising emulsifying ointment BP, isopropyl myristate, hydroxyethylcellulose, glycerol, phenoxyethanol, propylene glycol and water.

In another embodiment nicotinamide, or a precursor or derivative thereof, or a pharmaceutical composition comprising nicotinamide, or a precursor or derivative thereof, can be formulated with a delivery vehicle. For example, in a suitable embodiment, a pharmaceutical composition of the invention may comprise liposomes in which at least some of the nicotinamide, or a precursor or derivative thereof, is incorporated.

By way of further example, suitable delivery vehicles include those selected from the group consisting of: liposomes, amphoteric and cationic liposomes, niosomes, lipophilic formulations, aqueous-alcoholic solutions, hydrophilic formulations, water-in-oil nanoemulsions, nanoparticles of various sizes, microparticles of various sizes, polystyrene microspheres of various sizes, titanium dioxide particles and solid lipid particles.

Suitably liposomes employed in the compositions or methods of the invention may be ones that have a particular affinity for hair/hair follicles. By way of example suitable liposomes include, amphoteric and cationic liposomes and niosomes.

In a suitable embodiment a pharmaceutical composition of the invention may be formulated for use in combination with a device which enhances transdermal penetration of the pharmaceutical composition. The pharmaceutical composition may thus be applied to the area requiring hair growth inhibition prior to use of the device, or concurrently with use of the device. Merely by way of example such devices may include devices which produce electrical current such as galvanic devices, lasers, devices which produce vibrations such as ultrasound, and massage devices which enhance blood circulation around the hair follicles.

In a preferred embodiment, a pharmaceutical composition of the invention may be formulated for use in combination with a device used for the removal of hair by mechanical or chemical methods.

"Effective amounts" and doses

Without detracting from the considerations above regarding the amounts of nicotinamide, or a precursor or derivative thereof, that may be provided by a pharmaceutical composition of the invention, the following paragraphs also provide useful guidance as to the selection of an effective amount of such an agent, whether in the context of the uses, methods or compositions of the invention.

Suitably an effective amount of nicotinamide, or a precursor or derivative thereof, may be an amount sufficient to obtain a desired extent of inhibition of hair growth. Suitably the amount may be sufficient to obtain at least 20% inhibition of hair growth, at least 50% inhibition of hair growth, or at least 75% (or more) inhibition of hair growth as compared to a suitable control.

Suitably an effective amount of nicotinamide, or a precursor or derivative thereof, may be an amount sufficient to obtain a desired decrease in proliferation of matrix keratinocytes, progenitor cells and hair follicle keratinocytes cells associated with hair follicles. Suitably the amount may be sufficient to obtain at least 25% decrease in proliferation, at least 50% decrease in proliferation, or at least 75% (or more) decrease in proliferation of such cells as compared to a suitable control.

Suitably an effective amount nicotinamide, or a precursor or derivative thereof may be an amount sufficient to obtain a desired increase in apoptosis of matrix keratinocytes, progenitor cells and hair follicle keratinocytes cells associated with hair follicles. Suitably the amount may be sufficient to obtain at least 20% increase in apoptosis, at least 50% increase in apoptosis, or at least 75% (or more) increase in apoptosis of such cells as compared to a suitable control.

Suitably an effective amount of nicotinamide, or a precursor or derivative thereof, may be an amount sufficient to obtain a desired increase in catagen among hair follicles. Suitably the amount may be sufficient to obtain at least 20% increase in catagen, at least 50% increase in catagen, or at least 75% (or more) increase in catagen among hair follicles as compared to a suitable control.

Dosing regimens

The inventors have found that the agents, methods, and compositions of the invention are particularly effective in embodiments in which a subject is provided with an effective amount of nicotinamide, or a precursor or derivative thereof, on a daily basis. In another embodiment a subject can be provided with an effective amount of nicotinamide, or a precursor or derivative thereof on a daily basis for two weeks, followed by twice a week application for as long as the subject deems it necessary. In another embodiment a subject is provided with an effective amount of nicotinamide, or a precursor or derivative as and when the subject deems it necessary.

When the uses, methods, or compositions of the invention are to be employed in the inhibition of hair regrowth after hair removal they may be used before and/or after the hair removal has taken place. The uses, methods, or compositions of the invention may be used for the prophylactic inhibit of hair regrowth after hair removal, in which case they may be used prior to hair removal (and optionally further used after hair removal has occurred).

