Treatment of skin condition

Abstract

To provide a pharmaceutical composition for use in a method of treating a skin condition.SOLUTION: Provided is a pharmaceutical composition for use in a method of treating a skin condition of a subject. The method comprises administering intradermally to the subject a botulinum neurotoxin: herein, following administration, the botulinum neurotoxin induces secretion of one or more sebaceous lipid(s) selected from a squalene, a fatty acid, a cholesterol, and a wax ester to an epidermal layer of skin; and the skin condition is selected from a group consisting of xerosis, dehydrated, dry or cracked skin, eczema, atopic eczema, type 2 Gaucher disease, Sjogren-Larsson syndrome, ichthyosis, lamellar ichthyosis, X-linked ichthyosis, bullous ichthyosiform erythroderma, essential FFA deficiency, aged dry skin, anhidrotic ectodermal dysplasia, Netherton syndrome, actinic keratosis, carbuncle, cellulitis, skin cancer, and lichen pilaris.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to the treatment of skin conditions with Clostridial neurotoxins. [Background technology]

[0002] Skin is the largest organ in the mammalian body, providing protection from external irritants and infections and a barrier to prevent water loss. Skin consists of the epidermis (on the outside of the skin), the dermis, and the subcutaneous tissue. The outermost layer of the epidermis, the stratum corneum (SC), consists of large, flattened, enucleated keratinocytes and a lipid-rich extracellular matrix. The main classes of lipids found in the SC include fatty acids, cholesterol, squalene, and wax esters. The lipids originate (are excreted) from the sebaceous glands located in the skin, which excrete a mixture of these lipids in the form of sebum. These lipids are therefore called sebaceous lipids.

[0003] The presence of these lipids is essential for maintaining a moist, supple and healthy skin barrier. Indeed, healthy skin relies on an optimal lipid composition to form a barrier that provides protection, prevents excessive water loss, supports intercellular communication and regulates skin homeostasis and inflammation. Loss of individual lipids in the lipid composition of the skin can have significant consequences for skin health and is related to inflammatory skin conditions. Changes in the composition of SC lipids can lead to a breakdown or interference with the function of the skin barrier, resulting in transepidermal water loss (TEWL). Increases in TEWL have been observed in many skin diseases, such as atopic dermatitis (AD) and psoriasis. Thus, loss of these lipids (a decrease in the quality of the lipid composition of sebum) can lead to or predispose a person to developing skin conditions.

[0004] When these lipids (in the form of sebum) are produced in excess, they can lead to oily skin and acne. Thus, currently available skin treatments (such as retinoid-based treatments) focus on lowering the level of sebum at the expense of maintaining sufficient levels of skin-protecting lipids, with associated side effects such as accelerated skin aging, skin irritation (e.g., redness), dehydration, and infection. There is a significant lack of treatments to increase / maintain important sebaceous lipid levels (e.g., toward maintaining a balanced level that allows skin protection and hydration without causing acne or oily skin). Current treatments (such as hormone treatments using hormones that affect the secretion of sebum from the sebaceous glands) typically have systemic (non-skin-specific) effects, which can lead to undesirable off-target effects.

[0005] Thus, there is an increasing need for alternative / improved methods for treating skin conditions, particularly those resulting from insufficient sebaceous gland lipids (eg, insufficient sebum quality).

[0006] The present invention addresses this problem by providing an alternative and / or improved means for inducing sebaceous lipid secretion / excretion into the epidermis. Summary of the Invention

[0007] The present invention is based on the surprising finding that intradermal administration of Clostridium neurotoxin (such as botulinum neurotoxin) leads to an increase in the level of important skin-protecting sebaceous lipids in epidermal sebum.Clostridium neurotoxin does not necessarily substantially change the overall sebum level, which is advantageous, since increased sebum secretion is believed to be the main feature involved in the pathophysiology of acne.Therefore, the present invention leads to an improvement in sebum quality, such that sebum contains a higher relative percentage of desired lipids that have both therapeutic and cosmetic applications.

[0008] This is surprising because it was understood that clostridial neurotoxins act by inhibiting secretion of molecules (e.g., by cleaving polypeptides involved in secretory vesicles attached to cell membranes) rather than inducing or increasing secretion / excretion. For example, prior art methods involving subcutaneous administration of botulinum toxin were reported to result in reduced sebum levels in the skin (as opposed to the currently observed effects provided by intradermal administration, which elevates the levels of some specific sebaceous gland lipids in the skin without substantially changing the overall levels of sebum). The site of intradermal administration (between the epidermis and dermis) is more peripheral than the site of subcutaneous administration (i.e., subcutaneous layer, below the dermis). The composition (e.g., chemical composition) of these layers is different, and it is hypothesized (regardless of theory) that live clostridial neurotoxins may act on the sebaceous glands via different modes of action depending on the layer of skin injected, leading to different effects. The present invention has led to the observation of a previously unknown effect of intradermal administration of a clostridial neurotoxin (e.g., BoNT / A), namely the induction (e.g., increase) of secretion / excretion of certain sebaceous gland lipids as alluded to above. Without being bound by theory, it is believed that live clostridial neurotoxins (e.g., when administered intradermally) may (favorably) interact with enzymes that catalyze the production of these lipids.

[0009] References above to the terms "important skin-protecting sebaceous lipids," "desirable lipids," and "specific sebaceous lipids" refer to one or more of squalene, fatty acids, cholesterol, and wax esters, as explained in more detail below. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Each broad aspect of the invention includes any of the following: - a method for treating a skin condition, comprising administering a Clostridial neurotoxin to a patient, preferably comprising administering a Clostridial neurotoxin intradermally to a patient; - a Clostridial neurotoxin for use in a method of treating a skin condition, said method comprising administering the Clostridial neurotoxin to a patient, preferably comprising administering the Clostridial neurotoxin intradermally to the patient; - Use of a Clostridial neurotoxin in the manufacture of a medicament for treating a skin condition in a patient, preferably for intradermal administration to the patient.

[0011] Each broad aspect of the invention also includes any of the following: - non-therapeutic use of a Clostridial neurotoxin for the cosmetic treatment of the skin, preferably comprising administering the Clostridial neurotoxin intradermally to a patient; - a non-therapeutic use of a Clostridial neurotoxin to promote skin rejuvenation, preferably comprising administering the Clostridial neurotoxin intradermally to a patient; - a cosmetic method for the (cosmetic) treatment of the skin, comprising administering a Clostridial neurotoxin to a patient, preferably comprising administering a Clostridial neurotoxin intradermally to a patient.

[0012] Following administration, the Clostridial neurotoxin may induce the secretion of one or more sebaceous lipids into the epidermal layer of the skin. In other words, the Clostridial neurotoxin may treat a condition by inducing the secretion of one or more sebaceous lipids into the epidermal layer of the skin.

[0013] Examples of such sebaceous lipids include squalene, fatty acids, cholesterol, and wax esters. The skin condition is preferably a condition associated with a reduced level of sebaceous lipids (e.g., one or more of squalene, fatty acids, cholesterol, and wax esters) on the epidermal layer of the patient's skin, for example, said level of sebaceous lipids is reduced relative to the level of sebaceous lipids of subjects without the skin condition. Examples of sebaceous lipids involved in skin conditions and such conditions are also described in Sahle et al. (Skin Pharmacol Physiol 2015; 28: 42-55), which is incorporated herein by reference.

[0014] With respect to the non-therapeutic uses and / or cosmetic methods described herein, the cosmetic effect is preferably provided by increasing the level of sebaceous lipids (e.g., one or more of squalene, fatty acids, cholesterol, and wax esters) on the epidermal layer of the patient's skin, e.g., said level of sebaceous lipids is elevated relative to the level of sebaceous lipids on the epidermal layer of a subject not administered a Clostridial neurotoxin (or said level of sebaceous lipids is elevated relative to the level of sebaceous lipids on the epidermal layer of the patient prior to administration of a Clostridial neurotoxin).

[0015] Preferably, following administration, the Clostridial neurotoxin induces: - secretion (e.g. excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, and / or - Retention of tachykinin peptides (such as substance P) in the dermis of the skin.

[0016] The therapeutic or cosmetic effect may be brought about by inducing, following administration of a Clostridial neurotoxin to a patient: - secretion (e.g. excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, and / or - Retention of tachykinin peptides (such as substance P) within the dermis of the skin.

[0017] Thus, the present invention provides a method for treating a skin condition, said method comprising administering a Clostridial neurotoxin intradermally to a patient, wherein following administration, the Clostridial neurotoxin - secretion (e.g. excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, and / or - Inducing retention of tachykinin peptides (such as substance P) in the dermis of the skin.

[0018] One aspect of the invention is the administration of a Clostridial neurotoxin to a patient following intradermal administration of the neurotoxin. - secretion (e.g. excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, and / or - retention of tachykinin peptides (such as substance P) within the dermis of the skin, thereby providing a method for treating skin conditions.

[0019] Another aspect of the invention provides a Clostridial neurotoxin for use in a method of treating a skin condition, said method comprising administering a Clostridial neurotoxin intradermally to a patient, wherein following administration, the Clostridial neurotoxin - secretion (e.g. excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, and / or - Inducing retention of tachykinin peptides in the dermis of the skin.

[0020] One aspect of the present invention is - secretion (e.g. excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, and / or - retention of tachykinin peptides in the dermis of the skin, said method comprising administering the Clostridial neurotoxin intradermally to a patient.

[0021] Advantageously, the present invention also finds utility in the cosmetic field, as induction of sebaceous lipid secretion / excretion into the epidermis as described herein can improve the appearance / youthfulness of the skin and allow for skin rejuvenation.

[0022] Thus, a further aspect of the invention provides a non-therapeutic use of a Clostridial neurotoxin for the cosmetic treatment of the skin, the Clostridial neurotoxin (preferably following intradermal administration to a patient) comprising: - secretion (e.g. excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, and / or - Inducing retention of tachykinin peptides in the dermis of the skin.

[0023] One aspect of the present invention is - secretion (e.g. excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, and / or - retention of tachykinin peptides in the dermis of the skin; and non-therapeutic use of a Clostridial neurotoxin for cosmetic treatment of the skin by inducing, preferably, the method comprising administering the Clostridial neurotoxin intradermally to a patient.

[0024] Preferably, the non-therapeutic use and / or cosmetic method of the present invention comprises promoting skin rejuvenation by inducing the secretion (e.g. excretion) of one or more sebaceous lipids into the epidermal layer of the skin, preferably said one or more sebaceous lipids being selected from squalene, fatty acids, cholesterol and wax esters.

[0025] Another aspect of the present invention is - secretion (e.g. excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, and / or - retention of tachykinin peptides in the dermis of the skin; and non-therapeutic use of a Clostridial neurotoxin to promote skin rejuvenation by inducing the retention of tachykinin peptides in the dermis of the skin, preferably the method comprising intradermally administering the Clostridial neurotoxin to a patient.

[0026] Another embodiment provides a cosmetic method for the (cosmetic) treatment of the skin, wherein (preferably following intradermal administration to a patient) a Clostridial neurotoxin is - secretion (e.g. excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, and / or - Inducing retention of tachykinin peptides in the dermis of the skin.

[0027] One aspect of the invention is a method for treating a clostridial neurotoxin comprising administering to a patient a clostridial neurotoxin, preferably administering to a patient intradermally to said patient, - secretion (e.g. excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, and / or - retention of tachykinin peptides in the dermis of the skin, to provide a cosmetic method for the (cosmetic) treatment of the skin by inducing

[0028] One aspect of the invention is a method for treating a clostridial neurotoxin comprising administering to a patient a clostridial neurotoxin, preferably administering to a patient intradermally to said patient, - secretion (e.g. excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, and / or - To provide a cosmetic method for promoting skin rejuvenation by inducing retention of tachykinin peptides in the dermis of the skin.

[0029] An important advantage of employing Clostridial neurotoxins for therapeutic or cosmetic treatments is that they do not cause the unwanted side effects of current skin treatments such as retinoid creams, For example, the inventors have observed that intradermal injection of botulinum neurotoxin does not cause the skin erythema or scaling (e.g., peeling) seen following topical treatment with common retinoid creams.

[0030] Various (optional) embodiments of the present invention are described below. It is noted that each of the following embodiments is applicable to any of the methods, Clostridial neurotoxins for use, or non-therapeutic uses (e.g., cosmetic methods) described herein.

[0031] In one embodiment, the epidermal layer (eg, into which sebaceous lipids are secreted) is one or more selected from the stratum basale, stratum spinosum, stratum granulosum, or stratum corneum.

[0032] References to "sebum" typically refer to the oily / waxy substance produced by the sebaceous glands of the skin that serves to coat, moisturize, and protect the skin. Sebum is a complex mixture of fatty acids, sugars, waxes, and other natural chemicals that form a protective barrier against the evaporation of water. For example, sebum (e.g., human sebum) typically contains fatty acids (about 57%, the main components of which are triglycerides), wax esters (about 26%), squalene (about 12%), and cholesterol (about 4.5%).

[0033] Regardless of theory, it is believed that the increases in certain sebum components (e.g., squalene, fatty acids, cholesterol, and wax esters) observed following intradermal administration of a Clostridial neurotoxin are due to changes in the composition (e.g., relative levels of each) of these sebum components, rather than an increase in the total overall sebum levels, i.e., it has been observed that the relative levels of these lipids to total (overall) sebum levels may be increased following intradermal administration of a Clostridial neurotoxin.

[0034] Advantageously, therefore, an unexpected technical effect of the present invention is the increase in sebum quality (e.g., by increasing the levels of components that contribute to the beneficial properties of sebum on the skin). By improving sebum quality, rather than increasing the total sebum level, it is possible to treat skin conditions without inadvertently causing (or increasing the likelihood of) oily skin and / or other skin conditions, such as acne.

[0035] In one embodiment, the patient does not have oily skin (eg, oily facial skin) before and / or after administration of the Clostridial neurotoxin.

[0036] The term "oily skin" refers to a condition resulting from an excess production of sebum (e.g., from sebaceous glands beneath the skin's surface). Patients with oily skin have 2 Levels greater than 180 mg of sebum per cm (>180 mg / cm 2 ) and may contain sebum (e.g., on facial skin). Sebum levels (oily skin patients) may be ≥ 185 mg / cm 2 , ≥ 190mg / cm 2 , or ≥ 195 mg / cm 2 The level may refer to an average level of sebum (e.g., an average level on facial skin). The level may preferably refer to a level (e.g., an average level) of sebum present on a defined area of ​​the face, such as the forehead, nasolabial folds, and / or nose.

[0037] As mentioned elsewhere herein, sebum (eg, facial sebum) may be measured using a Sebumeter (SM8151, CL-Electronics, Cologne, Germany).

[0038] Throughout this specification any reference to levels of sebum may refer to levels of sebum measurable using a sebum meter.

[0039] In one embodiment, the patient does not have an overproduction of sebum (e.g., total sebum levels) after administration of the Clostridial neurotoxin. In other words, in one embodiment, the patient does not suffer from an overproduction of sebum (e.g., total sebum levels) after administration of the Clostridial neurotoxin.

[0040] In one embodiment, the patient has a skin area of ​​≦180 mg / cm 2 of skin (e.g., facial skin). 2 Such patients may be said to be patients without oily skin. The level of sebum may refer to the level in the area of ​​the patient's skin where the patient has the most sebum (e.g., since sebum levels tend to vary across different areas of the skin), e.g., the facial area such as the forehead, nasolabial folds, and / or nose. Due to the variation in levels across sebum, patients may have 20 mg / cm2 of skin (e.g., facial skin). 2 to ≦180 mg / cm 2 The skin may have sebum.

[0041] In one embodiment, the patient is a non-acne patient.

[0042] The term "acne-prone" refers to the presence of acne-prone skin in a patient (and thus "non-acne-prone" refers to the absence of acne-prone skin in a patient). Acne-prone skin refers to skin that has an excess of oil / sebum in the pores (e.g., typically resulting when the body's hormones are overstimulated). This, combined with the accumulation of bacteria and dead skin cells, can lead to inflammation, rashes, and severe breakouts. Some typical symptoms of acne-prone skin are inflamed pustular breakouts (age spots), severe blackheads and clogged pores, oily, rough and thick skin texture.

[0043] The treatment can be effective on the patient's face, e.g., the forehead, nasolabial folds, and / or nose (bridge and / or tip of the nose). For example, a Clostridial neurotoxin can be administered (e.g., administered intradermally) to the patient's face, e.g., the forehead, nasolabial folds, and / or nose (bridge and / or tip of the nose).

[0044] In one embodiment, the treatment (e.g., induction of sebaceous gland lipid secretion) can be effective on non-facial areas of the skin. For example, the Clostridial neurotoxin can be administered to a non-facial area of ​​the patient's skin (e.g., non-facial skin). For example, the Clostridial neurotoxin can be administered to the hands, feet, neck, scalp, and / or back (e.g., hands, feet, and / or back). The back area includes the upper back and both shoulders.

[0045] In one embodiment, following administration of a Clostridial neurotoxin, there is no induction of an increase in the level (e.g., total level) of sebum on the patient's skin (e.g., epidermal layer) relative to a reference standard, which corresponds to the level of sebum on the patient's skin (e.g., epidermal layer) without administration of the Clostridial neurotoxin (or the reference standard corresponds to the level of sebum on the patient's skin (e.g., epidermal layer) prior to administration of the Clostridial neurotoxin).

[0046] Additionally or alternatively, following administration of the Clostridial neurotoxin, no reduction in the level (e.g., total level) of sebum on the patient's skin (e.g., epidermal layer) is induced relative to a reference standard, which corresponds to the level of sebum on the patient's skin (e.g., epidermal layer) that has not been administered the Clostridial neurotoxin (or the reference standard corresponds to the level of sebum on the patient's skin (e.g., epidermal layer) prior to administration of the Clostridial neurotoxin).

[0047] In other words, in one embodiment, following administration of a Clostridial neurotoxin, the level of sebum on the patient's skin (e.g., epidermal layer) is unchanged relative to a reference standard, which corresponds to the level of sebum on the patient's skin (e.g., epidermal layer) without administration of a Clostridial neurotoxin (or the reference standard corresponds to the level of sebum on the patient's skin (e.g., epidermal layer) prior to administration of a Clostridial neurotoxin).

[0048] The term "no reduction in the level of sebum on the skin" means that there is no substantial reduction in the level of sebum on the skin. The term "substantially" as used herein in the context of the term "no reduction in the level of sebum on the skin" preferably means that there is no statistically significant reduction. The (non-substantial) reduction may be less than 5%, 2%, 1%, or 0.5% reduction. More preferably, the term "no reduction in the level of sebum on the skin" as used herein means that there is no reduction at all in the level of sebum on the skin (e.g., epidermal layer) (i.e., the reduction in the level is 0%).

[0049] Similarly, the term "no increase in the level of sebum on the skin" means that there is no substantial increase in the level of sebum on the skin. The term "substantially" as used herein in the context of the term "no increase in the level of sebum on the skin" preferably means that there is no statistically significant increase. The (non-substantial) increase may be less than 5%, 2%, 1%, or 0.5%. More preferably, the term "no increase in the level of sebum on the skin" as used herein means that there is no increase at all in the level of sebum on the skin (e.g., epidermal layer) (i.e., the level is reduced by 0%).

[0050] The term "the level of sebum on the patient's skin (e.g., epidermal layer) is unchanged" means that the level of sebum on the skin (e.g., epidermal layer) is not substantially changed (e.g., there is no increase or decrease). The term "substantially" as used herein in the context of the term "the level of sebum on the patient's skin (e.g., epidermal layer) is unchanged" preferably means that there is no statistically significant change. The change (not substantial) may be less than 5%, 2%, 1%, or 0.5%. More preferably, the term "the level of sebum on the patient's skin (e.g., epidermal layer) is unchanged" as used herein means that the level of sebum on the skin (e.g., epidermal layer) is not changed at all (i.e., the change in the level is 0%).

[0051] In one embodiment, following administration of the Clostridial neurotoxin, the level of sebum on the patient's skin (e.g., epidermal layer) increases by <30%, <25%, <20%, <15%, <10%, <5%, or <1% (e.g., <20%, <15%, <10%, <5%, or <1%). Preferably, following administration of the Clostridial neurotoxin, the level of sebum on the patient's skin (e.g., epidermal layer) may increase by <10%. The increase in the level of sebum may be compared to a reference standard, preferably the reference standard corresponding to the level of sebum on the patient's skin (e.g., epidermal layer) not administered with the Clostridial neurotoxin. Additionally or alternatively, the reference standard may correspond to the level of sebum on the patient's skin (e.g., epidermal layer) before administration of the Clostridial neurotoxin.

[0052] In one embodiment, following administration of the Clostridial neurotoxin, the patient has no elevated level of erythema and / or scaling on the skin relative to a reference standard, the reference standard corresponding to the level of erythema and / or scaling on the skin of a patient (having said skin condition) who has not been administered the Clostridial neurotoxin. Additionally or alternatively, the reference standard may correspond to the level of erythema and / or scaling on the patient's skin prior to administration of the Clostridial neurotoxin.

[0053] The term "no increase in the level of erythema and / or scaling on the skin" means that there is substantially no increase in the level of erythema and / or scaling on the skin. The term "substantially" as used herein in the context of the term "no increase in the level of erythema and / or scaling on the skin" preferably means that there is no statistically significant increase. The (non-substantial) increase may be less than 5%, 2%, 1%, or 0.5% increase. More preferably, the term "no increase in the level of erythema and / or scaling on the skin" as used herein means that there is no increase at all in the level of erythema and / or scaling on the skin (i.e., the decrease in the level is 0%).

[0054] The term "erythema" refers to a skin condition manifested as redness of the skin or mucous membranes caused by congestion (increased blood flow) in the superficial capillaries. It may result from injury, infection, or inflammation of the skin.

[0055] The term "scaly" refers to the appearance of dry, cracked, or flaky skin. Also known as desquamation, scaly skin typically occurs when the outer layer of skin, called the epidermis, begins to flake and shed. Scaly skin can occur when an injury or medical condition damages the outer layer of skin.

[0056] Preferably, following administration of the Clostridial neurotoxin, there is no change (e.g., increase or decrease) in the levels of one or more of the following parameters: - sebaceous gland surface; - the utricle surface (the utricle is, for example, a structure within the epidermis of the skin, filled with a firm plug of keratinocytes similar to that of an open human comedone); - epidermal thickness of the skin on the back; - Inflammation of the dermis; - keratinocyte proliferation; - fibroblast proliferation; - proliferation of sebaceous glands; - IL-1α levels in the dermis.

[0057] The change is compared to the level of said one or more parameters in a reference standard, the reference standard corresponding to the level of said one or more parameters in a subject / patient not administered a Clostridial neurotoxin. Additionally or alternatively, the reference standard may correspond to the level of said one or more parameters in the subject / patient prior to administration of a Clostridial neurotoxin.

[0058] "Sebaceous lipids" are so called because they are the portion of sebum secreted by the sebaceous glands. "Sebaceous glands" are microscopic exocrine glands in the skin that secrete sebum (an oily or waxy substance) to lubricate and waterproof mammalian skin and hair. In humans, sebaceous glands occur in greatest numbers on the face and scalp, but also on all parts of the skin except the palms of the hands and the soles of the feet. The form of secretion by sebaceous glands is called holocrine.

[0059] As used herein, the term "secretion" may be used interchangeably with the term "excretion". This is because sebum (and therefore sebaceous lipids) are produced by cells of the sebaceous gland called sebocytes, which break down to release their lipid / sebum content (typically to the base of the hair follicle before the lipid / sebum slowly travels up the follicle towards the skin surface). Thus, these cells may be considered to "excrete" the lipid / sebum into the epidermis layer. For example, the term "excretion" may be used to encompass secretion and / or excretion.

[0060] Throughout the specification, any reference to the level of sebaceous lipids described herein preferably refers to the level of said sebaceous lipids at or near the administration site of Clostridial neurotoxin. For example, any reference to the level of sebaceous lipids described herein may refer to the level of said sebaceous lipids (e.g., on the skin) at ≦1 cm, ≦2 cm, ≦4 cm, ≦6 cm, ≦8 cm, ≦10 cm, ≦12 cm, ≦16 cm, ≦18 cm, or ≦20 cm from the administration site of Clostridial neurotoxin. Any reference to the level of sebaceous lipids described herein may refer to the level of said sebaceous lipids at ≦2 cm, ≦4 cm, ≦6 cm, ≦8 cm, or ≦10 cm (preferably ≦4 cm) from the administration site of Clostridial neurotoxin.

[0061] One aspect of the invention provides a method for treating a skin condition, the method comprising administering a Clostridial neurotoxin intradermally to a patient, wherein following administration, the Clostridial neurotoxin: - release (e.g. secretion / excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, Induce.

[0062] One aspect of the invention involves administering to a patient intradermally a Clostridial neurotoxin followed by: - release (e.g. secretion / excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, The present invention provides a method for treating a skin condition by inducing

[0063] Another aspect of the invention provides a Clostridial neurotoxin for use in a method of treating a skin condition, said method comprising administering the Clostridial neurotoxin intradermally to a patient, wherein following administration, the Clostridial neurotoxin - release (e.g. secretion / excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, Induce.

[0064] One aspect of the present invention is - release (e.g. secretion / excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, The present invention provides a Clostridial neurotoxin for use in a method of treating a skin condition by inducing a cutaneous response, the method comprising administering the Clostridial neurotoxin intradermally to a patient.

[0065] A further aspect of the invention provides a non-therapeutic use of a Clostridial neurotoxin for the cosmetic treatment of the skin, wherein (preferably following intradermal administration to a patient) the Clostridial neurotoxin is - release (e.g. secretion / excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, Induce.

[0066] A further aspect of the invention provides a non-therapeutic use of a Clostridial neurotoxin to promote skin rejuvenation, the Clostridial neurotoxin (preferably following intradermal administration to a patient) comprising: - release (e.g. secretion / excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, Induce.

[0067] A further aspect of the invention is a method for treating a clostridial neurotoxin comprising administering to a patient a clostridial neurotoxin, preferably following intradermal administration of the clostridial neurotoxin to a patient, - release (e.g. secretion / excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, The present invention provides a non-therapeutic use of a Clostridial neurotoxin for the cosmetic treatment of the skin by inducing

[0068] A further aspect of the invention is a method for treating a clostridial neurotoxin comprising administering to a patient a clostridial neurotoxin, preferably following intradermal administration of the clostridial neurotoxin to a patient, - release (e.g. secretion / excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, The present invention provides a non-therapeutic use of a Clostridial neurotoxin to promote skin rejuvenation by inducing

[0069] Another aspect provides a cosmetic method for the (cosmetic) treatment of the skin, wherein (preferably following intradermal administration to a patient) a Clostridial neurotoxin is - release (e.g. secretion / excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, Induce.

[0070] Another aspect provides a cosmetic method for promoting skin rejuvenation, the method comprising (preferably following intradermal administration to a patient) administering a Clostridial neurotoxin to: - release (e.g. secretion / excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, Induce.

[0071] Another embodiment comprises administering to the patient a Clostridial neurotoxin, preferably following intradermal administration of the Clostridial neurotoxin to the patient, - release (e.g. secretion / excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, The present invention provides a cosmetic method for the (cosmetic) treatment of the skin by inducing

[0072] Another embodiment comprises administering to the patient a Clostridial neurotoxin, preferably following intradermal administration of the Clostridial neurotoxin to the patient, - release (e.g. secretion / excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin, To provide a cosmetic method for promoting skin rejuvenation by inducing

[0073] The term "release" as used herein includes the secretion of sebaceous lipids as well as the discharge of sebaceous lipids. For example, the term "release" as used herein may refer to the discharge (of sebaceous lipids). In addition, or alternatively, the term "release" as used herein may refer to the secretion (of sebaceous lipids).

[0074] The terms "release," "secretion," and "excretion" of sebaceous lipids refer to the release / secretion / excretion (respectively) from the sebaceous glands (eg, sebocytes) of a patient.

[0075] The term "treat" or "treatment" as used herein includes preventative treatment (e.g., to prevent the onset of a skin condition) as well as corrective treatment (treatment of a patient already suffering from a skin condition). In preferred embodiments, the term "treat" or "treatment" as used herein means corrective treatment. The term "treat" or "treatment" includes treating both the skin condition and its symptoms. In some embodiments, the term "treat" or "treatment" refers to the symptoms of a skin condition.

[0076] Thus, the Clostridial neurotoxin may be administered to a patient in a therapeutically effective amount or a prophylactically effective amount.

[0077] A "therapeutically effective amount" is any amount of a Clostridial neurotoxin that, when administered alone or in combination to a patient to treat a skin condition (or symptom), is sufficient to effect treatment of the skin condition or symptom.

[0078] A "prophylactically effective amount" is an amount of any Clostridial neurotoxin that, when administered alone or in combination to a patient, inhibits or delays the onset or recurrence of a skin condition (or symptom). In some embodiments, a prophylactically effective amount completely prevents the onset or recurrence of a skin condition. By "inhibiting" onset, it is meant either reducing the likelihood of onset of a skin condition (or symptom thereof) or preventing onset completely.

[0079] Preferably, a therapeutically and / or prophylactically effective amount is an amount that does not lead to muscle paralysis. The term "muscle paralysis" preferably refers to long-term muscle paralysis, since transient muscle paralysis may occur for a short period following administration.

[0080] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammalian subject. In one embodiment, a "patient" is a human, a companion animal (e.g., a pet such as a dog, cat, and / or rabbit), a farm animal (e.g., a pig, sheep, cow, and / or goat), and / or a horse. In a preferred embodiment, the subject is a human.

[0081] The term "non-therapeutic use" refers to the use of a Clostridial neurotoxin for cosmetic purposes, as opposed to therapeutic applications. The term "cosmetic method" refers to a method in which a Clostridial neurotoxin is administered for cosmetic purposes, as opposed to therapeutic applications. In each aspect and embodiment of the present invention related to the "non-therapeutic" use, the subject (e.g., to whom the Clostridial neurotoxin is administered) preferably does not have a skin condition, e.g., the subject may not have a skin condition described herein. The non-therapeutic use is preferably for improving the aesthetic appearance (e.g., youthfulness) of the skin, e.g., in elderly subjects, noting that endogenous sebaceous gland lipid secretion typically decreases with age. In each aspect and embodiment of the present invention related to the "cosmetic method", the subject (e.g., to whom the Clostridial neurotoxin is administered) preferably does not have a skin condition, e.g., the subject may not have a skin condition described herein. The cosmetic method is preferably for improving the aesthetic appearance (eg, youthfulness) of the skin, for example in elderly subjects, noting that endogenous sebaceous lipid secretion typically decreases with age.

[0082] In each method of the invention, the patient may not have been previously diagnosed with a skin condition. Alternatively, the patient may have been previously diagnosed with a skin condition. The patient may be a patient who exhibits disease risk factors or who is asymptomatic for the skin condition. The patient may be a patient who has suffered or is at risk for developing the skin condition. In one embodiment, the patient has been previously treated for the skin condition.

[0083] The skin condition can be any skin condition that can be alleviated (or prevented) by the presence of higher quality sebum on the skin. The "higher quality sebum" level of one of the sebum or sebaceous lipids (e.g., squalene, fatty acids, cholesterol, and one or more sebaceous lipids selected from wax esters) is elevated.

[0084] Preferably, the skin condition to be treated is accompanied by the presence of a decreased level of one or more sebaceous lipids (e.g., one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters) on the epidermal layer of the patient compared to the level of one or more sebaceous lipids on the epidermal layer of a healthy patient (e.g., a patient not having a skin condition).

[0085] Preferably, the skin condition is caused by a decrease in the level of said one or more sebaceous lipids (e.g., one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters) in the epidermal layer of the patient compared to the level of said one or more sebaceous lipids in the epidermal layer of a subject not having the skin condition.

[0086] Preferably, the skin condition is one that is inhibited (e.g., treated or prevented) by the presence of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters on the epidermal layer of the skin.

[0087] In one embodiment, the Clostridial neurotoxin is administered to a patient having sebum containing fatty acids at a concentration of <50% (v / v), <45% (v / v), <40% (v / v), <35% (v / v), <30% (v / v), <25% (v / v), <20% (v / v), <15% (v / v), or <10% (v / v). In some embodiments, the Clostridial neurotoxin is administered to a patient having sebum containing fatty acids at a concentration of <45% (v / v), <40% (v / v), <35% (v / v), <30% (v / v), <25% (v / v). The Clostridial neurotoxin may be administered to a patient having sebum containing fatty acids at a concentration of <45% (v / v). Preferably, the Clostridial neurotoxin may be administered to a patient having sebum that contains fatty acids at a concentration of ≦40% (v / v).

[0088] In one embodiment, the Clostridial neurotoxin is administered to a patient having sebum containing wax esters at a concentration of ≦20% (v / v), ≦15% (v / v), ≦10% (v / v), or ≦5% (v / v). In some embodiments, the Clostridial neurotoxin is administered to a patient having sebum containing fatty acids at a concentration of ≦15% (v / v), ≦10% (v / v), or ≦5% (v / v). The Clostridial neurotoxin may be administered to a patient having sebum containing wax esters at a concentration of ≦15% (v / v). Preferably, the Clostridial neurotoxin may be administered to a patient having sebum containing wax esters at a concentration of ≦10% (v / v).

[0089] In one embodiment, the Clostridial neurotoxin is administered to a patient having sebum containing squalene at a concentration of ≦10% (v / v), ≦8% (v / v), ≦6% (v / v), 4% (v / v), or ≦2% (v / v). In some embodiments, the Clostridial neurotoxin is administered to a patient having sebum containing squalene at a concentration of ≦8% (v / v), ≦6% (v / v), or ≦4% (v / v). The Clostridial neurotoxin may be administered to a patient having sebum containing squalene at a concentration of ≦8% (v / v). Preferably, the Clostridial neurotoxin may be administered to a patient having sebum containing squalene at a concentration of ≦6% (v / v).

[0090] In one embodiment, the Clostridial neurotoxin is administered to a patient having sebum containing cholesterol at a concentration of ≦4% (v / v), ≦3% (v / v), ≦2% (v / v), or ≦1% (v / v). In some embodiments, the Clostridial neurotoxin is administered to a patient having sebum containing cholesterol at a concentration of ≦3% (v / v), ≦2% (v / v), or ≦1% (v / v). The Clostridial neurotoxin may be administered to a patient having sebum containing cholesterol at a concentration of ≦3% (v / v). Preferably, the Clostridial neurotoxin may be administered to a patient having sebum containing cholesterol at a concentration of ≦2% (v / v).

[0091] In one embodiment, the skin condition is a disorder associated with abnormal secretion of sebaceous lipids (e.g., one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters) into the epidermal layer of a patient. Preferably, the patient has a lower level of said sebaceous lipids on the epidermal layer than a subject without the skin condition. In other words, in one embodiment, the level of said one or more sebaceous lipids on the epidermal layer (e.g., stratum corneum) of the patient (before administration of Clostridium neurotoxin) is lower (e.g., preferably at least 5%, 10%, 15%, or 20% lower, more preferably at least 10% lower) than the level of said one or more sebaceous lipids on the epidermal layer of a subject without the skin condition.

[0092] By way of example, conditions such as xerosis, psoriasis, atopic dermatitis, and ichthyosis may involve depletion of these sebaceous gland lipids.

[0093] In one embodiment, the skin condition is one or more selected from acne (e.g., acne vulgaris), atopic dermatitis, Netherton syndrome, psoriasis, itch, dehydrated skin (e.g., dry or cracked skin), actinic keratosis, rosacea, carbuncle, eczema (e.g., atopic eczema), cellulitis, dermatitis, skin cancer, lichen pilaris, skin aging (e.g., normal and / or premature aging), burns, scars, Type 2 Gaucher disease, Sjogren-Larsson syndrome, lamellar ichthyosis, X-linked ichthyosis, bullous congenital ichthyosiform erythroderma, essential free fatty acid (FFA) deficiency, aging dry skin, and anhidrotic ectodermal dysplasia.

[0094] In one embodiment, the skin condition is one or more selected from acne, atopic dermatitis, Netherton syndrome, psoriasis, dehydrated skin (e.g., dehydrated skin or cracked skin), actinic keratosis, rosacea, carbuncle, eczema (e.g., atopic eczema), cellulitis, dermatitis, skin cancer, lichen pilaris, skin aging (e.g., normal and / or premature aging), burns, or scars. For example, the skin condition may be one or more selected from acne, atopic dermatitis, dermatitis, psoriasis, eczema, or dehydrated skin (e.g., dry skin or cracked skin). Additionally or alternatively, the skin condition may be one or more selected from dermatitis, psoriasis, or eczema.

[0095] In one embodiment, the skin condition is one or more selected from acne (e.g., acne vulgaris), atopic dermatitis, psoriasis, pruritus, xerosis, dehydrated skin (e.g., dry or cracked skin), eczema (e.g., atopic eczema), type 2 Gaucher disease, Sjogren-Larsson syndrome, lamellar ichthyosis, X-linked ichthyosis, bullous congenital ichthyosiform erythroderma, essential free fatty acid (FFA) deficiency, aging dry skin, and anhidrotic ectodermal dysplasia. For example, the skin condition may be one or more selected from atopic dermatitis, psoriasis, xerosis, dehydrated skin (e.g., dry or cracked skin), eczema (e.g., atopic eczema), type 2 Gaucher disease, Sjogren-Larsson syndrome, lamellar ichthyosis, X-linked ichthyosis, bullous congenital ichthyosiform erythroderma, essential FFA deficiency, aging dry skin, and anhidrotic ectodermal dysplasia.

[0096] Preferably, the skin condition may be one or more selected from xerosis, psoriasis, atopic dermatitis, and ichthyosis (more preferably xerosis).

[0097] Additionally or alternatively, the Clostridial neurotoxin may be administered to a patient who has been exposed to one or more factors (preferably external factors) that affect the composition of sebaceous lipids, such as chemicals used for cleaning and sanitation purposes, environmental pollutants, and / or ingredients of pharmaceuticals. The term "affecting the composition of sebaceous lipids" refers to reducing the level of sebaceous lipids (e.g., one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters) on the epidermal layer of the patient.

[0098] Advantageously, the Clostridial neurotoxin may be administered after (or before or simultaneously with) an alternative (e.g., first) skin treatment that may reduce the quality of sebum on the patient's skin, e.g., predispose (or potentially predispose) the patient to the development of a skin condition as a side effect. The term "reducing the quality of sebum on the patient's skin" preferably refers to reducing the level of sebaceous lipids (e.g., one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters) on the epidermal layer of the patient.

[0099] For example, a Clostridial neurotoxin may be administered following (or in conjunction with, or at the same time as) a laser treatment, chemical peel, and / or acne treatment (e.g., adapalene treatment, which typically reduces sebum levels), preferably to enhance skin repair following said alternative treatments.

[0100] In one embodiment, a Clostridial neurotoxin is administered to a patient whose skin has been exposed to an agent that causes a decrease in the level of one or more sebaceous lipids (e.g., one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters) in the epidermal layer of the patient compared to the level of said one or more sebaceous lipids in the epidermal layer of an unexposed subject.

[0101] Clostridial neurotoxins may be administered to patients whose skin has been exposed to chemical contaminants, such as those that can act as contact allergens or irritants causing allergic or non-allergic contact dermatitis. Examples of such chemicals include metals, soaps, fragrances, preservatives, botanicals, and paraphenylenediamine.

[0102] Clostridial neurotoxins may be administered to patients whose skin has been exposed to high heat (e.g., >30° C.) and / or moisture, e.g., levels of heat and / or moisture that may affect the composition of sebaceous lipids, which may lead to loss of moisture, dry skin, and increased sweating that causes itching.

[0103] In one embodiment, the patient is undergoing (or about to undergo) a treatment (e.g., a skin treatment) to reduce the level of one or more sebaceous lipids (e.g., one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters) on the epidermal layer of the skin, e.g., relative to the level of said one or more sebaceous lipids in a reference standard, the reference standard corresponding to the level of said one or more sebaceous lipids on the epidermal layer of a patient not undergoing said treatment (e.g., a skin treatment), or the reference standard corresponding to the level of said one or more sebaceous lipids on the epidermal layer of a patient prior to undergoing said treatment (e.g., a skin treatment). Examples of such treatments may include laser treatments, chemical peels, and / or acne treatments (e.g., adapalene treatments).

[0104] The term "one or more sebaceous lipids" preferably means that at least two, three, or four of the indicated sebaceous lipids (e.g., selected from squalene, fatty acids, cholesterol, and wax esters) are induced following administration of a Clostridial neurotoxin. Thus, the secretion of at least two, three, or four sebaceous lipids may be induced by a Clostridial neurotoxin. More preferably, the term "one or more sebaceous lipids" means that the secretion of all of the indicated sebaceous lipids (e.g., squalene, fatty acids, cholesterol, and wax esters) is induced following administration of a Clostridial neurotoxin.

[0105] In one embodiment, the sebaceous lipids ("one or more sebaceous lipids") are one or more (e.g., two or more, three or more, or four or more) selected from fatty acids, squalene, cholesterol, and wax esters. In one embodiment, the sebaceous lipids are one or more (e.g., two or more, three or more, or four or more) selected from squalene, fatty acids, cholesterol, and wax esters. In one embodiment, the sebaceous lipids are one or more (e.g., two or more, three or more, or four or more) selected from cholesterol, fatty acids, squalene, and wax esters. In one embodiment, the sebaceous lipids are one or more (e.g., two or more, three or more, or four or more) selected from wax esters, fatty acids, squalene, and cholesterol.

[0106] In one embodiment, the sebaceous lipid is a fatty acid. Examples of fatty acids include free fatty acids, diglycerides (e.g., glycerides that consist of two fatty acid chains covalently bound to a single glycerol molecule via ester bonds), triglycerides (e.g., esters obtained from glycerol and three fatty acid chains), ceramides, and cholesteryl esters. For example, the fatty acid can be a triglyceride and / or a free fatty acid. The fatty acid can preferably be a triglyceride.