The invention will now be further described with reference to the following Experimental Results and accompanying drawings in which:

Figure 1 illustrates the method by which hair shaft elongation measurements were obtained in the studies set out below;

Figure 2 is a graph illustrating the effects of different concentrations of nicotinamide on hair growth (n=5 individual patients, minimum 60-80 hair follicles/data point);

Figure 3 is a bar chart illustrating the effects of different concentrations of nicotinamide on hair follicle cycle phase (n=5 individual patients, minimum 60-80 hair follicles/data point);

Figure 4 sets out a bar chart and fluorescence photomicrographs illustrating the effects of different concentrations of nicotinamide on hair follicle cell proliferation and apoptosis; Figure 5 sets out a bar chart and photomicrographs illustrating the effects of different concentrations of nicotinamide on hair follicle proliferation analysed in situ;

Figure 6 sets out a graph and photomicrographs illustrating the effects of different concentrations of nicotinamide on hair follicle proliferation analysed in situ;

Figure 7 sets out graphs illustrating the lack of effects of PEA on hair shaft elongation and hair follicle cycle phase as measured in the model used for the studies set out below;

Figure 8 sets out a bar chart and fluorescence photomicrographs illustrating that PEA does not alter hair follicle cell proliferation or apoptosis;

Figure 9 sets out graphs illustrating the effects of nicotinamide, alone (n=5 individual patients, minimum 60-80 hair follicles/data point), or in combination with PEA (n=2 individual patients, minimum 18-24 hair follicles/data point) on hair shaft elongation (*P<0.05 - significant versus control);

Figure 10 is a bar chart illustrating the effects of different concentrations of nicotinamide on mast cell degranulation;

Figure 1 1 sets out bar charts illustrating that PEA alone does not significantly hair follicle mast cell number or mast cell degranulation;

Figure 12 sets out bar charts illustrating effects of combination treatments comprising both nicotinamide and PEA on interfollicular mast cell degranulation and mast cell numbers (data are mean +/- SEM of 1-4 scalp skin organ cultures from separate donors);

Figure 13 sets out bar charts illustrating effects of combination treatments comprising both nicotinamide and PEA on hair follicle mast cell degranulation and mast cell numbers (data are mean +/- SEM of 1-4 scalp skin organ cultures from separate donors). Experimental Results

The effects of nicotinamide on hair growth inhibition

The effects of nicotinamide on hair shaft elongation were studied in cultured human hair follicles and human scalp skin organ cultures. Cultured hair follicles (whether isolated or in scalp skin) were treated with either nicotinamide or control vehicle for 6 days. Fig 1 shows an example of how hair shaft elongation measurements were obtained. As seen in Fig 1 , the hair shaft incubated with control vehicle increased in length over time, while the hair shaft incubated with nicotinamide showed no change in length over a 6 day period.

The results in Fig 2, show the effects of nicotinamide treatment at two different concentrations, 200μΜ and 10mM. Both concentrations inhibited hair growth, as shown by the decrease in hair shaft elongation. A statistically significant decrease was observed in response to the 10mM nicotinamide treatment when compared to the vehicle control. As seen in Fig 2, 10mM nicotinamide treatment reduced hair shaft elongation by more than 70% at each time point investigated throughout the course of the 6 day treatment (for example on day 3 by 72.4% and on day 6 by 73.6%). In contrast, while 200μΜ treatment demonstrated inhibition of hair growth, the magnitude of effect was non-significant.

Investigation of mechanisms by which nicotinamide inhibits hair growth

In order to identify the mechanisms by which nicotinamide inhibits hair growth, the hair follicle cycle phase was analysed (Fig 3). In the vehicle treated control group the proportion of hair follicles in the catagen and anagen phase was approximately the same, with 55% of hair follicles in the anagen phase and 45% of hair follicles in the catagen phase. Treatment of hair follicles with 200μΜ nicotinamide increased the proportion of hair follicles in the catagen phase to approximately 60% and reduced the percentage of hair follicles in the anagen phase to 40%. Treatment of hair follicles with 10mM nicotinamide significantly increased the percentage of hair follicles in the catagen phase, to at least 80%, and reduced the percentage of hair follicles in the anagen phase to less than 20%. 200μΜ treatment demonstrated an induction of catagen, but the effect was non-significant. These data suggest that nicotinamide inhibits hair growth by inducing hair follicle catagen phase.

Additional analysis investigated hair follicle cell proliferation (assessed by Ki67 staining) and hair follicle cell apoptosis (assessed by TUNEL staining) (Fig 4) in response to a 6 day treatment with 200μΜ or 10mM nicotinamide. As shown in Fig 4, treatment with either nicotinamide concentration reduced the number of hair follicle cells undergoing proliferation and significantly increased the number of hair follicle cells undergoing apoptosis when compared to the control group. The results were statistically significant when the 10mM nicotinamide concentration was used.

Moreover, hair follicle proliferation was analysed in situ, on human scalp skin organ cultures (Fig 5 and 6). The results show that topical administration of an experimental pharmaceutical composition comprising 10mM nicotinamide over a 6 day period reduced the percentage of proliferating hair follicle cells from approximately 68% in the control hair follicle to less than 20%. Additionally, another experimental pharmaceutical composition comprising nicotinamide and PEA was tested (Figure 6) and no effect on hair follicle cell proliferation was observed upon a 3 day treatment. In summary, the results presented in Fig 5 & 6 suggest that nicotinamide treatment inhibits hair growth through decreasing hair follicle cell proliferation, increasing apoptosis and promoting the catagen phase.

The effects of PEA on the inhibition of hair growth, hair growth cycle phase and hair follicle cell proliferation and apoptosis, and the effects of an experimental pharmaceutical composition comprising nicotinamide and PEA on the inhibition of hair growth

The purpose of this assay was to determine whether adding PEA to an experimental pharmaceutical composition comprising nicotinamide would further effect nicotinamide induced hair growth inhibition. Hair shaft growth and hair growth cycle phase were analysed in response to treatment with an experimental pharmaceutical composition comprising 30μΜ PEA but no nicotinamide (Fig 7). The data show that PEA has no effect on either of these parameters. Hair follicle cell proliferation and apoptosis assessment also showed no significant change between the control and PEA treated group (Fig 8). These data suggest that PEA has no impact upon hair growth.

Further, inhibition of hair growth was measured during a 6 day treatment using an experimental pharmaceutical composition comprising 10mM nicotinamide and 30μΜ PEA to determine if combining nicotinamide and PEA would influence the effect of nicotinamide on hair growth inhibition. The results of this study are shown in Fig 9. The data illustrate that 10mM nicotinamide treatment significantly reduces hair shaft elongation at all time points measured, but suggest that the combination of nicotinamide and PEA does not alter the effect of nicotinamide on hair growth inhibition. The effects of an experimental pharmaceutical composition comprising nicotinamide and/or PEA on interfollicular and hair follicle mast cell degranulation, and interfollicular and hair follicle mast cell numbers

Mast cell degranulation in response to a 6 day treatment with an experimental pharmaceutical composition comprising either 10mM or 200μΜ nicotinamide was assessed by mast cell tryptase immunostaining (Fig 10). The results show that treatment with 10mM nicotinamide significantly increased the percentage of mast cell degranulation when compared to the control treatment. The use of an experimental pharmaceutical composition comprising PEA (without nicotinamide) had no significant effect on mast cell degranulation or mast cell numbers (Fig 11).

Unexpectedly, when samples were treated with an experimental pharmaceutical composition comprising both nicotinamide and PEA a reduction in interfollicular mast cell degranulation and total mast cell number (Fig 12) was observed. Interfollicular mast cell degranulation induced by nicotinamide alone was significantly increased (as compared to controls) at days 3 and 6. When the composition comprising both nicotinamide and PEA was used a significant reduction in interfollicular mast cell degranulation (as compared to nicotinamide treatment alone) was observed at days 1 , 3, and 6. Both nicotinamide alone, and the combination of nicotinamide and PEA, significantly decreased total interfollicular mast cell numbers (as compared to controls) at days 3 and 6. The combination of nicotinamide and PEA also significantly decreased total interfollicular mast cell numbers as compared to nicotinamide alone.

While nicotinamide treatment non-significantly increased hair follicle mast cell degranulation at all time points investigated, treatment with a combination of nicotinamide and PEA brought about a (non-significant) reduction of hair follicle mast cell degranulation during the first three days of a six day treatment, and a reduction in total hair follicle mast cell numbers upon a 6 day treatment was also noticed (Fig 13). These data suggest that an experimental pharmaceutical composition comprising nicotinamide and PEA is beneficial for inhibiting hair growth, while simultaneously reducing mast cell number and degranulation, which could be associated with irritation.