[0107] The free fatty acid may include or consist of the following formula (eg, with one or more substitutions): [ka]

[0108] The free fatty acid may include or consist of the following formula (eg, with one or more substitutions): [ka]

[0109] The triglyceride may comprise or consist of the formula: [ka]

[0110] Many fatty acids produced by sebaceous glands have two unique features: they are branched fatty acids and they are highly unsaturated. When triglycerides are included, they occupy the predominant proportion in sebum (e.g., about 57%). It is believed that the fatty acids of sebum are important for the delivery of lipid-soluble antioxidants to the skin surface and for enabling the pro-inflammatory and anti-inflammatory effects exerted by certain molecules. In one embodiment, the fatty acid can be a branched fatty acid. In addition, or alternatively (preferably, in addition), the fatty acid can be a highly unsaturated fatty acid.

[0111] Preferred examples of fatty acids include linoleic acid (a polyunsaturated omega-6 fatty acid) and sapienic acid (eg, cis-6 hexadecanoic acid).

[0112] Sebaceous lipids (e.g. fatty acids) may be defined by reference to their retention time on a gas chromatography (GC) column. "Retention time" (RT) is a measure of the time (preferably in minutes) it takes for a solute (e.g. lipid) to pass through a chromatographic column while a sample containing the analyte is subjected to gas chromatography analysis (preferably gas chromatography with flame ionization detection). Retention time is calculated as the time from injection (into the GC column) of the sample (e.g., in this application, sebum / sebaceous lipids) to detection of the analyte. Said detection is typically by means of a defined peak in the output of a gas chromatogram, the area of ​​which allows detection of the level of sebaceous lipids.

[0113] Gas chromatography (GC) analysis may be performed using a GC / flame ionization detection (FID) instrument such as a 7890B (Agilent Technologies), and data acquisition may optionally be performed using Empower, version 3471 (Waters Corporation).

[0114] The GC analysis may include: - Helium carrier gas; - GC column with dimensions of 30m x 0.25mm x 0.1μm; - Injector temperature of about 350°C, split injection mode; - GC column temperature gradient: 80℃ to 240℃: 10℃ / min; 240℃ to 320℃: 5℃ / min; 320°C to 350°C at 2°C / min; and 350°C for 20 minutes; - Flame ionization detection (FID) detector temperature of 250°C; - FID hydrogen flow rate of 35mL / min; - 350mL / min FID air flow rate; and / or - FID generation helium flow rate of 25mL / min.

[0115] Preferably, the GC analysis comprises subjecting 1 μl of sebum to GC analysis with helium carrier gas (35 cm / sec) using a GC column containing 5% phenyl and 95% dimethylpolysiloxane and having dimensions of 30 m×0.25 mm×0.1 μm (e.g., a Zebron ZB-SHT GC); - Injection is by split mode injection (e.g. 4mm ID, liner, straight, inactivated, no packing) - Column temperature gradient: 80℃ to 240℃: 10℃ / min; 240℃ to 320℃: 5℃ / min; 320℃-350℃: 2℃ / min; 350°C for 20 minutes, including - Lipid detection: · Detector temperature of 250°C; ·35ml / min FID hydrogen flow rate; ·350ml / min FID air flow rate; ·25ml / min FID generation helium flow rate; This is done using a flame ionization detector (FID), which shows:

[0116] In one embodiment, the fatty acid is a fatty acid having a retention time, e.g., on a GC column, of about 15.5 to 16 minutes (e.g., about 15.7 or 15.8 minutes), about 20 to 20.2 minutes (e.g., about 20 minutes), and / or about 22 to 22.2 minutes (e.g., about 22.1 minutes). Preferably, the fatty acid may be a fatty acid having a retention time (e.g., on a GC column) of about 22 to 22.2 minutes, more preferably about 22.1 minutes.

[0117] Retention times may be expressed in absolute terms or relative terms (relative to a reference / standard analyte). For example, fatty acids may be defined by reference to their "relative retention time" (RRT) on a gas chromatographic column. RRT is an expression of the retention time of the analyte (e.g., lipid) relative to the retention time of the reference / standard analyte (e.g., RRT=standard RT / sample RT). The analyte and reference / standard analyte are preferably subjected to GC analysis simultaneously. For example, a sample (e.g., sebaceous gland lipids) subjected to GC analysis may contain both the analyte (e.g., fatty acid) and the reference / standard analyte.

[0118] The reference / standard analyte may preferably be, for example, squalene.

[0119] For example, comparing the retention time of fatty acid (FA4) to that of squalene (ST1) in the gas chromatogram output of FIG. 17A (shown in a stereogram in FIG. 17B), we see that the retention time of squalene is about 25 min and the retention time of fatty acid (FA4) is 22.1 min. This demonstrates that the RTT of fatty acid (relative to the retention time of squalene) can be 25 min / 22.1 min=about 1.13 min.

[0120] In one embodiment, the fatty acid is: - about 1.5 to 1.6 minutes for the squalene retention time (e.g., about 1.6 or 1.58 minutes); - about 1.23 to 1.25 minutes (e.g., about 1.25 minutes) for the squalene retention time; and / or - a fatty acid having a relative retention time of about 0.9 to about 1.13 minutes relative to the retention time of squalene; Preferably, the retention times (eg relative retention times) are determinable by gas chromatography.

[0121] The fatty acid may be one that has a retention time of between 1 and 1.15 minutes (eg, about 1.13 minutes) relative to the retention time of squalene (eg, as can be determined by gas chromatography).

[0122] Preferably, the fatty acid may be a fatty acid having a retention time of 0.9 to 1.14 minutes (e.g., about 1.13 minutes) relative to the retention time of squalene (e.g., as can be determined by gas chromatography), more preferably, about 1.12 to 1.14 minutes (e.g., about 1.13 minutes) relative to the retention time of squalene (e.g., as can be determined by gas chromatography).

[0123] The term "fatty acid" includes ceramides (typically composed of fatty acids and sphingosine). For example, increasing the level of fatty acids / ceramides, such as ceramide 1 linoleic acid, can be beneficial in treating skin conditions, including atopic dermatitis. Ceramide 1 linoleic acid is the major reservoir of linoleic acid in the epidermis, and changes in its level are associated with subcutaneous disorders, such as atopic dermatitis. It undergoes dramatic seasonal changes and declines with age, and the treatment of the present invention can be used to combat these seasonal and age-related changes.

[0124] The ceramide may comprise or consist of the following formula (eg, with one or more further substitutions): [ka]

[0125] As used herein, the term "fatty acid" also includes "cholesteryl esters," which are esters of cholesterol, where an ester bond is formed between the carboxylic acid group of the fatty acid and the hydroxyl group of cholesterol.

[0126] The cholesteryl ester can be, for example, a cholesteryl ester having a retention time of about 40 to 42 minutes (eg, about 41 minutes) on a GC column.

[0127] The cholesteryl ester may be a cholesteryl ester having a relative retention time relative to the retention time of squalene (e.g., as can be determined by gas chromatography) of 0.55 to 0.65 minutes, 0.57 to 0.63 minutes, or 0.59 to 0.61 minutes (e.g., about 0.6 minutes).

[0128] Preferably, the cholesteryl ester may be one having a relative retention time (eg, as determinable by gas chromatography) of 0.59 to 0.625 minutes (eg, about 0.6 minutes) relative to the retention time of squalene.

[0129] The cholesteryl ester may include or consist of the following formula (eg, with one or more further substitutions): [ka]

[0130] In one embodiment, the sebaceous lipids are wax esters.

[0131] Wax esters are typically the second most abundant lipid in sebum, accounting for approximately 26% of (normal) sebum in humans. "Wax esters" are esters of fatty acids and fatty alcohols (e.g., a wax ester is formed by the combination of one fatty acid and one fatty alcohol). Wax esters are important for reducing water loss from the skin due to evaporation, and therefore dehydration of the skin.

[0132] In one embodiment, the wax ester is a wax ester having a retention time, for example, on a GC column, of about 30-32 minutes (e.g., about 31 minutes), about 35-36 minutes (e.g., about 35.5 minutes), and / or about 37-39 minutes (e.g., about 38 minutes).

[0133] Preferably, the wax ester may be a wax ester having a retention time relative to the retention time of squalene (e.g., as can be determined by gas chromatography) of 0.78 to 0.83 minutes (e.g., about 0.8 minutes), about 0.69 to 0.71 minutes (e.g., about 0.7 minutes), and / or about 0.64 to 0.67 minutes (e.g., about 0.74 minutes).

[0134] The wax ester may comprise or consist of the formula: [ka]

[0135] In one embodiment, the sebaceous lipid is squalene.

[0136] Squalene (as referred to in this disclosure) preferably comprises (eg, with one or more further substitutions) or consists of the following structure: [ka]

[0137] Squalene is unique to sebum (i.e., it is not found in any other secretion of the body) and typically accounts for about 12% of (normal) sebum in humans. Squalene is a linear intermediate that precedes cholesterol in its biosynthesis. More specifically, in the sebaceous gland, the squalene produced is not converted to lanostenol, halting the completion of the biosynthetic pathway leading to cholesterol. Possible explanations for the buildup of squalene in the sebaceous gland have been hypothesized, including overexpression / increased activity of squalene synthase in the cells, or reduced levels / activity of enzymes involved in the conversion of squalene to cholesterol. Regardless of theory, it has been hypothesized that intradermal administration of Clostridial neurotoxins may beneficially interact with one or more of the mechanisms that lead to the buildup of squalene in the sebaceous gland.

[0138] Squalene, nature's most important hydrating substance, helps maintain the hydration of the most superficial layers of the epidermis. In addition, squalene has antioxidant properties against solar radiation and, consequently, skin cancer, since it reduces the production of free radicals at the level of sun-exposed skin. Shark liver oil, rich in squalene and alkylglycerols, has been reported to be protective against bacterial and fungal infections, especially in patients with atopic dermatitis and xerosis-related lesions. When administered topically (e.g., in topical / over-the-counter preparations), squalene penetrates the skin quickly and effectively, providing a healthy return of firmness and suppleness.

[0139] The squalene may, for example, have a retention time on a GC column of about 24 to 26 minutes (eg, about 25 minutes).

[0140] In one embodiment, the sebaceous lipid is cholesterol.

[0141] Cholesterol (as referred to in this disclosure) preferably comprises (eg, with one or more further substitutions) or consists of the following structure: [ka]

[0142] Cholesterol is the least abundant sebaceous lipid in sebum, accounting for about 2-4.5% of the total sebaceous lipids in humans (together with its esters). By increasing the level of sebaceous cholesterol, the present invention may make it possible to combat conditions associated with the age-related decrease in cholesterol in the skin. The natural cholesterol level in the skin begins to decrease dramatically from the mid-fifties onwards, inducing a breakdown of the lipid barrier. This leads to increased dehydration, accelerates cracking of the skin and increases the likelihood of infection.

[0143] The cholesterol can, for example, have a retention time on a GC column of about 24.2 to 24.6 minutes (eg, about 24.4 minutes).

[0144] Cholesterol may have a relative retention time relative to the retention time of squalene (eg, as can be determined by gas chromatography) of 0.95 to 1.1 minutes, 0.97 to 1.07 minutes, 1 to 1.05 minutes, or 1.01 to 1.03 minutes (eg, about 1.02 minutes).

[0145] Preferably, cholesterol will have a relative retention time of 1.01 to 1.03 minutes (eg, about 1.02 minutes) relative to the retention time of squalene (eg, as can be determined by gas chromatography).

[0146] The term "induce the secretion of" in the context of one or more sebaceous lipids. "Induce the excretion of" and "induce the release of" mean that the level of one or more sebaceous lipids in sebum is increased. Preferred levels of such increase are outlined below along with a reference / control where such increase is quantifiable. For the avoidance of doubt, each of the following embodiments remain intended to relate to any of the methods of treatment, Clostridial neurotoxins for use, non-therapeutic uses, and cosmetic methods described herein.

[0147] The term "promoting skin rejuvenation" is preferably quantified by a corresponding increase in the level of one or more sebaceous lipids (e.g., selected from squalene, fatty acids, cholesterol, and wax esters) of sebum. For example, the term "skin rejuvenation" preferably refers to promoting the youthful / young appearance of skin through the effects of sebaceous lipids described herein, including, for example, improving skin hydration, softness, etc. Thus, the following embodiment outlining the level of increase in sebaceous lipids may be combined with any non-therapeutic use and / or cosmetic method herein to quantify the promotion of skin rejuvenation. For example, if the level of one or more sebaceous lipids of sebum is increased by at least 10%, it may be said that skin rejuvenation is increased by the corresponding level (10%).

[0148] In one embodiment, following administration of a Clostridial neurotoxin, the level of one or more sebaceous lipids (e.g., selected from squalene, fatty acids, cholesterol, and wax esters) in sebum (e.g., the patient's sebum) may be increased. For example, the level of one or more sebaceous lipids (e.g., selected from squalene, fatty acids, cholesterol, and wax esters) in sebum (e.g., the patient's sebum) may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (preferably at least 60%). Preferably, the increase is compared to the level of the one or more sebaceous lipids in the sebum of a subject not administered a Clostridial neurotoxin. Additionally or alternatively, the increase may be compared to the level of the one or more sebaceous lipids in the sebum of a patient (e.g., the patient's sebum) before administration of a Clostridial neurotoxin.

[0149] For example, following administration of a Clostridial neurotoxin according to the present invention: - the level of fatty acids in sebum (e.g. the patient's sebum) may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% (preferably at least 60%); - the level of wax esters in sebum (e.g., the patient's sebum) may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (preferably at least 60%); - the level of squalene in sebum (e.g., the patient's sebum) may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (preferably at least 60%); and / or - The level of cholesterol in sebum (e.g. the patient's sebum) may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% (preferably at least 60%).

[0150] Preferably, the increase is compared to the level of the one or more sebaceous lipids in the sebum of a subject not administered a Clostridial neurotoxin. Additionally or alternatively, the increase may be compared to the level of the one or more sebaceous lipids in the patient's sebum (e.g., the patient's sebum) prior to administration of a Clostridial neurotoxin.

[0151] In one embodiment, after administration of the Clostridial neurotoxin, the level of fatty acids in sebum (e.g., the patient's sebum) is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130%. In some embodiments, after administration of the Clostridial neurotoxin, the level of fatty acids in sebum (e.g., the patient's sebum) is increased by at least 20%, 30%, 40%, 50%, 60%, or 70%. For example, after administration of the Clostridial neurotoxin, the level of fatty acids in sebum (e.g., the patient's sebum) may be increased by at least 10%. Preferably, the level of fatty acids in sebum (e.g., the patient's sebum) may be increased by at least 50%. The increase in the level of fatty acids in sebum (e.g., the patient's sebum) may be compared to the level of fatty acids in sebum of a subject not administered the Clostridial neurotoxin. Additionally or alternatively, said increase in the level of fatty acids in sebum (e.g., the patient's sebum) may be an increase compared to the level of fatty acids in the patient's sebum prior to administration of the Clostridial neurotoxin. Throughout this specification, comparison of lipid levels preferably refers to a comparison at the site of administration of the Clostridial neurotoxin.

[0152] In one embodiment, after administration of the Clostridial neurotoxin, the level of wax esters in sebum (e.g., the patient's sebum) is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130%. In some embodiments, after administration of the Clostridial neurotoxin, the level of wax esters in sebum (e.g., the patient's sebum) is increased by at least 20%, 30%, 40%, 50%, 60%, or 70%. For example, after administration of the Clostridial neurotoxin, the level of wax esters in sebum (e.g., the patient's sebum) can be increased by at least 10%. Preferably, the level of wax esters in sebum (e.g., the patient's sebum) can be increased by at least 50%. The increase in the level of wax esters in sebum (e.g., the patient's sebum) can be compared to the level of wax esters in sebum of a subject not administered the Clostridial neurotoxin. Additionally or alternatively, the increase in the level of wax esters in sebum (e.g., the patient's sebum) may be an increase compared to the level of wax esters in the patient's sebum prior to administration of the Clostridial neurotoxin. Throughout this specification, comparison of lipid levels preferably refers to a comparison at the site of administration of the Clostridial neurotoxin.

[0153] In one embodiment, after administration of the Clostridial neurotoxin, the level of squalene in sebum (e.g., the patient's sebum) is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130%. In some embodiments, after administration of the Clostridial neurotoxin, the level of squalene in sebum (e.g., the patient's sebum) is increased by at least 20%, 30%, 40%, 50%, 60%, or 70%. In some embodiments, after administration of the Clostridial neurotoxin, the level of squalene in sebum (e.g., the patient's sebum) is increased by at least 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, or 160%. For example, after administration of the Clostridial neurotoxin, the level of squalene in sebum (e.g., the patient's sebum) may be increased by at least 10%. Preferably, the level of squalene in sebum (e.g., the patient's sebum) may be increased by at least 50%. The increase in the level of squalene in sebum (e.g., the patient's sebum) may be compared to the level of squalene in sebum of a subject not administered the Clostridial neurotoxin. Additionally or alternatively, the increase in the level of squalene in sebum (e.g., the patient's sebum) may be compared to the level of squalene in the patient's sebum before administration of the Clostridial neurotoxin. Throughout this specification, the comparison of lipid levels preferably refers to a comparison at the site of administration of the Clostridial neurotoxin.

[0154] In one embodiment, after administration of the Clostridial neurotoxin, the level of cholesterol in sebum (e.g., the patient's sebum) is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130%. In some embodiments, after administration of the Clostridial neurotoxin, the level of cholesterol in sebum (e.g., the patient's sebum) is increased by at least 20%, 30%, 40%, 50%, 60%, or 70%. In some embodiments, after administration of the Clostridial neurotoxin, the level of cholesterol in sebum (e.g., the patient's sebum) is increased by at least 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, or 160%. For example, after administration of a Clostridial neurotoxin, the level of cholesterol in sebum (e.g., the patient's sebum) may be increased by at least 10%. Preferably, the level of cholesterol in sebum (e.g., the patient's sebum) may be increased by at least 50%. The increase in the level of cholesterol in sebum (e.g., the patient's sebum) may be compared to the level of cholesterol in sebum of a subject not administered a Clostridial neurotoxin. Additionally or alternatively, the increase in the level of cholesterol in sebum (e.g., the patient's sebum) may be compared to the level of cholesterol in the patient's sebum before administration of a Clostridial neurotoxin. Throughout this specification, a comparison of lipid levels preferably refers to a comparison at the site of administration of a Clostridial neurotoxin.

[0155] Administration of a Clostridial neurotoxin may increase the proportion of sebum that is contributed by fatty acids, wax esters, cholesterol, and / or squalene.

[0156] In one embodiment, following administration of a Clostridial neurotoxin according to the present invention: - the relative proportion of fatty acids in sebum (e.g., the patient's sebum) may be at least about 60%, 65%, 70%, 75%, or 80% (e.g., elevated from a typical value such as 57%); - the relative proportion of wax esters in sebum (e.g., the patient's sebum) may be at least about 30%, 35%, 40%, 45%, or 50% (e.g., increased from a typical value such as 26%); - the relative proportion of squalene in sebum (e.g., the patient's sebum) may be at least about 15%, 20%, 25%, 30%, 35%, or 40% (e.g., elevated from a typical value such as 12%); and / or The relative proportion of cholesterol in sebum (e.g., the patient's sebum) may be at least about 5%, 10%, 15%, 20%, or 25% (e.g., elevated from a typical value such as 4.5%).

[0157] For example, following administration of a Clostridial neurotoxin according to the present invention: - the relative proportion of fatty acids in sebum (e.g., the patient's sebum) may be about 60%, 65%, 70%, 75% or 80% (e.g., elevated from a typical value such as 57%); - the relative proportion of wax esters in sebum (e.g., the patient's sebum) may be about 30%, 35%, 40%, 45%, or 50% (e.g., elevated from a typical value such as 26%); the relative proportion of squalene in sebum (e.g., the patient's sebum) may be about 15%, 20%, 25%, 30%, 35%, or 40% (e.g., elevated from a typical value such as 12%); and / or The relative proportion of cholesterol in sebum (eg, the patient's sebum) may be about 5%, 10%, 15%, 20% or 25% (eg, elevated from a typical value such as 4.5%).

[0158] The term "relative proportion" refers to the total volume of that sebaceous lipid in sebum relative to the total volume of sebum. The relative proportion is preferably measured as a percentage of the volume of sebaceous lipid relative to the volume of sebum (v / v).

[0159] The "relative proportion" comparison may refer to the sebum surrounding the site of administration of the Clostridial neurotoxin, for example within 5 cm, 4 cm, 3 cm, 2 cm, or 1 cm of the administration site.

[0160] In one embodiment, after administration of the Clostridial neurotoxin, the relative proportion of fatty acids in sebum (e.g., the patient's sebum) may be at least about 60%, 65%, 70%, 75%, or 80%. In some embodiments, after administration of the Clostridial neurotoxin, the relative proportion of fatty acids in sebum (e.g., the patient's sebum) may be at least about 65%, 70%, 75%, or 80%. For example, after administration of the Clostridial neurotoxin, the relative proportion of fatty acids in sebum (e.g., the patient's sebum) may be at least about 70%. The relative proportion of fatty acids in sebum may be elevated from a typical value (e.g., a basal value), such as about 57%.

[0161] In one embodiment, after administration of the Clostridial neurotoxin, the relative proportion of wax esters in sebum (e.g., the patient's sebum) may be at least about 30%, 35%, 40%, 45%, or 50%. In some embodiments, after administration of the Clostridial neurotoxin, the relative proportion of wax esters in sebum (e.g., the patient's sebum) may be at least about 35%, 40%, 45%, or 50%. For example, after administration of the Clostridial neurotoxin, the relative proportion of wax esters in sebum (e.g., the patient's sebum) may be at least about 40%. The relative proportion of wax esters in sebum may be elevated from a typical value (e.g., a baseline value), such as about 26%.

[0162] In one embodiment, after administration of the Clostridial neurotoxin, the relative proportion of squalene in sebum (e.g., the patient's sebum) may be at least about 15%, 20%, 25%, 30%, 35%, or 40%. In some embodiments, after administration of the Clostridial neurotoxin, the relative proportion of squalene in sebum (e.g., the patient's sebum) may be at least about 20%, 25%, 30%, 35%, or 40%. For example, after administration of the Clostridial neurotoxin, the relative proportion of squalene in sebum (e.g., the patient's sebum) may be at least about 25%. The relative proportion of squalene in sebum may be elevated from a typical value (e.g., a basal value), such as about 12%.

[0163] In one embodiment, after administration of the Clostridial neurotoxin, the relative percentage of cholesterol in sebum (e.g., the patient's sebum) may be at least about 5%, 10%, or 15%. In one embodiment, after administration of the Clostridial neurotoxin, the relative percentage of cholesterol in sebum (e.g., the patient's sebum) may be at least about 5%, 10%, 15%, 20%, or 25%. In one embodiment, after administration of the Clostridial neurotoxin, the relative percentage of cholesterol in sebum (e.g., the patient's sebum) may be at least about 10%, 15%, 20%, or 25%. For example, after administration of the Clostridial neurotoxin, the relative percentage of cholesterol in sebum (e.g., the patient's sebum) may be at least about 10%. The relative percentage of cholesterol in sebum may be elevated from a typical value (e.g., a basal value), such as about 4.5%.

[0164] In one embodiment, after administration of a Clostridial neurotoxin, the relative proportion of one or more sebaceous lipids (e.g., selected from squalene, fatty acids, cholesterol, and wax esters) of sebum (e.g., the patient's sebum) may be increased. For example, the relative proportion of one or more sebaceous lipids (e.g., selected from squalene, fatty acids, cholesterol, and wax esters) of sebum (e.g., the patient's sebum) may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (preferably at least 60%). Preferably, the increase is compared to the relative proportion of the one or more sebaceous lipids of the patient's sebum (e.g., the patient's sebum) that has not been administered a Clostridial neurotoxin. Additionally or alternatively, the increase may be compared to the relative proportion of the one or more sebaceous lipids of the patient's sebum (e.g., the patient's sebum) before administration of a Clostridial neurotoxin.

[0165] For example, following administration of a Clostridial neurotoxin according to the present invention: - the relative proportion of fatty acids in sebum (e.g. the patient's sebum) may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% (preferably at least 60%); - the relative proportion of wax esters in sebum (e.g. the patient's sebum) may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% (preferably at least 60%); - the relative proportion of squalene in sebum (e.g., the patient's sebum) may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (preferably at least 60%); and / or - the relative proportion of cholesterol in sebum (e.g. the patient's sebum) may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% (preferably at least 60%).

[0166] In one embodiment, after administration of the Clostridial neurotoxin, the relative ratio of fatty acids in sebum (e.g., the patient's sebum) is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130%. In some embodiments, after administration of the Clostridial neurotoxin, the relative ratio of fatty acids in sebum (e.g., the patient's sebum) is increased by at least 20%, 30%, 40%, 50%, 60%, or 70%. For example, after administration of the Clostridial neurotoxin, the relative ratio of fatty acids in sebum (e.g., the patient's sebum) can be increased by at least 10%. Preferably, the relative ratio of fatty acids in sebum (e.g., the patient's sebum) can be increased by at least 50%. The increase in the relative proportions of fatty acids in sebum (e.g., the patient's sebum) may be compared to the relative proportions of fatty acids in sebum of a subject not administered a Clostridial neurotoxin. Additionally or alternatively, the increase in the relative proportions of fatty acids in sebum (e.g., the patient's sebum) may be compared to the relative proportions of fatty acids in the patient's sebum prior to administration of a Clostridial neurotoxin.

[0167] In one embodiment, after administration of the Clostridial neurotoxin, the relative proportion of wax esters in sebum (e.g., the patient's sebum) is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130%. In some embodiments, after administration of the Clostridial neurotoxin, the relative proportion of wax esters in sebum (e.g., the patient's sebum) is increased by at least 20%, 30%, 40%, 50%, 60%, or 70%. For example, after administration of the Clostridial neurotoxin, the relative proportion of wax esters in sebum (e.g., the patient's sebum) can be increased by at least 10%. Preferably, the relative proportion of wax esters in sebum (e.g., the patient's sebum) can be increased by at least 50%. The increase in the relative proportion of wax esters in sebum (e.g., the patient's sebum) can be compared to the relative proportion of wax esters in sebum of a subject not administered a Clostridial neurotoxin. Additionally or alternatively, the increase in the relative proportion of wax esters in sebum (e.g., the patient's sebum) can be compared to the relative proportion of wax esters in the patient's sebum prior to administration of a Clostridial neurotoxin.

[0168] In one embodiment, after administration of the Clostridial neurotoxin, the relative proportion of squalene in sebum (e.g., the patient's sebum) is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130%. In some embodiments, after administration of the Clostridial neurotoxin, the relative proportion of squalene in sebum (e.g., the patient's sebum) is increased by at least 20%, 30%, 40%, 50%, 60%, or 70%. In some embodiments, after administration of the Clostridial neurotoxin, the relative proportion of squalene in sebum (e.g., the patient's sebum) is increased by at least 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, or 160%. For example, after administration of a Clostridial neurotoxin, the relative proportion of squalene in sebum (e.g., the patient's sebum) may be increased by at least 10%. Preferably, the relative proportion of squalene in sebum (e.g., the patient's sebum) may be increased by at least 50%. The increase in the relative proportion of squalene in sebum (e.g., the patient's sebum) may be compared to the relative proportion of squalene in the sebum of a subject not administered a Clostridial neurotoxin. Additionally or alternatively, the increase in the relative proportion of squalene in sebum (e.g., the patient's sebum) may be compared to the relative proportion of squalene in the patient's sebum before administration of a Clostridial neurotoxin.

[0169] In one embodiment, after administration of the Clostridial neurotoxin, the relative proportion of cholesterol in sebum (e.g., the patient's sebum) is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, or 130%. In some embodiments, after administration of the Clostridial neurotoxin, the relative proportion of cholesterol in sebum (e.g., the patient's sebum) is increased by at least 20%, 30%, 40%, 50%, 60%, or 70%. In some embodiments, after administration of the Clostridial neurotoxin, the relative proportion of cholesterol in sebum (e.g., the patient's sebum) is increased by at least 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, or 160%. For example, after administration of a Clostridial neurotoxin, the relative ratio of cholesterol in sebum (e.g., the patient's sebum) may be increased by at least 10%. Preferably, the relative ratio of cholesterol in sebum (e.g., the patient's sebum) may be increased by at least 50%. The increase in the relative ratio of cholesterol in sebum (e.g., the patient's sebum) may be compared to the relative ratio of cholesterol in sebum of a subject not administered a Clostridial neurotoxin. Additionally or alternatively, the increase in the relative ratio of cholesterol in sebum (e.g., the patient's sebum) may be compared to the relative ratio of cholesterol in the patient's sebum before administration of a Clostridial neurotoxin.

[0170] Induction of sebaceous lipid secretion (following administration of a Clostridial neurotoxin) may be measured by detecting an increase in the level of sebaceous lipids on the patient's skin surface (e.g., the epidermal layer), preferably measured relative to a reference standard that has not been administered the Clostridial neurotoxin.

[0171] The increase in sebaceous lipids can be determined by any suitable method known to those skilled in the art, with particular reference to the examples described herein. For example, lipid levels can be measured by subjecting a sebum sample to gas chromatography analysis to generate a chromatographic output, and detecting the area of ​​one or more defined peaks in the chromatogram, the one or more defined peaks corresponding to fatty acids, wax esters, squalene, and / or cholesterol. The area of ​​the one or more defined peaks provides a detection of the level of the corresponding lipid in the sebum. In each such method, the sebum sample can be collected (e.g., collected) by contacting a collection means with the surface of the skin. Examples of such collection means include cotton pads, glass cones (or cylinders), glass disks, paper, and / or hydrophobic films. Preferably, the collection means is a glass disk.

[0172] Preferably, the "sebum sample" is a lipid extract, which comprises lipids extracted (e.g., purified) from sebum. Suitable methods for lipid extraction are described in the Examples.

[0173] Alternatively, or in addition, immunological methods may be used to measure the levels of the molecules described herein. For example, levels of tachykinin peptides (e.g., substance P) can be measured by subjecting isolated skin samples to an immunological assay, such as an ELISA assay.

[0174] Tachykinin peptides are neuropeptides typically 10 to 12 amino acid residues long, the best known being substance P. Genes that produce tachykinin peptides code for precursor proteins called preprotachykinins, which are processed by post-translational proteolytic processes into smaller peptides (i.e., tachykinin peptides). These genes also code for multiple subtype forms of a diverse set of peptides. Tachykinin peptides have been implicated in inflammation.

[0175] In one embodiment, following administration, the Clostridial neurotoxin may induce retention of a tachykinin peptide (preferably substance P) within the dermis of the skin. In one embodiment, the tachykinin peptide is one or more selected from substance P, neurokinin A, neuropeptide K, or neuropeptide gamma (preferably substance P).

[0176] In one embodiment, following administration, the Clostridial neurotoxin may induce the secretion (e.g., excretion) of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin and may induce retention of a tachykinin peptide (preferably substance P) within the dermis of the skin.

[0177] "Substance P" is a peptide that is a member of the tachykinin neuropeptide family. It is a neuropeptide that functions as both a neurotransmitter and a neuromodulator. Substance P and the closely related neurokinin A (NKA) are produced from a polyprotein precursor after alternative splicing of the preprotachykinin A gene.

[0178] Substance P as referred to herein may comprise an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% (e.g. at least 95%) sequence identity to the sequence of SEQ ID NO: 17. Preferably, substance P as referred to herein may comprise or consist of the amino acid sequence of SEQ ID NO: 17.

[0179] It may be particularly advantageous to induce both lipid secretion and retention of tachykinin peptides (preferably substance P). Depletion or inhibition of the main sebaceous lipids in the skin / stratum corneum (which may be caused by various environmental and physical factors such as soap, dry air, and age) is one of the etiological factors that creates dryness and barrier disruption in skin conditions. As a result, the skin loses moisture, dries, cracks, and cracks, and allows the entry of allergens, toxins, and microorganisms that may inflame and irritate the skin. Inflammation may then cause further disruption of barrier function. This may be alleviated by either induction of sebaceous lipid secretion or retention of tachykinin peptides by the method of the present invention, and indeed by both lipid secretion and retention of tachykinin peptides. Thus, the present invention may prevent the loss of sebaceous lipid barrier function which can lead to skin conditions such as severe dryness, itchiness, and cracking which in turn can lead to secondary skin conditions such as herpes, warts, warts, Staphylococcus aureus, Streptococcus, Pseudomonas aeruginosa, fungi, yeast, and tuberculosis.

[0180] Induction of sebaceous lipid secretion (and / or reduction in erythema / scaling) may be measured in human patients / subjects (e.g., analysis may be performed on isolated sebum samples obtained from human patients / subjects).

[0181] Conveniently, the induction of sebaceous lipid secretion (and / or reduction in erythema / scaling) may be measured in a suitable model organism such as mice.

[0182] A particularly suitable model organism is the mutant hr rh-JRhino mice are (hairless) rhino mice. Rhino mice have spleen cells with poor response to T-dependent antigens and have increased numbers of THY1-positive epidermal dendritic cells. As a result, rhino mice are hairless by 5 weeks and have wrinkled, thick skin. Rhino mice are now a widely accepted model organism for the study of skin conditions (and the effect of therapeutic / cosmetic agents on said skin conditions). Importantly, rhino mice continue to produce sebum (e.g., at effectively native levels), which can be readily retrieved from the skin surface (e.g., via a glass cone) due to the hairless nature of the mice.

[0183] Further details of the Clostridial neurotoxins encompassed by the present invention are provided below, along with background information on the technology.

[0184] Bacteria of the genus Clostridia produce highly potent and unique protein toxins that are capable of poisoning neurons and other cells to which they are delivered. Examples of such clostridial toxins include the neurotoxins (TeNT) produced by C. tetani and those produced by C. botulinum (BoNT) serotypes A to G, as well as those produced by C. baratii and C. butyricum.

[0185] Clostridial neurotoxins cause muscle paralysis by inhibiting cholinergic transmission in the peripheral nervous system, especially at the neuromuscular junction, and can therefore be lethal. In nature, clostridial neurotoxins are synthesized as single-chain polypeptides, which are post-translationally modified by a proteolytic cleavage event to form two polypeptide chains linked together by a disulfide bond. Cleavage occurs at a specific cleavage site, often referred to as the active site, which is located between the cysteine ​​remnants that result in the interchain disulfide bond. It is this two-chain form that results in the active form of the toxin. The two chains are designated the heavy chain (H chain), which has a molecular weight of approximately 100 kDa, and the light chain (L chain), which has a molecular weight of approximately 50 kDa. The H chain is linked to an N-terminal translocation component (H chain), which is linked to an N-terminal translocation component (H chain). Ndomain) and the C-terminal targeting component (H C The cleavage sites are the L chain and the H chain. N It is located between the domain.

[0186] The mode of action of clostridial neurotoxins depends on five distinct steps: (1)H C (2) binding of the domain to the plasma membrane of its target neuron, followed by internalization of the bound toxin into the cell via endosomes; (3) H N (4) proteolytic cleavage by the L chain of intercellular transport proteins known as SNARE proteins, resulting in a non-cytotoxic protease function; and (5) inhibition of cellular secretion from target cells.

[0187] In this chain of events, SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are essential for intracellular vesicle fusion and thus secretion of molecules via vesicle transport from cells. Examples of SNARE proteins present in neurons include SNAP-25, VAMP, or syntaxin, among others. The non-cytotoxic protease function of the L chain, on the other hand, is a zinc-dependent endopeptidase activity, which exhibits a high degree of substrate specificity for SNARE proteins. Thus, once delivered to the target neuronal cell, the non-cytotoxic proteases of clostridial neurotoxins inhibit the release of neurotransmitters by cleaving their substrate SNARE proteins, which results in neuroparalysis. Thus, clostridial neurotoxins act by inhibiting secretion.

[0188] Thanks to their unique properties, clostridial neurotoxins such as botulinum toxins have been successfully employed in a wide range of therapeutic applications, especially against movement disorders and autonomic failure, e.g. to restore the activity of overactive nerve endings to normal levels. So far, at least seven antigenically distinct BoNT serotypes have been described: BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, and BoNT / G (Rossetto, O. et al., "Botulinum neurotoxins: genetic, structural and mechanistic insights." Nature Reviews Microbiology 12.8 (2014): 535-549). In addition to the seven major serotypes, there are also naturally occurring chimeric serotypes, such as BoNT / D-C (Moriishi et al., "Mosaic structures of neurotoxins produced from Clostridium botulinum types C and D organisms," Biochim Biophys Acta. (1996):1307:123-126.).

[0189] Additionally, an eighth serotype, BoNT / X, was recently identified (Zhang et al., “Identification and characterization of a novel botulinum neurotoxin,” Nature Communications, vol 8, article no: 14130 (2017)). The term "clostridial neurotoxin" may also include newly discovered members of the botulinum neurotoxin protein family expressed by non-clostridial microorganisms, such as the Enterococcus-encoded toxin with closest sequence identity to BoNT / X, the Weissella oryzae-encoded toxin called BoNT / Wo that cleaves VAMP2 in W89-W90 (NCBI Reference Seq: WP_027699549.1), the Enterococcus faecium-encoded toxin that cleaves VAMP2 and SNAP25 (GenBank: OTO22244.1), and the Chryseobacterium pipero-encoded toxin (NCGI Reference Seq: WP_034687872.1).

[0190] Despite this diversity, BoNT / A remains the serotype of choice in treatment with three commonly available commercial formulations (Botox®, Dysport®, and Xeomin®), while only one BoNT / B product is commercially available (Neurobloc® / Myobloc®). To date, these BoNT / A and BoNT / B products, which are toxins purified from Clostridium strains, are the only two BoNT serotypes currently approved by regulatory agencies for use in humans for applications including, inter alia, spasticity, bladder dysfunction, or hyperhidrosis (for BoNT / A) (see, e.g., https: / / www.medicines.org.uk / emc / medicine / 112, https: / / www.medicines.org.uk / emc / medicine / 870, https: / / www.medicines.org.uk / emc / medicine / 2162, all of which are incorporated herein by reference in their entireties) and cervical dystonia (for BoNT / B) (see, e.g., https: / / www.medicines.org.uk / emc / medicine / 20568, all of which are incorporated herein by reference in their entireties).

[0191] In contrast to cytotoxic proteases (e.g., ricin, diphtheria toxin, Pseudomonas aeruginosa exotoxin) that act by killing their natural target cells, clostridial neurotoxins are non-cytotoxic proteases that act by temporarily disabling the cellular functions of their natural target cells. Importantly, non-cytotoxic proteases do not kill the natural target cells on which they act. In addition to clostridial neurotoxins (e.g., botulinum neurotoxins, commercially available under names such as Dysport®, Neurobloc®, and Botox®), some of the best-known examples of non-cytotoxic proteases include the lgA proteases (see, e.g., WO 99 / 032272) and the anthraase proteases (see, e.g., WO 2011 / 022357).

[0192] The term "clostridial neurotoxin" as used herein refers to any polypeptide that enters neurons and inhibits neurotransmitter release. This process includes binding of the neurotoxin to low or high affinity receptors, internalization of the neurotoxin, translocation of the endopeptidase portion of the neurotoxin to the cytoplasm, and enzymatic modification of the neurotoxin substrate. More specifically, the term "neurotoxin" includes any polypeptide produced by Clostridium bacteria that enters neurons and inhibits neurotransmitter release (clostridial neurotoxin), and such polypeptides produced by recombinant or chemical techniques. It is this two-chain form that provides the active form of the toxin. The two chains are designated the heavy chain (H chain), which has a molecular weight of about 100 kDa, and the light chain (L chain), which has a molecular weight of about 50 kDa. Preferably, the clostridial neurotoxin is a botulinum neurotoxin (BoNT).

[0193] BoNT serotypes A through G can be distinguished based on inactivation by specific neutralizing antisera using serotype classification correlated with percentage sequence identity at the amino acid level. BoNT proteins of a particular serotype are further divided into various subtypes based on percentage amino acid sequence identity.

[0194] The clostridial neurotoxin of the present invention may be selected from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, and TeNT (tetanus neurotoxin). Preferably, the clostridial neurotoxin is a botulinum neurotoxin, such as a botulinum neurotoxin selected from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, and BoNT / X. For example, the clostridial neurotoxin may be selected from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, and BoNT / G.

[0195] In one embodiment, the Clostridial neurotoxin may be BoNT / A. An exemplary BoNT / A neurotoxin amino acid sequence is set forth as SEQ ID NO: 1 (UniProt Accession Number: A5HZZ9). Another exemplary BoNT / A neurotoxin amino acid sequence is set forth as SEQ ID NO: 13 (UniProt Accession Number: P10845). In another embodiment, the Clostridial neurotoxin may be BoNT / B. An exemplary BoNT / B neurotoxin amino acid sequence is set forth as SEQ ID NO: 2 (UniProt Accession Number: B1INP5). In another embodiment, the Clostridial neurotoxin may be BoNT / C. An exemplary BoNT / C neurotoxin amino acid sequence is set forth as SEQ ID NO: 3 (UniProt Accession Number: P18640). In another embodiment, the Clostridial neurotoxin may be BoNT / D. An exemplary BoNT / D neurotoxin amino acid sequence is set forth as SEQ ID NO: 4 (UniProt Accession Number: P19321). In another embodiment, the Clostridial neurotoxin may be BoNT / E. An example of a BoNT / E neurotoxin amino acid sequence is set forth as SEQ ID NO: 5 (Accession Number: WP_003372387). Another example of a BoNT / E neurotoxin amino acid sequence is set forth as SEQ ID NO: 16 (UniProt Accession Number: Q00496). In another embodiment, the Clostridial neurotoxin may be BoNT / F. An example of a BoNT / F neurotoxin amino acid sequence is set forth as SEQ ID NO: 6 (UniProt Accession Number: Q57236) or SEQ ID NO: 9 (UniProt / UniParc Accession Number: UPI0001DE3DAC). In another embodiment, the Clostridial neurotoxin may be BoNT / G. An example of a BoNT / G neurotoxin amino acid sequence is set forth as SEQ ID NO: 7 (Accession Number: WP_039635782). In another embodiment, the Clostridial neurotoxin may be BoNT / D-C. An example of a BoNT / D-C neurotoxin amino acid sequence is set forth as SEQ ID NO: 8 (Accession Number: BAM65681). In another embodiment, the Clostridial neurotoxin may be BoNT / X. An exemplary BoNT / X neurotoxin amino acid sequence is provided as SEQ ID NO: 11 (Accession Number: BAQ12790.1). In another embodiment, the Clostridial neurotoxin may be TeNT. An exemplary TeNT neurotoxin amino acid sequence is provided as SEQ ID NO: 12 (UniProt Accession Number: P04958).

[0196] In one embodiment, the Clostridial neurotoxin consists of or comprises an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:11. In one embodiment, the Clostridial neurotoxin consists of or comprises an amino acid sequence of any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:11.

[0197] As used throughout this specification, the term "percentage sequence identity" between two or more amino acid sequences refers to a function of the number of identical amino acids at identical positions shared by both aligned amino acid sequences. Thus, as used herein, % identity can be calculated as the number of identical amino acids at each position in the alignment divided by the total number of amino acids in the aligned sequences, multiplied by 100. The calculation of % sequence identity may also take into account the number of gaps and the length of each gap that need to be introduced to optimize the alignment of two or more sequences. The comparison of sequences and the determination of the percentage identity between two or more sequences can be performed using certain mathematical algorithms, in particular global alignment mathematical algorithms (such as those described in Needleman and Wunsch, J. Mol. Biol. 48(3), 443-453, 1972), which will be familiar to those skilled in the art.

[0198] In one embodiment, a Clostridial neurotoxin consists of or comprises an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 1. For example, a Clostridial neurotoxin may consist of or comprise an amino acid sequence having at least 90%, (more preferably at least 95%) sequence identity to SEQ ID NO: 1.

[0199] In one embodiment, the Clostridial neurotoxin consists of or comprises the amino acid sequence of SEQ ID NO:1 (eg, BoNT / A).

[0200] In one embodiment, the Clostridial neurotoxin consists of or comprises an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 13. For example, the Clostridial neurotoxin may consist of or comprise an amino acid sequence having at least 90%, (more preferably at least 95%) sequence identity to SEQ ID NO: 13. In one embodiment, the Clostridial neurotoxin consists of or comprises the amino acid sequence of SEQ ID NO: 13 (e.g., BoNT / A).

[0201] The term "Clostridial neurotoxin" is intended to encompass hybrid and chimeric Clostridial neurotoxins. In one embodiment, the Clostridial neurotoxin is a chimeric neurotoxin.

[0202] A hybrid Clostridial neurotoxin comprises at least a portion of a light chain from one Clostridial neurotoxin or subtype and at least a portion of a heavy chain from another Clostridial neurotoxin or Clostridial neurotoxin subtype. In one embodiment, a hybrid Clostridial neurotoxin may comprise the entire light chain from one Clostridial neurotoxin subtype and a heavy chain from another Clostridial neurotoxin subtype. In another embodiment, a chimeric Clostridial neurotoxin may comprise a portion of a heavy chain (e.g., a binding domain) from one Clostridial neurotoxin subtype together with another portion of a heavy chain from another Clostridial neurotoxin subtype. Also, or alternatively, a therapeutic element may comprise each light chain portion from each of the different Clostridial neurotoxins. The hybrid or chimeric Clostridial neurotoxins are useful, for example, as a means of delivering the therapeutic benefits of the Clostridial neurotoxins to subjects who have immune resistance to a particular Clostridial neurotoxin subtype, who may have a lower than average concentration of receptors for a particular Clostridial neurotoxin heavy chain binding domain, or who may have antiprotease mutations in membrane or vesicular toxin substrates (e.g., SNAP-25, VAMP, and syntaxin). Hybrid and chimeric Clostridial neurotoxins are described in U.S. Patent No. 8,071,110, which is incorporated herein by reference in its entirety. Thus, in one embodiment, the Clostridial neurotoxin (or fragment thereof) of the present invention is a hybrid Clostridial neurotoxin or a chimeric Clostridial neurotoxin.

[0203] As may be used herein, the term "chimeric neurotoxin" refers to a neurotoxin that contains one or more domains derived from a first neurotoxin and one or more domains derived from a second neurotoxin. For example, a chimeric neurotoxin may contain an LH domain derived from a first neurotoxin serotype or subtype. N H from the domain and the second neurotoxin serotype or subtype C Other examples of chimeric neurotoxins include an LH domain derived from a first neurotoxin serotype or subtype. N H CNH from the domain and the second neurotoxin serotype or subtype CC A further example of a chimeric neurotoxin is a neurotoxin that contains an LH domain from a first neurotoxin serotype or subtype. N A chimeric neurotoxin is a neurotoxin that comprises a domain and an activation loop from a second neurotoxin serotype or subtype. Examples of chimeric neurotoxins are provided in WO2017191315 and WO2016156113, both of which are incorporated herein by reference in their entirety.

[0204] As used herein, the term "H C "H domain" refers to a functionally defined region of the neurotoxin heavy chain having a molecular weight of about 50 kDa that enables the neurotoxin to bind to a receptor located on the surface of a target cell. C The domain is composed of two structurally distinct subdomains, each with a molecular weight of approximately 25 kDa, called the H CN Subdomains (H C N-terminal part of the domain) and "H CC Subdomains (H CC Domain also named H C H CC The domain is capable of binding to a Clostridial neurotoxin protein receptor.

[0205] As used herein, the term "LH N The domain is H C The neurotoxic region is distinct from the domains, the endopeptidase domain (the "L" or "light chain") and the domain responsible for translocation of the endopeptidase to the cytoplasm (the H of the heavy chain). N The term refers to the neurotoxic region consisting of the endopeptidase domain ("L" or "light chain"), which is capable of cleaving SNARE proteins.

[0206] Exemplary L, H N , H CN , and H CC The domains are shown in Table 1. Table 1 - Exemplary L, H N , H CN , and H CCEach domain [Table 1]

[0207] The above identified reference sequences should be considered as a guideline as slight variations may occur depending on the subserotype, for example, U.S. Patent Application Publication No. 2007 / 0166332 (hereby incorporated by reference in its entirety) describes each of the slightly different clostridial sequences.

[0208] The term "activation loop" refers to a polypeptide domain that contains a proteolytic cleavage site. Activation loops of neurotoxins have been described in the art, such as in WO2016156113, which is incorporated herein by reference in its entirety.

[0209] For example, a chimeric neurotoxin may contain H from a second neurotoxin. C LH from the first neurotoxin covalently bound to the domain N domain, preferably each of said first and second neurotoxins is different; The above LH N The C-terminal amino acid residue of the domain is the LH domain of the first neurotoxin. N Domain and H C 3. Domain and 10 corresponding to the first amino acid residue of the helix, and The above H C The N-terminal amino acid residues of the domain correspond to the LH domain of the second neurotoxin. N Domain and H C 3. Domain and 10 It corresponds to the second amino acid residue in the helix.

[0210] In one embodiment, the clostridial neurotoxin is H from BoNT / B. C domain and the LH from BoNT / A, BoNT / C, BoNT / D, BoNT / E, BoNT / F, or BoNT / G (e.g., BoNT / A) N It is a chimeric neurotoxin containing the domain.

[0211] For example, in one embodiment, H C The domain consists of or comprises an amino acid sequence corresponding to amino acid residues 860 to 1291 of SEQ ID NO:2 (e.g., BoNT / B), or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto; N The domain is: - amino acid residues 1 to 872 of SEQ ID NO:1 (e.g., BoNT / A), or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto; - amino acid residues 1 to 867 of SEQ ID NO: 3, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto; - amino acid residues 1 to 863 of SEQ ID NO: 4, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto; - amino acid residues 1 to 846 of SEQ ID NO: 5, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto; - amino acid residues 1 to 865 of SEQ ID NO: 6, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto; - amino acid residues 1 to 864 of SEQ ID NO: 7, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto; - amino acid residues 1 to 863 of SEQ ID NO: 8, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto; and - consisting of or comprising an amino acid sequence selected from the group consisting of amino acid residues 1 to 862 of SEQ ID NO: 9, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto;

[0212] In one embodiment, the clostridial neurotoxin of the present invention comprises the light chain and translocation domain of BoNT / A and the BoNT / B receptor binding domain (H C domain) or a part thereof (e.g., the LH domain of BoNT / A N -BoNT / B H C A suitable chimeric and / or hybrid Clostridial neurotoxin may be one taught in WO 2017 / 191315 A1, which is incorporated herein by reference. The chimeric Clostridial neurotoxin may comprise (preferably consist of) the sequence of SEQ ID NO: 10.

[0213] BoNT / A LH N The domain is BoNT / B H C The chimeric BoNT / A may be covalently linked to the domain. The chimeric BoNT / A is also referred to herein as a "BoNT / AB" or a "BoNT / AB chimera."

[0214] LH N The C-terminal amino acid residue of the domain is the LH of BoNT / A. N Domain and H C Domain fragmentation 3 10 may correspond to the first amino acid residue of the helix, and H C The N-terminal amino acid residue of the domain is the LH of BoNT / B. N Domain and H C Domain fragmentation 3 10 It may correspond to the second amino acid residue of the helix.

[0215] "BoNT / A LH N Domain and H C Domain fragmentation 3 10References herein to "the first amino acid residue of the helix" include N Domain and H C Domain fragmentation 3 10 The N-terminal residue of the helix. N Domain and H C Domain fragmentation 3 10 References herein to "the second amino acid residue of the helix" include N Domain and H C Domain fragmentation 3 10 It refers to the amino acid residue following the N-terminal residue of the helix.

[0216] "3 10 A "helix" is a type of secondary structure found in proteins and polypeptides along with alpha helices, beta sheets, and reverse helices. 3 10 The amino acids in the helix are arranged in a right-handed helical conformation, where one full turn is completed by three residues and ten atoms that break intramolecular hydrogen bonds between them. Each amino acid has ten atoms in a ring formed by hydrogen bonding, corresponding to a turn of 120 degrees in the helix (i.e., the helix has three residues per turn) and a translation of 2.0 Å (=0.2 nm) along the axis of the helix. Most importantly, the NH group of an amino acid forms a hydrogen bond with the C=O group of the amino acid three residues before it, and this repeated i+3→i hydrogen bond is repeated three times. 10 Define a spiral. 3 10 A helix is ​​a standard concept in structural biology that is well known to those skilled in the art.

[0217] These three 10 The helix corresponds to the four residues that form the actual helix and two cap (or transition) residues, one at each end of these four residues. N Domain and H C Splitting the Domain 3 10 The "helix" consists of these six residues.

[0218] Through performing structural analysis and sequence alignment, LHN Domain and H C Splitting the Domain 3 10 The helix was identified. 10 The helix is ​​located at its N-terminus (i.e., LH N in the C-terminal portion of the domain) and an α-helix in its C-terminus (i.e. C It is surrounded by β-strands (at the N-terminal part of the domain). 10 The first (N-terminal) residue of the helix (the cap or transition residue) also corresponds to the C-terminal residue of this α-helix.

[0219] LH N Domain and H C Splitting the Domain 3 10 The helices can be determined, for example, from the publicly available crystal structures of botulinum neurotoxins, such as 3BTA (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=3BTA) and 1EPW (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=1EPW) for botulinum neurotoxins A1 and B1, respectively.

[0220] Publicly available computational modeling and alignment tools, such as the homology modeling servers LOOPP (Learning, Observing, and Outputting Protein Patterns, http: / / loopp.org), PHYRE (Protein Homology / analogY Recognition Engine, http: / / www.sbg.bio.ic.ac.uk / phyre2 / ), and Rosetta (https: / / www.rosettacommons.org / ), the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ), the alignment program Clustal Omega (http: / / www.clustal.org / omega / ), and the Internet Resources for Molecular and Cell Many other tools / services, such as those listed in the Internet Resources for Molecular and Cellular Biologists (http: / / molbiol-tools.ca / ), also support the use of LH in other neurotoxins. N Domain and H C Splitting the Domain 3 10 It can be used to determine the location of the helix. N / H CN The region surrounding the junction is highly structurally conserved, making it an ideal region for superimposing different serotypes.

[0221] For example, the following method is a 3D method for other neurotoxins. 10 Can be used to determine the sequence of the helix: 1. The structural homology modeling tool LOOP (http: / / loopp.org) was used to obtain predicted structures of other BoNT serotypes based on the BoNT / A1 crystal structure (3BTA.pdb); 2. The structure (pdb) file obtained in this way is CN The N-terminal end of the domain and (H N The nucleotide sequence was then edited to include only the approximately 80 residues preceding this (which is part of the H domain), thereby resolving the highly structurally conserved H N / H CN " held territory; 3. The protein superposition server SuperPose ( http: / / wishart.biology.ualberta.ca / superpose / ) was used to superimpose each serotype onto the 3BTA.pdb structure; 4. The overlapping pdb files are the H of BoNT / A1. C 3 at the start of the domain 10 were examined to find the location of the helix and subsequently identified the corresponding residues in other serotypes; 5. Other BoNT serotype sequences were aligned with Clustal Omega to ensure that corresponding residues were correct.

[0222] LH determined by this method N , H C , and 3 10 Examples of each domain of the helix are shown below: [Table 2]

[0223] Using structural analysis and sequence alignment, LH N Domain and H C Splitting the Domain 3 10 The helix followed by a β-strand is a conserved structure in all botulinum and tetanus neurotoxins, and LH N Domain and H C Splitting the Domain 3 10 It has been found to begin at the eighth residue (eg, residue 879 in BoNT / A1) when starting from the first residue of the helix.

[0224] BoNT / AB chimera is a H-linked C LH from BoNT / A covalently bound to the domain N may include a domain, ·LH N The C-terminal amino acid residue of the domain is H C corresponds to the eighth amino acid residue N-terminal to the β-strand at the start (N-terminus) of the domain, and H C The N-terminal amino acid residue of the domain is H of BoNT / B. C It corresponds to the seventh amino acid residue N-terminally following the β-strand at the start (N-terminus) of the domain.

[0225] BoNT / AB chimera is a H-linked C LH from BoNT / A covalently binds to the domain N may include a domain, ·LH N The C-terminal amino acid residue of the domain is the LH of BoNT / A. N corresponds to the C-terminal amino acid residue of an alpha helix located at the end (C-terminus) of the domain, and H C The N-terminal amino acid residue of the domain is the LH of BoNT / B. N It corresponds to the amino acid residue adjacent to the C-terminal amino acid residue of the α-helix located at the end (C-terminus) of the domain.

[0226] The rationale for the design process of the BoNT / AB chimera was to ensure that the secondary structure was unknown, thereby minimizing any changes to the tertiary structure and function of each domain. Regardless of theory, the hypothesis is that the 3 10 Not disrupting the four central amino acid residues of the helix is ​​said to ensure an optimal conformation for the chimeric neurotoxin, thereby allowing the chimeric neurotoxin to function to its full potential.

[0227] In a preferred embodiment, the clostridial neurotoxin is H from BoNT / B. CDomain and LH from BoNT / A N It is a chimeric neurotoxin containing the domain.

[0228] LH from BoNT / A N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO:1 or a polypeptide sequence having at least 70% sequence identity thereto. N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO:1 or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the LH domain from BoNT / A N The domain corresponds to amino acid residues 1 to 872 of SEQ ID NO:1.

[0229] H from BoNT / B C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO:2 or a polypeptide sequence having at least 70% sequence identity thereto. C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO:2 or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. C The domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO: 2. C The domain consists of or comprises amino acid residues corresponding to amino acid residues 860 to 1291 of SEQ ID NO:2 or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, and N The domain comprises an amino acid sequence corresponding to amino acid residues 1 to 872 of SEQ ID NO:1 or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0230] BoNT / AB chimera is the LH of BoNT / A. N Domain and H of BoNT / B CIt may contain domains. LH N The domain may correspond to amino acid residues 1 to 872 of BoNT / A (SEQ ID NO:1), C The domain may correspond to amino acid residues 860 to 1291 of BoNT / B (SEQ ID NO:2).

[0231] A Clostridial neurotoxin having one or more modifications (preferably in the amino acid sequence of the heavy chain) is referred to herein as a "modified Clostridial neurotoxin."

[0232] BoNT / B H C The H domain has the effect of increasing the binding affinity of BoNT / B neurotoxin to human SytII compared to the native BoNT / B sequence. CC It may further comprise at least one amino acid residue substitution, addition, or deletion in the subdomain. CC Suitable amino acid residue substitutions, additions, or deletions in the subdomains are disclosed in WO 2013 / 180799 and WO 2016 / 154534 (both of which are incorporated by reference herein).

[0233] For example, clostridial neurotoxins can bind to H from BoNT / B. C domain (e.g., a clostridial neurotoxin is a BoNT / B or H domain from BoNT / B) C In each embodiment, the clostridial neurotoxin is a chimeric neurotoxin containing a "modified heavy chain" (H C The neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (e.g., in the H domain), and preferably, the modified heavy chain binds to the target neuronal cell with higher (or lower) affinity than the original neurotoxin. C Such modifications of the domain may alter the binding of H to gangliosides in target neurons. CC Modification of amino acid residues in the ganglioside-binding site of the domain and / or H that can alter binding to protein receptors on target neurons CCThis may include modifications of amino acid residues in the protein receptor binding sites of the domains. Examples of such modified neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are incorporated herein by reference in their entireties.

[0234] In one embodiment of the modified Clostridial neurotoxin according to the present invention, H from BoNT / B is CC The domain is the natural H domain of the BoNT serotype. CC It is modified relative to the domain.

[0235] In a preferred embodiment, H from BoNT / B neurotoxin CC The domain is the H of native BoNT / B. CC The H domain for human SytII is CC The at least one amino acid residue mutation that increases the binding affinity of the domain is preferably, however, at least one amino acid residue mutation that increases the binding affinity of the naturally occurring BoNT / BH domain. CC The H domain is at least 50% different from the H domain of human SytII. CC Increases the binding affinity of the domain.

[0236] BoNT / BH CC Such suitable amino acid residue mutations in domains are described in the art in International Publication No. WO2013180799 and U.S. Patent Application No. 2016154534, both of which are incorporated herein by reference in their entireties.

[0237] In particular, natural BoNT / BH CC The BoNT / BH domain is at least 50% more effective against human SytII. CCThe at least one amino acid residue mutation suitable for increasing the binding affinity of the domain is an amino acid residue substitution, addition, or deletion selected from the group consisting of 1118M, 1183M, 1191M, 1191I, 1191Q, 1191T, 1199Y, 1199F, 1199L, 1201V, 1191C, 1191V, 1191L, 1191Y, 1199W, 1199E, 1199H, 1178Y, 1178Q, 1178A, 1178S, 1183C, 1183P, and any combination thereof. CC The at least one amino acid residue mutation in the domain comprises a substitution, addition or deletion of two amino acid residues selected from the group consisting of 1191M and 1199L, 1191M and 1199Y, 1191M and 1199F, 1191Q and 1199L, 1191Q and 1199Y, 1191Q and 1199F, 1191M and 1199W, 1191M and 1178Q, 1191C and 1199W, 1191C and 1199Y, 1191C and 1178Q, 1191Q and 1199W, 1191V and 1199W, 1191V and 1199Y, or 1191V and 1178Q. CC The at least one amino acid residue mutation in the BoNT / BH domain consists of three amino acid residue substitutions, additions, or deletions: 1191M, 1199W, and 1178Q. CC The at least one amino acid residue mutation in the domain consists of two amino acid residue substitutions, additions, or deletions: 1191M and 1199Y.

[0238] In a more preferred embodiment, the natural BoNT / BH CC The BoNT / BH domain is at least 50% more effective against human SytII. CCThe at least one amino acid residue mutation suitable for increasing the binding affinity of the domain is an amino acid residue substitution selected from the group consisting of V1118M, Y1183M, E1191M, E1191I, E1191Q, E1191T, S1199Y, S1199F, S1199L, S1201V, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, and any combination thereof. CC The at least one amino acid residue mutation in the domain comprises two amino acid residue substitutions selected from the group consisting of E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q. Nevertheless, preferably, the BoNT / BH CC The at least one amino acid residue mutation in the BoNT / BH domain consists of three amino acid residue substitutions, E1191M, S1199W, and W1178Q. CC The at least one amino acid residue mutation in the domain consists of two amino acid residue substitutions, E1191M and S1199Y.

[0239] In other words, BoNT / BH CCSuitable amino acid residue substitutions, additions, or deletions in the subdomains include substitution mutations selected from the group consisting of V1118M, Y1183M, E1191M, E1191I, E1191Q, E1191T, S1199Y, S1199F, S1199L, S1201V, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, and combinations thereof. CC Suitable amino acid residue substitutions, additions, or deletions in the subdomain further include combinations of two substitution mutations selected from the group consisting of E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q. CC Suitable amino acid residue substitutions, additions or deletions in the subdomains also include the combination of the three substitution mutations: E1191M, S1199W, and W1178Q.

[0240] Preferably, BoNT / BH CC Suitable amino acid residue substitutions, additions or deletions in the subdomains include the combination of two substitution mutations: E1191M and S1199Y.

[0241] The modifications may be modifications relative to unmodified BoNT / B shown as SEQ ID NO:2, and the numbering of the amino acid residues is determined by aligning with SEQ ID NO:2. Since the presence of a methionine residue at position 1 of SEQ ID NO:2 is optional, one of skill in the art would take into consideration the presence / absence of the methionine residue when determining the numbering of the amino acid residues. For example, if SEQ ID NO:2 contains a methionine, the numbering of the positions would be as defined above (e.g., E1191 would be E1191 in SEQ ID NO:2). Alternatively, if a methionine is absent in SEQ ID NO:2, the numbering of the amino acid residues would be modified by -1 (e.g., E1191 would be E1190 in SEQ ID NO:2). Similar considerations apply to the presence / absence of a methionine at position 1 of the other polypeptide sequences described herein, and one of skill in the art would readily determine the correct numbering of the amino acid residues using techniques routine in the art.

[0242] In a preferred embodiment, the modified BoNT / BH CC The domain is amino acid residues 1082 to 1291 of SEQ ID NO:2 (natural BoNT / BH CC domain), or an amino acid sequence having at least 70%, preferably at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0243] In one embodiment, the Clostridial neurotoxins of the invention can be both chimeric and modified as described above. For example, in a preferred embodiment, the Clostridial neurotoxin comprises (or consists of) the amino acid sequence SEQ ID NO: 10, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0244] In one embodiment, the Clostridial neurotoxins of the present invention can be both chimeric and modified as described above. For example, in a preferred embodiment, the Clostridial neurotoxin has the amino acid sequence SEQ ID NO: 10 (e.g., BoNT / AB MY), or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0245] The modified Clostridial neurotoxin may have one or more modifications in the amino acid sequence of the light chain, such as modifications in the substrate binding or catalytic domains that can alter or modify the SNARE protein specificity of the modified L chain. Examples of such modified Clostridial neurotoxins are described in WO 2010 / 120766 and U.S. Patent Application Publication No. 2011 / 0318385, both of which are incorporated herein by reference in their entireties.

[0246] The modified clostridial neurotoxin may contain one or more modifications that increase or decrease the biological activity and / or biological persistence of the modified clostridial neurotoxin. For example, the modified clostridial neurotoxin may contain a leucine-based or tyrosine-based motif that increases or decreases the biological activity and / or biological persistence of the modified clostridial neurotoxin. Suitable leucine-based motifs include xDxxxLL, xExxxIL, xExxxIL, and xExxxLM, where x is any amino acid. Suitable tyrosine-based motifs include Yxx-Hy, where Hy is a hydrophobic amino acid. Examples of modified clostridial neurotoxins that contain leucine-based and tyrosine-based motifs are described in International Publication No. WO 2002 / 08268, the entirety of which is incorporated herein by reference.

[0247] As described below, a modified Clostridial neurotoxin (or a Clostridial neurotoxin fragment) may include one or more modifications that increase the isoelectric point of the Clostridial neurotoxin when compared to an equivalent unmodified Clostridial neurotoxin lacking said one or more modifications. Suitable modified Clostridial neurotoxins are described above and in WO 2015 / 004461 A1 and WO 2016 / 110662 A1, which are incorporated herein by reference. Exemplary sequences include SEQ ID NOs: 61 and 42, as described herein.

[0248] The modified Clostridial neurotoxin may preferably comprise or consist of the sequence of SEQ ID NO:15.

[0249] In one embodiment, the modified Clostridial neurotoxin may preferably comprise or consist of the sequence of SEQ ID NO:10.

[0250] In a particularly preferred embodiment, the clostridial neurotoxin of the present invention is a modified BoNT / A or a fragment thereof (preferably, a BoNT / AH CThe modified BoNT / A or fragment thereof may include a modification at one or more amino acid residues selected from ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277. Such modified BoNT / As or fragments thereof can demonstrate reduced or absent side effects compared to the use of known BoNT / As. The enhanced tissue retention properties of the modified BoNT / As of the present invention can also provide increased efficacy and / or duration of action compared to known clostridial toxin therapies and can allow for reduced dosages to be used (or increased dosages without any additional adverse effects), thus providing further advantages.

[0251] The modifications may be modifications relative to the unmodified BoNT / A shown as SEQ ID NO:1, and the numbering of the amino acid residues is determined by alignment with SEQ ID NO:1. Since the presence of a methionine residue at position 1 of SEQ ID NO:1 (as well as each SEQ ID NO: corresponding to a modified BoNT / A polypeptide or fragment thereof described herein) is optional, one of skill in the art will take into consideration the presence / absence of the methionine residue when determining the amino acid residue numbering. For example, if SEQ ID NO:1 contains a methionine, the numbering of the positions will be as defined above (e.g., ASN886 will be ASN886 in SEQ ID NO:1). Alternatively, if a methionine is absent in SEQ ID NO:1, the numbering of the amino acid residues should be corrected by -1 (e.g., ASN886 will be ASN885 in SEQ ID NO:1). Similar considerations apply to the presence / absence of a methionine at position 1 of the other polypeptide sequences described herein, and one of skill in the art will readily determine the correct amino acid residue numbering using techniques routine in the art.

[0252] The amino acid residues indicated for modification above are surface-exposed amino acid residues.

[0253] A modified BoNT / A or fragment thereof may include a modification at one or more amino acid residues selected from ASN886, ASN930, ASN954, SER955, GLN991, ASN1025, ASN1026, ASN1052, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277.

[0254] The term "one or more amino acid residues" when used in the context of a modified BoNT / A or a fragment thereof preferably means at least 2, 3, 4, 5, 6, or 7 of the indicated amino acid residues. Thus, a modified BoNT / A may contain at least 2, 3, 4, 5, 6, or 7 (preferably 7) modifications at the indicated amino acid residues. A modified BoNT / A or a fragment thereof may contain 1 to 30, 3 to 20, or 5 to 10 amino acid modifications. More preferably, the term "one or more amino acid residues" when used in the context of a modified BoNT / A or a fragment thereof means all of the indicated amino acid residues.

[0255] Preferably, beyond one or more amino acid modifications at the indicated amino acid residues, the modified BoNT / A or fragment thereof does not contain any additional amino acid modifications when compared to SEQ ID NO:1.

[0256] Modifications are: i. replacement of acidic surface-exposed amino acid residues with basic amino acid residues; ii. replacement of acidic surface exposed amino acid residues with uncharged amino acid residues; iii. replacement of uncharged surface exposed amino acid residues with basic amino acid residues; iv. insertion of a basic amino acid residue; and v. deletion of acidic surface-exposed amino acid residues;

[0257] The modifications set forth above result in a modified BoNT / A or fragment thereof having an increased positive surface charge and an increased isoelectric point when compared to the corresponding unmodified BoNT / A or fragment thereof.

[0258] Isoelectric point (pl) is an intrinsic property of a particular protein. As is well known in the art, proteins are made up of unique sequences of amino acids (also called amino acid residues when in a protein). Each amino acid in the standard set of 20 has a different side chain (or R group), meaning that each amino acid residue in a protein exhibits different chemical properties such as charge and hydrophobicity. These properties can be influenced by the surrounding chemical environment such as temperature and pH. The overall chemical characteristics of a protein depend on the sum of these various factors.

[0259] Certain amino acid residues (described in more detail below) have ionizable side chains that can exhibit a charge depending on the pH of their surroundings. Whether or not this side chain is charged at a particular pH depends on the pKa of the associated ionizable moiety, which is the negative logarithm of the acid dissociation constant (Ka) for a particular proton from the conjugate base.

[0260] For example, acidic residues such as aspartic acid and glutamic acid have side chain carboxylic acid groups with pKa values ​​of approximately 4.1 (the exact pKa value may depend on temperature, ionic strength, and the microenvironment of the ionizable group). Thus, these side chains exhibit a negative charge at a pH of 7.4 (often referred to as "physiological pH"). At lower pH values, these side chains lose their charge by adding a proton.

[0261] Conversely, basic residues such as lysine and arginine have nitrogen-containing side groups with pKa values ​​of about 10 to 12. Thus, these side chains are positively charged at a pH of 7.4. At higher pH values, these side chains lose a proton and lose their charge.

[0262] Thus, the total (net) charge of a protein molecule depends on the number of acidic and basic residues present in the protein (and their degree of surface exposure) as well as the surrounding pH. Changing the surrounding pH changes the total charge of the protein. Thus, for each protein, there is a specific pH at which the number of positive and negative charges is equal and the protein exhibits no overall net charge. This point is known as the isoelectric point (pl). The isoelectric point is a standard concept in protein biochemistry that is well known to those skilled in the art.

[0263] Thus, the isoelectric point (pl) is defined as the pH value at which a protein exhibits a zero net charge. An increase in pl means that a higher pH value is required for the protein to exhibit a zero net charge. Thus, an increase in pl represents an increase in the net positive charge of the protein at a particular pH. Conversely, a decrease in pl means that a lower pH value is required for the protein to exhibit a zero net charge. Thus, a decrease in pl represents a decrease in the net positive charge of the protein at a particular pH.

[0264] Methods for determining the pl of a protein are known in the art and will be familiar to those skilled in the art. As an example, the pl of a protein can be calculated from the average pKa value of each amino acid present in the protein ("calculated pl"). This calculation can be performed using computer programs known in the art, such as the Compute pl / MW Tool from ExPASy (https: / / web.expasy.org / compute_pl / ), which is a preferred method for calculating pl according to the present invention. Comparison of pl values ​​between different molecules should be performed using the same calculation techniques / programs.

[0265] Where appropriate, the calculated pl of a protein can be experimentally confirmed using the technique of isoelectric focusing ("observed pl"). This technique uses electrophoresis to separate proteins by their pl. Isoelectric focusing is typically performed using a gel with an immobilized pH gradient. When an electric field is applied, the protein migrates through the pH gradient until it reaches a pH at which it has a zero net charge, which becomes the pl of the protein. The results provided by isoelectric focusing are typically relatively low resolution in nature, and therefore the inventors believe that the results provided by calculated pl (as described above) are more suitable for use.

[0266] Throughout this specification, unless otherwise stated, "pl" means "calculated pl."

[0267] The pl of a protein can be increased or decreased by changing the number of basic and / or acidic groups displayed on its surface. This can be achieved by modifying one or more amino acids of the protein. For example, an increase in pl can be brought about by decreasing the number of acidic residues or by increasing the number of basic residues.

[0268] The modified BoNT / A or fragment thereof of the present invention can have a pl value that is at least 0.2, 0.4, 0.5, or 1 pl unit higher than the pl of the unmodified BoNT / A (e.g., SEQ ID NO: 1) or fragment thereof. Preferably, the modified BoNT / A or fragment thereof can have a pl of at least 6.6, e.g., at least 6.8.

[0269] The properties of the 20 common amino acids are given in the table below: [Table 3]

[0270] The following amino acids are considered to be charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).

[0271] At pH 7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) are negatively charged, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) are positively charged. Aspartic acid and glutamic acid are called acidic amino acid residues. Arginine and lysine are called basic amino acid residues.

[0272] The following amino acids are considered to be uncharged and polar (meaning they can participate in hydrogen bonding): asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan.

[0273] The following amino acids are considered to be uncharged hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.

[0274] In an amino acid insertion, an additional amino acid residue (one that is not normally present) is incorporated into a BoNT / A polypeptide sequence or fragment thereof, thus increasing the total number of amino acid residues in said sequence. In an amino acid deletion, an amino acid residue is removed from a Clostridial toxin amino acid sequence, thus decreasing the total number of amino acid residues in said sequence.

[0275] Preferably, the modification is a substitution, which advantageously maintains the same number of amino acid residues in the modified BoNT / A or fragment thereof. In an amino acid substitution, an amino acid residue that forms part of the BoNT / A polypeptide sequence or fragment thereof is replaced with a different amino acid residue. The replacing amino acid residue may be one of the twenty standard amino acids mentioned above. Alternatively, the replacing amino acid in an amino acid substitution may be a non-standard amino acid (an amino acid that is not part of the standard set of twenty). By way of example, the replacing amino acid may be a basic non-standard amino acid, such as L-ornithine, L-2-amino-3-guanidinopropionic acid, or the D-isomers of lysine, arginine, and ornithine. Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using an E. coli auxotrophic expression host.

[0276] In one embodiment, the substitutions are selected from substitutions of acidic amino acid residues with basic amino acid residues, substitutions of acidic amino acid residues with uncharged amino acid residues, and substitutions of uncharged amino acid residues with basic amino acid residues, hi one embodiment, the substitutions are substitutions of acidic amino acid residues with uncharged amino acid residues, where an acidic amino acid residue is replaced by its corresponding uncharged amide amino acid residue (i.e., aspartic acid is replaced by asparagine, glutamic acid is replaced by glutamine).

[0277] Preferably, the basic amino acid residue is a lysine or arginine residue. In other words, the substitution is with lysine or arginine. Most preferably, the modification is with lysine.

[0278] Preferably, the modified BoNT / A or fragment thereof for use in the present invention is Clostridial toxin H CNThe modified BoNT / A or fragment thereof preferably has a pl of at least 6.6. The modified BoNT / A or fragment thereof preferably has a modification of at least four amino acids selected from ASN886, ASN930, ASN954, SER955, GLN991, ASN1025, ASN1026, and ASN1052, the modification comprising a substitution of the amino acid with a lysine or arginine residue. For example, the modified BoNT / A or fragment thereof may have a modification of at least five amino acids selected from ASN886, ASN930, ASN954, SER955, GLN991, ASN1025, ASN1026, ASN1052, and GLN1229, the modification comprising a substitution of the amino acid with a lysine or arginine residue.

[0279] Methods for modifying proteins by substitution, insertion, or deletion of amino acid residues are known in the art. By way of example, amino acid modifications can be introduced by modification of the DNA sequence encoding the polypeptide (e.g., encoding the unmodified BoNT / A or a fragment thereof). This can be achieved using standard molecular cloning techniques, such as site-directed mutagenesis, in which a short stretch of DNA (oligonucleotide) coding for the desired amino acid is used to replace the original coding sequence using a polymerase enzyme, or by insertion / deletion of parts of the gene with various enzymes (e.g., ligases and restriction endonucleases). Alternatively, modified gene sequences can be chemically synthesized.

[0280] Thus, a modified Clostridial neurotoxin may have one or several such amino acid modifications (e.g., substitutions) as compared to native BoNT / A, which increase the isoelectric point of the polypeptide. Without being bound by theory, it is believed that the increased net positive charge promotes electrostatic interactions between the polypeptide and anionic extracellular components, thereby promoting binding between the polypeptide and cell surfaces, thus increasing retention and duration of action at the site of administration.

[0281] For the above-mentioned repaired BoNT / A polypeptide (e.g., SEQ ID NO: 15), one way in which these advantageous properties (representing an increased therapeutic index) can be defined is in terms of the safety factor of the modified BoNT / A. In this regard, the unwanted effects of the clostridial neurotoxin (caused by diffusion of the toxin away from the administration site) can be experimentally evaluated by measuring the percentage weight loss in a relevant animal model (e.g., mice, where weight loss is detected within 7 days of administration). Conversely, the desired on-target effects of the clostridial neurotoxin can be experimentally evaluated by the digit abduction score (DAS) test, which is a measurement of muscle paralysis. The DAS test can be performed by injection of 20 μl of the clostridial neurotoxin formulated in gelatin phosphate buffer into the gastrocnemius / soleus muscle complex of mice, followed by evaluation of the digit abduction score using the method of Aoki (Aoki KR, Toxicology 39: 1815-1820; 2001). In the DAS assay, mice are briefly suspended by their tails to elicit a specific startle response in which the mice extend their hind limbs and abduct their hind digits. Following injection of a clostridial neurotoxin, the varying degrees of digit abduction are scored on a 5-point scale (0=normal to 4=maximal reduction in digit abduction and leg extension).

[0282] The safety factor of a Clostridial neurotoxin may then be expressed as the ratio between the amount of toxin required for a 10% reduction in body weight (measured at peak effect within the first 7 days after dosing in mice) and the amount of toxin required for a score of 2 on the DAS. Thus, a high safety factor score is desirable, which indicates a toxin capable of effectively paralyzing target muscles with few unwanted off-target effects. The modified BoNT / A of the present invention may have a safety factor higher than that of a comparable unmodified (native) botulinum toxin (e.g., SEQ ID NO: 1).

[0283] Thus, in one embodiment, a modified BoNT / A of the invention has a safety factor of at least 8 (e.g., at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50), which is the dose of toxin (pg / mouse) required for about a 10% change in body weight, relative to the DAS ED. 50 (pg / mouse) [ED 50 = dose required to produce a DAS score of 2].

[0284] In one embodiment, the modified BoNT / A of the invention has a safety factor of at least 10. In one embodiment, the modified BoNT / A or fragment thereof of the invention has a safety factor of at least 15.

[0285] Clostridial neurotoxins comprising at least 70% sequence identity to SEQ ID NO:15 are described in WO 2015 / 004461 A1, which is incorporated by reference in its entirety.

[0286] In one embodiment, a Clostridial neurotoxin comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:15 and / or comprising a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:14 comprises substitutions at one or more (preferably 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more, more preferably all) of positions 930, 955, 991, 1026, 1052, 1229, and 886. The numbering of the positions corresponds to the positions of SEQ ID NO:1 and can be determined by aligning the polypeptide sequence to SEQ ID NO:1 (unmodified / native BoNT / A). Since the presence of a methionine residue at position 1 of SEQ ID NO:1 is optional, one skilled in the art would take into account the presence / absence of the methionine residue when determining the numbering of the amino acid residues. For example, if SEQ ID NO:1 contains a methionine, the numbering of the positions would be as defined above (e.g., position 886 would be ASN886 of SEQ ID NO:1). Alternatively, in the absence of methionine in SEQ ID NO:62, the numbering of the amino acid residues should be corrected by -1 (e.g., position 886 becomes ASN 885 in SEQ ID NO:1). Similar considerations apply to the presence / absence of methionine at position 1 of the other polypeptide sequences described herein, and the skilled artisan will readily determine the correct numbering of the amino acid residues using techniques routine in the art.

[0287] Preferably, a Clostridial neurotoxin comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 15 and / or comprising a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 14 comprises a lysine or arginine (more preferably, a lysine) at one or more of positions 930, 955, 991, 1026, 1052, 1229, and 886. In one embodiment, the Clostridial neurotoxin comprises a lysine or arginine (more preferably, a lysine) at at least two, three, four, five, six, or all of positions 930, 955, 991, 1026, 1052, 1229, and 886. Most preferably, the Clostridial neurotoxin comprises a lysine or arginine (more preferably, a lysine) at all of positions 930, 955, 991, 1026, 1052, 1229, and 886.

[0288] As described above, clostridial neurotoxins contain a targeting moiety (e.g., a C-terminal targeting moiety, H) that mediates receptor binding to target cells. C The clostridial neurotoxins typically contain a nucleoside-binding domain (Ganglioside Binding Domain), and may also have binding affinity for specific gangliosides present on target cells. For example, BoNT / A, BoNT / D, BoNT / E, BoNT / F, and TeNT bind to synaptic vesicle protein 2 (SV2), and BoNT / A can bind to all three of its isoforms (SV2A, SV2B, and SV2C), while BoNT / E can bind to the SV2A and SV2B isoforms. BoNT / B and BoNT / G bind to both synaptotagmin isoforms (I and II). Synaptotagmin and SV2 can be localized on synaptic vesicles and are exposed to the extracellular space when the vesicles fuse to the presynaptic membrane. It is during this period that the clostridial neurotoxins bind to their protein receptors. For further information on suitable receptors / gangliosides, see Binz and Rummel (Journal of Neurochemistry, Volume 109, Issue 6, June 2009), which is incorporated herein by reference.

[0289] The clostridial neurotoxins of the invention may be referred to based on the target receptor / ganglioside to which they bind. For example, the clostridial neurotoxins of the invention may bind to a receptor selected from SV2 (SV2A, SV2B, and / or SV2C) and synaptotagmin (synaptotagmin I and / or II).

[0290] In a preferred embodiment, the Clostridial neurotoxin binds to SV2. In one embodiment, the Clostridial neurotoxin binds to SV2A, SV2B, and / or SV2C. Preferably, the Clostridial neurotoxin binds to SV2A. In one embodiment, the Clostridial neurotoxin binds to SV2A, SV2B, and SV2C. The Clostridial neurotoxin binds to BoNT / AH. CC Domain (e.g., amino acids 1095 to 1296 of SEQ ID NO: 1), BoNT / DH CC Domain (e.g., amino acids 1083 to 1276 of SEQ ID NO:4), BoNT / EH CC domain (e.g., amino acids 1070 to 1252 of SEQ ID NO:5), and BoNT / FH CC A BoNTH domain selected from the amino acids 1088 to 1278 of SEQ ID NO:6 and / or amino acids 1077 to 1268 of SEQ ID NO:9. CC It may include a domain.

[0291] In a preferred embodiment, the Clostridial neurotoxin is BoNT / AH CC domain (e.g., amino acids 1095 to 1296 of SEQ ID NO:1).

[0292] In some embodiments, the Clostridial neurotoxin binds to Syt-I and / or Syt-II. CC The clostridial neurotoxin may comprise the BoNT / EH domain (e.g., amino acids 1082 to 1291 of SEQ ID NO:2). In some embodiments, the SV2A and / or SV2B (preferably, SV2A and SV2B).CC domain (eg, amino acids 1070 to 1252 of SEQ ID NO:5).

[0293] The Clostridial neurotoxins of the present invention can be produced using recombinant techniques. Thus, in one embodiment, the Clostridial neurotoxins of the present invention are recombinant Clostridial neurotoxins.

[0294] In one embodiment, the clostridial neurotoxin is associated with a BoNT complex protein, also known as a non-toxic neurotoxin associated protein (NAP). In other words, the clostridial neurotoxin is administered to a human patient together with or in combination with a BoNT complex protein. Thus, in one embodiment, the clostridial neurotoxin is complexed with one or more BoNT complex proteins.

[0295] In other embodiments, the Clostridial neurotoxin is free of (or is not accompanied by or in combination with) a BoNT complex protein.

[0296] Preferably, the Clostridial neurotoxin (e.g., for use as described herein) is part of a pharmaceutical composition together with at least one pharma- ceutically acceptable carrier. By "pharma-ceutically acceptable carrier" herein is meant any component that is compatible with the other components of the pharmaceutical composition, particularly the Clostridial neurotoxin, and is not harmful to a human patient. Pharmaceutically acceptable carriers can be selected based on the desired route of administration according to standard pharmaceutical practice, and can include, without limitation, excipients, diluents, adjuvants, propellants, and salts.

[0297] Thus, the present invention further relates to a pharmaceutical composition for use in treating a skin condition in a human patient, said composition comprising a Clostridial neurotoxin of the present invention and at least one pharma- ceutically acceptable carrier, and the dosage of Clostridial neurotoxin administered to the patient is as described above. Also included are corresponding uses and methods of treating a skin condition comprising administering the pharmaceutical composition of the present invention to a human patient.

[0298] The Clostridial neurotoxins of the present invention are preferably formulated for intradermal administration.

[0299] A preferred route of administration is via intradermal administration, preferably by intradermal administration it is meant intradermal injection.

[0300] Preferably, the BoNT / A treating the same skin condition is purified BoNT / A. As used herein, the term "purified BoNT / A" refers to botulinum neurotoxin type A purified from a clostridial strain that naturally produces botulinum neurotoxin type A (a naturally occurring clostridial strain). Purified BoNT / A may be associated with or free of complexing proteins. Examples of commercially available purified BoNT / A include Botox®, Dysport®, and Xeomin®.

[0301] Dosages of clostridial neurotoxins herein are preferably measured in nanograms.

[0302] The dosage of the clostridial neurotoxin according to the present invention is understood as the dosage of the active two-chain clostridial neurotoxin, i.e., without the amount of complex protein that the neurotoxin may be associated with. In other words, it refers to the dosage of the active two-chain clostridial neurotoxin, in which the neurotoxin is administered to a patient with or without the complex protein. As those skilled in the art are well aware, the active two-chain clostridial neurotoxin can bind to a membrane (e.g., cell membrane) receptor, translocate the light chain into the cytoplasm, and cleave SNARE proteins, while the complex protein does not exhibit such biological activity (i.e., is not "active").

[0303] Alternatively, the dosage of a Clostridial neurotoxin can be measured in "units" (U) of Clostridial neurotoxin.

[0304] Indeed, as those skilled in the art are well aware, the potency of a clostridial neurotoxin is related to the amount (e.g., nanograms) of neurotoxin required to achieve a LD50 (lethal dose 50) unit, with one LD50 unit defined as the median intraperitoneal lethal dose (measured in mice). However, currently commercially available BoNT pharmaceutical preparations contain different amounts of the 150 kD neurotoxin, but also different amounts of LD50 units. Moreover, in these preparations, the neurotoxin may or may not be associated with (i.e., combined with) a non-toxic neurotoxin-associated protein (NAP), also known as a complex protein. For simplicity of conversion (as reported in Field et al., "AbobotulinumtoxinA (Dysport), OnabotulinumtoxinA (Botox), and IncobotulinumtoxinA (Xeomin) Neurotoxin Content and Potential Implications for Duration of Response in Patients," Toxins 2018, 10(12), 535): - 100 units of Botox® (also known as onabotulinumtoxin A) contains approximately 0.9 ng of the 150 kD BoNT / A as well as complex proteins; - 500 units of Dysport® (also known as abobotulinum toxin A) contains approximately 2.69 ng of the 150 kD BoNT / A and complex proteins; - 100 units of Xeomin® (also known as incobotulinum toxin A) contains approximately 0.40 ng of the 150 kD BoNT / A as well as complex proteins.

[0305] Note that conversion values ​​may vary slightly. For example, conversion values ​​reported in Frevert, 2012 ("Content of botulinum neurotoxin in Botox® / Vistabel®, Dysport® / Azzalure®, and Xeomin® / Bocouture®"); Drugs R D. 2010;10(2):67-73) are as follows: - 100 units of Botox® (also known as onabotulinumtoxin A) contains approximately 0.73 ng of the 150 kD BoNT / A as well as complex proteins; - 100 units of Dysport® (also known as abobotulinum toxin A) contains approximately 0.65 ng of the 150 kD BoNT / A as well as complex proteins; - 100 units of Xeomin® (also known as incobotulinum toxin A) contains approximately 0.44 ng of the 150 kD BoNT / A and complex proteins; - 100 units of Neurobloc / Myobloc® (also known as rima botulinum toxin B) contains about 0.2 ng to about 1 ng of the 150 kD BoNT / B as well as complex proteins.

[0306] The amount of Clostridial neurotoxin can be determined by those skilled in the art by methods conventionally used in the art for quantifying proteins, preferably at the nanogram level, including, inter alia, mass spectrometry, such as isotope dilution mass spectrometry (Munoz et al., Quantification of protein calibrants by amino acid analysis using isotope dilution mass spectrometry, Anal. Biochem. 2011, 408, 124-131), or fluorescence assays (Poras, Detection and Quantification of Botulinum Neurotoxin Type A by a Novel Rapid In Vitro Fluorometric Assay, Appl Environ Microbiol. 2009 Jul; 75(13): 4382-4390).

[0307] Thus, the dosage ranges for administration of the Clostridial neurotoxins of the present invention are those to produce the desired therapeutic and / or prophylactic effect. It will be understood that the required dosage range will depend on the particular nature of the Clostridial neurotoxin or composition, the route of administration, the nature of the formulation, the age of the subject, the nature, extent or severity of the subject's condition, any contraindications, and the judgment of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimization.

[0308] In one embodiment, the dose of Clostridial neurotoxin is a flat dose. The flat dose may range from 50 pg to 250 μg, preferably 100 pg to 100 μg. In one embodiment, the flat dose is at least 50 pg, 100 pg, 500 pg, 1 ng, 50 ng. It may be 100 ng, 500 ng, 1 μg, or 50 μg. The dose may be a single flat dose.

[0309] Fluid dosage forms are typically prepared using a Clostridial neurotoxin and a sterile pyrogen-free solvent. Depending on the solvent and concentration used, the Clostridial neurotoxin can be either dissolved or suspended in the solvent. In preparing a solution, the Clostridial neurotoxin can be dissolved in the solvent, and the solution is made isotonic by adding sodium chloride if necessary, and sterilized by filtration through a sterile filter using an aseptic method before being filled into a suitable sterile vial or ampoule and sealed. Alternatively, if the stability of the solution is appropriate, the solution in the sealed container can be sterilized by processing with an autoclave. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending or emulsifying agents, and / or local anesthetics can be dissolved in the solvent.

[0310] Dry powders, dissolved or suspended in a suitable solvent prior to use, may be prepared by filling pre-sterilized contents into sterile containers using aseptic techniques in a sterile area. Alternatively, the contents may be dissolved in a suitable container using aseptic techniques in a sterile area. The product is then freeze-dried and the containers are aseptically sealed.

[0311] Parenteral suspensions suitable for the routes of administration described herein are prepared in substantially the same manner, except that the sterile ingredients are suspended in a sterile solvent instead of dissolved, and sterilization cannot be achieved by filtration. The ingredients may be isolated under sterile conditions, or alternatively, may be sterilized after isolation, for example by gamma irradiation.

[0312] Advantageously, a suspending agent, such as polyvinylpyrrolidone, is included in the composition to ensure uniform distribution of the components.

[0313] Administration according to the present invention may utilize a variety of delivery technologies, including microparticle, encapsulation, or pressurized aerosol injection.

[0314] Intradermal administration may include intradermal injection using a needle, such as a 30 gauge needle, preferably inserted into the dermis of the skin at an angle of about 5° to 15° relative to the surface of the skin (e.g., a flat surface). Preferably, the depth of injection (relative to the surface of the skin) is approximately 0.2 to 0.3 inches (preferably, approximately 0.25 inches). The term "flat surface" may refer to the contact area of ​​the skin.

[0315] Where a range of values ​​is provided herein, it will be understood that each intermediate value between the upper and lower limit of that range is also specifically disclosed to the tenth of a unit, unless the context clearly dictates otherwise. Every smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within that stated range is encompassed within the disclosure. It will be further understood that any range of numerical values ​​indicated herein by the expression "from a to b" means a range of numerical values ​​extending from a to b (i.e., including the precise endpoints a and b).

[0316] Moreover, the term "about" is to be understood herein as plus or minus (±) 5% of the numerical value of the number with which it is used, preferably ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%.

[0317] In preferred embodiments, the dosage of a Clostridial neurotoxin of the invention administered to treat a skin condition in a human patient (ie, a therapeutic or cosmetic dose) ranges from about 0.00025 ng to about 3 ng.

[0318] In a preferred embodiment, a therapeutic (or cosmetic) dose of a Clostridial neurotoxin ranges from about 0.0003 ng to about 2 ng, preferably from about 0.0004 ng to about 1.5 ng, from about 0.0005 ng to about 1 ng, and more preferably from about 0.0006 ng to about 0.5 ng of said Clostridial neurotoxin.

[0319] For example, the dosage of a clostridial neurotoxin, including BoNT / A, preferably ranges from about 0.001 ng to about 2 ng.

[0320] It will nevertheless be understood that the required dosage range will depend on the precise nature of the Clostridial neurotoxin, the maximum tolerated dose of the particular patient / subject (e.g., a human patient / subject), the condition of the skin, the route of administration, the nature of the formulation, the age of the patient, the nature, extent, or severity of the patient's condition, any contraindications, and the judgment of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimization.

[0321] When a reference standard is referred to herein for purposes of comparing the level of sebum (after administration) to the reference standard - the reference standard may correspond to the level of sebum on the skin (e.g., epidermal layer) of a patient who has not been administered a Clostridial neurotoxin. Additionally or alternatively, the reference standard may correspond to the level of sebum on the skin (e.g., epidermal layer) of a patient (e.g., a patient to whom a Clostridial neurotoxin has been administered) prior to administration of a Clostridial neurotoxin.

[0322] Each of the embodiments relating to various therapeutic uses of the invention are intended to apply equally to the methods of treatment, Clostridial neurotoxins of the invention, and vice versa.

[0323] term

[0324] 1. A method for treating a skin condition comprising intradermally administering to a patient a Clostridial neurotoxin, wherein following administration, the Clostridial neurotoxin: - inducing secretion of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin. 2. A Clostridial neurotoxin for use in a method of treating a skin condition, the method comprising administering a Clostridial neurotoxin intradermally to a patient, wherein following administration, the Clostridial neurotoxin: - a Clostridial neurotoxin that induces the secretion of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin. 3. Non-therapeutic use of a Clostridial neurotoxin for cosmetic treatment of the skin, said Clostridial neurotoxin comprising: - non-therapeutic use to induce secretion of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin. 4. The non-therapeutic use according to paragraph 3, wherein the Clostridial neurotoxin is administered by intradermal administration. 5. The method of claim 1 or the use of claim 2, wherein the skin condition is a condition involving abnormal sebaceous lipid levels, and the sebaceous lipids are present in the epidermal layer at a lower level than in the epidermal layer of a patient not having the skin condition. 6. The method of claim 1 or 5, or the use of a Clostridial neurotoxin according to claim 2 or 5, wherein the beneficiary skin condition is one or more conditions selected from acne, atopic dermatitis, Netherton syndrome, psoriasis, dehydrated skin (e.g., dry or cracked skin), actinic keratosis, rosacea, carbuncle, eczema, cellulitis, dermatitis, skin cancer, and lichen pilaris. 7. The method of any one of clauses 1, 5 or 6, or the Clostridial neurotoxin for use of any one of clauses 2, 5 or 6, wherein the skin condition is one or more conditions selected from psoriasis, eczema and dermatitis. 8. Following administration of said Clostridial neurotoxin: - no increase is induced in the level of sebum on the patient's skin relative to the reference standard, preferably no decrease is induced in the level of sebum on the patient's skin relative to the reference standard; - the reference standard corresponds to the level of sebum on the skin of a subject not administered the Clostridial neurotoxin. 9. The method, use, or non-therapeutic use of any one of the preceding clauses, wherein the patient does not have oily skin prior to and / or after administration of the Clostridial neurotoxin. 10. The method, use or non-therapeutic use of any one of the preceding claims, wherein said Clostridial neurotoxin is a BoNT / A neurotoxin. 11. The method, use or non-therapeutic use of a Clostridial neurotoxin according to any one of the preceding claims, wherein said Clostridial neurotoxin is a chimeric neurotoxin. 12. The method, use or non-therapeutic use of a Clostridial neurotoxin according to paragraph 11, wherein the chimeric neurotoxin is selected from the group consisting of BoNT / DC and BoNT / X. 13. The chimeric neurotoxin comprises an H from a second neurotoxin. C LH from the first neurotoxin covalently bound to the domain N Includes the domain, Preferably, said first and said second neurotoxins are different; The above LH N The C-terminal amino acid residue of the domain is the LH N Domain and H C Splitting the Domain 3 10 corresponds to the first amino acid residue of the helix, The above H C The N-terminal amino acid residue of the domain is the LH N Domain and H C Splitting the Domain 3 10 12. The method, use or non-therapeutic use of claim 11, wherein the clostridial neurotoxin corresponds to the second amino acid residue of the helix. 14. The method, use or non-therapeutic use of the Clostridial neurotoxin according to paragraph 13, wherein the first neurotoxin is BoNT / A and the second neurotoxin is BoNT / B. 15. The method, use or non-therapeutic use of any one of the preceding claims, wherein said intradermal administration comprises administration of a dose of Clostridial neurotoxin in the range of about 0.00025 ng to 3 ng to the administration site. 16. The method, use, or non-therapeutic use of any one of the preceding claims, wherein the intradermal administration comprises intradermal injection using a 30 gauge needle, preferably where the 30 gauge needle is inserted into the dermis of the skin at an angle of about 5° to 15° to the flat surface of the skin. 17. The method, use, or non-therapeutic use of any one of the preceding claims, wherein the epidermal layer is one or more selected from the stratum basale, stratum spinosum, stratum granulosum, or stratum corneum. 18. The method, use, or non-therapeutic use of any one of the preceding claims, wherein said epidermal layer is the stratum corneum.

[0325] sequence homology

[0326] Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, individual methods, and hybrid methods such as, for example, segmental approaches. Defined procedures for determining percent identity are routine procedures within the skill of the art. Global methods align the sequence from beginning to end of the molecule and determine the best alignment by adding up the scores of each residue pair and applying a gap penalty. Non-limiting methods include, for example, CLUSTAL W ("Clustal Double"), see, e.g., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice by Thompson et al., Julie D., 22(22) Nucleic Acids Research 4673-4680 (1994), and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. MoI. Biol. 823-838 (1996). Individual methods align sequences by identifying one or more conserved motifs that are shared by all of the input sequences.Non-limiting methods include, for example, Match-box (see, e.g., Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences by Eric Depiereux and Ernest Feytmans, 8(5) CABIOS 501 -509 (1992); Gibbs sampling (see, e.g., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment by C.E. Lawrence et al., 262(5131 ) Science 208-214 (1993); Align-M (see, e.g., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent by Ivo Van WaIIe et al. Sequences ("AlignEm - a new algorithm for multiple alignment of highly divergent sequences"), 20(9) Bioinformatics:1428-1435 (2004), including:

[0327] Thus, percentage sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio., 48: 603-16, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment score using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.), shown below (amino acids are shown by standard one-letter codes):

[0328] The "percentage sequence identity" between two or more sequences of nucleic acids or amino acids is a function of the number of identical positions shared by those sequences. Thus, % identity may be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. The calculation of % sequence identity may also take into account the number of gaps and the length of each gap that needs to be introduced to optimize the alignment of two or more sequences. The comparison of sequences between two or more sequences and the determination of percentage identity can be performed using certain mathematical algorithms such as BLAST ("blast"), which are well known to those skilled in the art.

number

[0329] Therefore, the percentage identity is calculated as follows: [total number of perfect matches × 100] / [length of the longer sequence + number of gaps introduced in the longer sequence to align the two sequences]

[0330] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions, or additions. These changes are preferably minor, such as substitutions of conserved amino acid residues (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide, as well as small deletions, typically from one to about 30 amino acids, and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, small linker peptides of up to about 20-25 residues, or affinity tags.

[0331] Conservative amino acid substitutions Basic: arginine, lysine, histidine Acidic: glutamic acid, aspartic acid Polar: glutamine, asparagine Hydrophobic: leucine, isoleucine, valine Aromatic: phenylalanine, tryptophan, tyrosine Small ones: glycine, alanine, serine, threonine, methionine

[0332] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) may be substituted for amino acid residues in the polypeptides of the invention. A limited number of non-conserved amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues. The polypeptides of the invention can also include non-naturally occurring amino acid residues.

[0333] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylated tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are carried out in a cell-free system containing E. coli S30 extracts and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, e.g., Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993. In the second method, translation is carried out in Xenopus oocytes by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:1991-8, 1996).In a third method, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the scope of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0334] A limited number of non-conserved amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues in the polypeptides of the invention.

[0335] Essential amino acids in the polypeptides of the invention can be identified by procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244:1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structures determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling involving mutation of putative contact site amino acids. See, e.g., de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identity of essential amino acids can also be inferred from analysis of homologies with related components of the polypeptides of the invention (eg, translocation components or protease enzyme components).

[0336] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions within a polypeptide, selecting functional polypeptides, and subsequently sequencing the mutagenized polypeptides to determine the spectrum of permissible substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0337] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions within a polypeptide, selecting functional polypeptides, and subsequently sequencing the mutagenized polypeptides to determine the spectrum of permissible substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0338] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY by Singleton et al., 20th ED., John Wiley and Sons, New York, (1994) and THE HARPER COLLINS DICTIONARY OF BIOLOGY by Hale & Marham, Harper Perennial, Y (1991) provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0339] The disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numeric ranges are inclusive of the numbers defining the range, and unless otherwise indicated, all nucleic acid sequences are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0340] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure.

[0341] Amino acids are referred to herein using their amino acid name, three-letter abbreviation, or one-letter abbreviation. As used herein, the term "protein" includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein." In some instances, the term "amino acid sequence" is synonymous with the term "peptide." In some instances, the term "amino acid sequence" is synonymous with the term "enzyme." The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional one-letter and three-letter codes for amino acid residues may be used. The three-letter codes for amino acids as defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is understood that a polypeptide may be encoded by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0342] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a Clostridial neurotoxin" includes a plurality of such Clostridial neurotoxins, a reference to "the Clostridial neurotoxin" includes a reference to one or more Clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0343] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims which follow herefrom.

[0344] The invention will now be described, by way of example only, with reference to the following figures and examples.

[0345] Embodiments of the invention will now be described, by way of example only, with reference to the following drawings and examples.

[0346] In all figures, Gp1 is group 1, Gp2 is group 2, Gp3 is group 3, Gp4 is group 4, Gp5 is group 5, Dysport is BoNT / A, U is units, and retention time is gas chromatography retention time. [Brief description of the drawings]

[0347] [Figure 1] (a) Image of a rhino mouse administered BoNT / A (Dysport) via intradermal injection on the back (the injection sites are the four darkened areas in the center of the back that appear to form the four corners of a square). (B) Levels of sebum measured later on the skin of the back of the rhino mouse after injection (or administration of the control substance adapalene). Dysport is BoNT / A. [Diagram 2]Figure 17 shows the levels of representative fatty acids in sebum recovered from the back skin of different groups of rhino mice 15 days after injection. The retention time of this fatty acid was 22.1 min (this fatty acid corresponds to the peak marked "FA4" in Figure 17B). The data shows the average of six samples with standard deviation. [Diagram 3] Figure 17 shows the levels of squalene in sebum collected from the back skin of different groups of rhino mice 15 days after injection. The retention time of squalene was 25 min (squalene corresponds to the peak marked "ST1" in Figure 17B). The data shows the average of six samples with standard deviation. [Figure 4] Figure 17 shows cholesterol levels in sebum recovered from the back skin of different groups of rhino mice after 15 days of injection. The retention time of cholesterol was 24.4 min (cholesterol corresponds to the peak marked "Chol" in Figure 17B). The data shows the average of six samples with standard deviation. [Diagram 5] (a)-(c) show the levels of three different wax esters in sebum recovered from the back skin of different groups of rhino mice 15 days after injection. The retention time of "Wax Ester 2" shown in FIG. 5A was 31 min. The retention time of "Wax Ester 3" shown in FIG. 5B was 35.5 min. The retention time of "Wax Ester 4" shown in FIG. 5C was 38 min. Wax Esters 2-3 correspond to the peaks marked "WX2-3" (respectively) in FIG. 17C. The data show the average of six samples with standard deviation. [Figure 6] FIG. 17 shows the levels of cholesteryl ester in sebum recovered from the back skin of different groups of rhino mice after 15 days of injection. The retention time of this cholesteryl ester was 41 min (this cholesteryl ester corresponds to the peak marked "CE" in FIG. 17D). The data shows the average of six samples with the standard deviation. [Figure 7]Figure 1 shows the levels of substance P (pg / g tissue) in back skin samples collected from the back skin of Rhino mice of different groups 15 days after injection. ***P<0.001 for the evolving agent (Gp1). One-way ANOVA followed by Dunnett's post-hoc test. [Figure 8] Figure 1 shows the development of (a) erythema and (b) scaling on the back skin of Rhino mice from different groups, scored on days 1, 5, 10 and 15. [Figure 9] Figure 1. Size of sebaceous gland surface in the back skin of Rhino mice from different groups 15 days after injection. ***P<0.001 vs vehicle (Gp1). One-way ANOVA followed by Dunnett's post-hoc test. [Figure 10] Figure 1 shows the size of the utricle surface of the back skin of Rhino mice from different groups 15 days after injection. ***P<0.001 vs vehicle (Gp1). One-way ANOVA followed by Dunnett's post-hoc test. [Figure 11] Figure 14. Back skin epidermis thickness of the back skin of Rhino mice from different groups 15 days after injection. ***P<0.001 vs vehicle (Gp1). One-way ANOVA followed by Dunnett's post-hoc test. [Figure 12] Figure 14. Levels of dermal inflammation in the back skin of Rhino mice from different groups 15 days after injection. ***P<0.001 vs vehicle (Gp1). One-way ANOVA followed by Dunnett's post-hoc test. [Figure 13] FIG. 1 shows the level of keratinocyte proliferation in the epidermis of the back of Rhino mice from different groups 15 days after injection. ***P<0.001 vs. vehicle (Gp1). One-way ANOVA followed by Dunnett's post-hoc test. [Figure 14] Figure 1 shows the levels of proliferation of fibroblasts in the dorsal dermis of Rhino mice from different groups 15 days after injection. ***P<0.001 vs vehicle (Gp1). One-way ANOVA followed by Dunnett's post-hoc test. [Figure 15]FIG. 1 shows the level of sebocyte proliferation in the back dermis of different groups of rhino mice 15 days after injection. [Figure 16] Figure 14. Levels of IL-1α (ng / g tissue) in back skin samples collected from the back skin of Rhino mice from different groups 15 days after injection. ***P<0.001 vs vehicle (Gp1). One-way ANOVA followed by Dunnett's post-hoc test. [Figure 17] 17A-17D show exemplary chromatograms generated during the lipidomics analysis described herein for each of groups 1-5. Chromatograms are shown in expanded views in FIGS. 17B-D. A is a median full length chromatogram obtained for each group in a sebum sample (e.g., lipid extract of a lipid sample containing sebaceous gland lipids). B is an expanded view of peaks corresponding to fatty acids (peak designated FA4), squalene (peak designated ST1), and cholesterol (peak designated Chol) obtained for each group in a sebum sample. C is an expanded view of peaks corresponding to wax / wax esters (peak designated WX1, WX2, WX3, and WX4) obtained for each group in a sebum sample. D is an expanded view of peaks corresponding to cholesteryl esters (peak designated CE) obtained for each group in a sebum sample.

[0348] Sequence Listing

[0349] Where the first Met amino acid residue or the corresponding first codon is shown in any of the following respective SEQ ID NOs, said residue / codon is optional.

[0350] SEQ ID NO:1 - BoNT / A1, Accession number A5HZZ9, amino acid sequence

[0351] SEQ ID NO:2 - BoNT / B1, Accession number B1INP5, amino acid sequence

[0352] SEQ ID NO:3 - BoNT / C1, Accession number P18640, amino acid sequence

[0353] SEQ ID NO:4 - BoNT / D, Accession number P19321, amino acid sequence

[0354] SEQ ID NO:5 - BoNT / E1, Accession number WP_003372387, amino acid sequence

[0355] SEQ ID NO:6 - BoNT / F1, Accession No. Q57236, amino acid sequence

[0356] SEQ ID NO:7 - BoNT / G, Accession number WP_039635782, amino acid sequence

[0357] SEQ ID NO:8 - BoNT / DC, Accession number BAM65681, amino acid sequence

[0358] SEQ ID NO:9 - BoNT / F7, amino acid sequence

[0359] SEQ ID NO: 10 - mrBoNT / AB (BoNT / AB MY ), amino acid sequence MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERD 49 TFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIY 99 STDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEEL 149 NLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEES 199 LEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNA 249 YYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNK 299 AKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIY 349 TEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAA 399 NFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKA 449 LNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLI 499 QQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYT 549 MFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATE 599 AAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYK 649 DDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNAL 699 SKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINY 749 QYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNS 799 MIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDI 849 PFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVE 899 VYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPK 949 YKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVF 999 FEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIA 1049 NGEIIFKLDGDIDRTQFIWMKYFSIFTELSQSNIEERYKIQSYSEYLKD 1099 FWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINY 1149 RDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYF 1199 KKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIG 1249 LIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKD 1299 EGWTE 1304

[0360] SEQ ID NO:11 - BoNT / X, amino acid sequence (GenBank: BAQ12790.1)

[0361] SEQ ID NO:12 - Polypeptide sequence, TeNT - UniProt P04958

[0362] SEQ ID NO:13 - Polypeptide sequence, BoNT / A - UniProt P10845 MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNN EYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTIT NNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELN EKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPR GSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA GVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAK LVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

[0363] SEQ ID NO:14 - nucleotide sequence, mrBoNT / A

[0364] SEQ ID NO:15 - Polypeptide sequence, mrBoNT / A

[0365] SEQ ID NO:16 - Polypeptide sequence, BoNT / E - UniProt Q00496

[0366] SEQ ID NO:17 - Substance P RPKPQQFFGLM EXAMPLES

[0367] The present invention will now be described, by way of example only, with reference to the following examples which serve to illustrate particular embodiments of the invention and are not intended to limit in any way the scope of the invention as defined by the claims. The aim of this study was to evaluate the efficacy of clostridial neurotoxins administered via the intradermal route in a rhino mouse model of skin conditions. Dysport (BoNT / A) was selected as the clostridial neurotoxin to be tested. Briefly, animals were injected (as a single dose on day 1) via the intradermal route (injected at 4 sites) with vehicle (saline) or three ascending doses of Dysport (0.1, 0.3, and 1 unit of Dysport per mouse) under anesthesia. A reference group received daily administration of adapalene 0.1% gel by topical administration for 15 days. During this period, animals were observed daily. Body weight and dorsal skin thickness were measured on days 1, 5, 10, and 15. At the same time, the mice were subjected to macroscopic observations (scoring of erythema and scaling). Sebum samples were collected only on day 15. On day 15, animals were ethically euthanized and skin samples were collected for histology hematoxylin and eosin saffron (HES, for structural / anatomical analysis of e.g. utricle size), Ki67 (proliferation marker) staining, ELISA, and lipidomics studies.

[0368] In conclusion, Dysport administered via the intradermal route at doses of 0.1 U and 0.3 U per mouse was well tolerated. The highest dose of Dysport (1 U per mouse) induced a temporary loss of muscle tone accompanied by a slight weight loss (this was due to diffusion of Dysport away from the intradermal injection site). The mice regained good health after 8 days. Dysport did not show any significant effect on the utricle surface area (see Figure 10), but the two highest doses induced a significant anti-inflammatory effect in the dermis (see Figure 12). Inflammation of the dermis (back skin area) was evaluated for each mouse on HES sections by counting the number of nuclei, a marker of inflammatory infiltrates. Furthermore, Dysport did not show any side effects commonly observed with topical retinoid therapeutics (e.g., adapalene), as well as epidermal hyperplasia and dermal inflammation. Dysport did not alter the levels of IL-1α cytokine (see Figure 16). On the other hand, we recorded a dose-dependent increase in the concentration of substance P in the dorsal skin (see Figure 17), suggesting that Dysport induces the accumulation of neuropeptides in the tissue, for example by inhibiting the exocytosis mechanism. Lipidomic analysis showed that Dysport significantly increased certain (desirable) lipid species exhibiting protective properties in the skin, such as fatty acids (Figure 2), squalene (Figure 3), cholesterol (Figure 4), wax esters (Figure 5), and cholesteryl (Figure 6). Overall sebum levels did not change significantly (Figure 1B).

[0369] material and method

[0370] mouse

[0371] Strain used: RHJ / LeJ (eg, Rhino Mouse), Gender: Female, Age: 6-8 weeks upon receipt, Weight: Approximately 20g upon arrival, Number: 30 + 2 spares.

[0372] This mouse strain is known to be a suitable model for evaluating the efficacy and pharmacodynamics of new drugs for skin conditions, and there is a significant amount of historical data available in the literature for comparative purposes.

[0373] Source: Animals were obtained from Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA, and imported by Charles River Laboratories, BP0109, 69592 L'Arbresle Cedex, France.

[0374] Housing: Animals were caged in groups of six. Feeding was provided inside the cage. Equipment and animal housing were regularly cleaned. Cages were identified with labels including, as a minimum, the study number and the identification of each animal. Minimum and maximum room temperature (target: 22 ± 2°C) and humidity (target: SO ± 20%) were recorded daily and retained as raw data. The light / dark cycle was 12 / 12 h.

[0375] Test item

[0376] BoNT / A (Dysport) was stored at +4° C. until use. Adapalene 0.1% gel was obtained from a commercial supplier (Differine®, Galderma France).

[0377] Reconstitution of Dysport (552U Dysport) (in 1 ml sterile saline = stock solution) and further dilutions (in 5 ml siliconized tubes, Vacutainer, BD Biosciences) were performed in sterile saline as follows: [Table 4] Group: group, Final neurotoxin concentration (units / mL): final neurotoxin concentration (units / mL), Initial solution: initial solution, Concentration of neurotoxin solution (units / mL): concentration of neurotoxin solution (units / mL), Neurotoxin solution (μL): neurotoxin solution (μL), Saline (μL): physiological saline (μL), Final(μL): final solution (μL), Diluted stock solution: diluted stock solution, Stock solution: stock solution

[0378] treatment

[0379] Dysport (test article) or saline (negative control) was injected via the intradermal route at four sites on the back (see Figure 1). The reference compound adapalene (positive control) was applied by the topical route (skin on the back). All administrations were given on the same day. The following doses were evaluated: [Table 5] Group: Group, Number of animals: Number of animals, Vehicle: Solvent, Formulation: Formulation, Concentration: Concentration, Dose per mouse: Dose per mouse, Volume: Volume, Saline: Physiological saline, Adapalene gel 0.1%: Adapalene gel 0.1%, id: Intradermal, Topic: Topical, Dysport dose: Dysport dose

[0380] Six animals were dosed per group on day 1 (30 doses).

[0381] Intradermal injection (BoNT) and application of reference compound (adapalene)

[0382] A 30G needle connected to a 100 μL microsyringe was used to inject blood into 4 sites (approximately 2 to 3 cm) on the skin of the back. 2Intradermal injections (groups 1, 3, 4, and 5) were performed under anesthesia (isoflurane) in a rectangular area of ​​4 × 20 μL, 80 μL per mouse, see Figure 1 (day 1 only).

[0383] Topical application of the reference compound (no anesthesia required) (Group 2) was performed on approximately 4 cm of skin, corresponding to a rectangle of approximately 1.5 cm to 2.5 cm . 2 After application of the gel (100 μL), the area was massaged smoothly with gloved fingers for 10 seconds.

[0384] Efficacy Endpoints

[0385] The following parameters were evaluated on four occasions: days 1 (before treatment), 5, 10 and 15 (before euthanasia): - Dorsal skin thickness using calipers at the application site, - Macroscopic description of the skin at the application site (if possible).

[0386] Further observations such as erythema or scaling were noted when demonstrated. These parameters were scored as shown below. [Table 6] score: score, erythema: erythema, scaling: scaling, Normal: normal, Slight: slight, Moderate: moderate, Important: important

[0387] Calibrated digital images of the areas of formulation application were taken on four occasions (days 1, 5, 10, and 15) prior to recording efficacy endpoints.

[0388] Sample collection and processing

[0389] On the 15th day (after injection / application), the mice were euthanized by cervical dislocation under isoflurane anesthesia. A sample of the dorsal skin was removed at the application site, and a portion of the tissue was identified for histological analysis and transferred to formalin for 48 hours, followed by EtOH. The other portion of the sample was frozen and stored at -80°C for ELISA testing.

[0390] For lipid analysis (performed on day 15), sebum was collected by applying a glass cone to an area of ​​dorsal skin for approximately 10 seconds and then removing it, and stored at −80°C until analysis.

[0391] Histological analysis

[0392] Histological analysis was processed by the ANEXPLO platform, Toulouse, France. After paraffin embedding, sections were made and HES (hematoxylin / eosin / saffron for structural / anatomical analysis, e.g., size of the sac) and Ki67 (proliferation marker) (immunohistochemical analysis) staining was performed on two different sections. Stained sections were digitized (microscope objective x20) and stored.

[0393] Image scanning, image analysis, and anatomopathological evaluation (via conventional surface measurements / nuclei counting using histology software) were performed with blinded data to allow objective analysis of the following parameters: - utricle surface measurements (HES); - sebaceous gland surface (HES); - epidermal thickness (HES); - Inflammation of the dermis (HES); - Proliferation of keratinocytes and fibroblasts (Ki67 only).

[0394] Lipidomic analysis

[0395] After lipid extraction, sebum samples (n=30) were analyzed for lipidomics to assess the composition of sebum lipids using gas chromatographic analysis. The following lipid families were analyzed: - Free fatty acids / monoglycerides; - Squalene; - Cholesterol; - Wax esters / diglycerides; - Cholesteryl esters / triglycerides.

[0396] Lipid extraction was performed as follows: - 2 ml of hexane was added onto the glass cone (which holds the collected sebum) in a 5 ml glass tube; - The tube was vortexed for 1 minute; - The solvent was separated and placed into a separate clean glass tube that was weighed prior to analysis; - The solvent was evaporated under a stream of nitrogen at +60°C; - Each tube was weighed for calculation of total sebum recovery (weighing scale was properly tared / balanced); - 700 μL of chloroform / isopropanol 1:1 was added to a pre-weighed dry tube; - The tube was vortexed for 1 minute; - 300 μL of water was added; - The tubes were vortexed for 1 minute and placed on an orbital shaker for 10 minutes; - The tubes were centrifuged at 3,000 g for 5 min at room temperature; - The organic phase was separated and placed in a clean glass tube; - The solvent was evaporated under a stream of nitrogen at +60°C; - The dry residue was extracted with 100 μL of ethyl acetate, placed in a GC vial for injection and stored at +4°C until analysis.

[0397] GC analysis was performed using a GC / FID system 7890B (Agilent), and data acquisition was performed using Empower3, ver. 3471 (Waters).

[0398] A Zebron, ZB-SHT, ref. 7HG-G015-02, capillary GC column with dimensions 30 m x 0.25 mm x 0.1 μm was used. The following GC method was used: - Injector temperature, 350°C / split mode injection; - Column temperature gradient: 80°C to 240°C at 10°C / min; 240°C to 320°C at 5°C / min; 320°C to 350°C at 2°C / min; 350°C for 20 minutes; - FID detector temperature, 250 °C; - FID hydrogen flow rate, 35 ml / min; - FID air flow rate, 350ml / min; - FID generation helium flow rate, 25 ml / min.

[0399] After acquisition, chromatogram averages per group were calculated and compared between groups. Peaks of interest were integrated and used for comparison between groups. More specifically, the (major) peaks in the chromatograms corresponding to the lipids of interest were integrated, and the peak area of ​​each compound (lipid) was determined by the peak area of ​​the relevant lipid standard (of known concentration).

[0400] ELISA test

[0401] Frozen skin samples (n=30) were analyzed using commercial kits for substance P (Enzo, substance P ELISA kit, France) and IL-1α (Mabtech AB, mouse IL-1α ELISA expression kit, France). ELISA assays were performed in duplicate on tissue extracts obtained from skin samples as suggested by the manufacturer.

[0402] Tissue extracts were obtained after mechanical grinding (Precellys®, 3 cycles, 6800 rpm, 30 sec, with beads, CKMixSO-R) in DPBS buffer supplemented with protease inhibitor cocktail (Roche, Complete, Mini Easy Pack, protease inhibitor cocktail tablets, France). After grinding, the extracts were centrifuged at 12,000×g for 10 min at 4° C. The supernatant was removed and an additional centrifugation was performed at 12,000×g for 10 min at 4° C. The supernatant was stored at −80° C. Before grinding, the samples were weighed and tissue dilution in buffer was applied identically for all samples (10 mg tissue in 0.1 ml buffer). ELISA assays for substance P were performed on 96-well plates. The analysis was performed according to the method given by the supplier and the following procedure.

[0403] 50 μL of tissue extract or standard or buffer (blank) was added to the appropriate wells with 50 μL of buffer, 50 μL of blue conjugate, and 50 μL of antibody and incubated at room temperature for 2 hours at 500 rpm. The plate was then emptied and washed three times with wash solution. After the final wash, all residual wash buffer was removed and 200 μL of p-Npp substrate solution was added to each well and incubated at room temperature for 1 hour. 50 μL of stop solution was then added to each well and absorbance was read at 405 nm. A single calibration standard curve was analyzed on the day of analysis. Blank subtraction from the standards and quantification were performed according to the supplier's recommendations.

[0404] Elisa assays for IL-1α were performed on 96-well plates. The assays were performed according to the methods provided by the supplier and the following procedure: - Anti-IL-1α mAb was coated into the wells at a concentration of 2 μg / ml per well overnight at 4°C; - Wash twice with PBS (200 μL / well); - Add 200 μL / well of PBST / BSA and incubate for 1 hour at room temperature; - Wash wells 5 times with PBST (200 μL / well); - Add 100 μL of tissue extract (diluted 1:1,000 in PBST / BSA) or standard or blank to appropriate wells and incubate for 2 hours at room temperature; - Wash 5 times with PBST (200 μL / well); - Add biotinylated mAb (100 μL / well) at a final concentration of 0.5 μg / ml and incubate for 1 hour at room temperature; - Wash the plate 5 times with PBST (200 μL / well); - Add streptavidin-ALP (100 μL / well) and incubate at room temperature for 1 hour; - Wash the plate 5 times with PBST (200 μL / well); - Add p-Npp substrate solvent (100 μL / well); - After the yellow coloration appears, measure the local concentration at 405 nm.

[0405] A single calibration standard curve was analyzed on the day of analysis.

[0406] The concentrations of substance P and IL-1α are shown relative to tissue weight (of the sample).

[0407] Example 1

[0408] Intradermal administration of BoNT / A did not change sebum levels on the epidermis

[0409] Sebum levels on the epidermis of mice from groups 1 to 5 (as described in the "Treatment" section of "Materials and Methods" above) were measured.

[0410] The sebum was collected by applying a glass cone to the dorsal skin area for approximately 10 seconds and then removing it, which was subsequently weighed on a precision scale (the scale was balanced / tared with the same glass cone prior to contacting the skin to collect the sebum). The resulting weight measurement corresponded to the weight of sebum collected (thus providing a reading of the level of sebum on the mouse's skin).

[0411] There was no statistically significant difference in sebum levels between Group 1 (treated with vehicle only) and any of the BoNT / A-treated groups (Groups 3-5) (see FIG. 1). The only group that showed a change (e.g., a decrease) in sebum levels was Group 2 (treated with adapalene).

[0412] FIG. 1 shows the mean (and standard deviation) of six samples.

[0413] Example 2

[0414] BoNT / A induces increased levels of fatty acids in epidermal sebum

[0415] The following table outlines the peak areas (e.g., chromatogram peak areas) of fatty acids from the sebaceous glands of subjects in sebum samples. Intradermal administration of BoNT / A (at levels immediately following dosing) induced an increase in the excretion of fatty acids into the epidermis, whereas adapalene did not. This data is shown in the bar graph in FIG. 2. [Table 7] group: group, vehicle: solvent, Adapalene: adapalene, Animal ID: animal ID, Peak area (μV*sec), Mean: mean, SD: standard deviation

[0416] Example 3

[0417] BoNT / A induces increased levels of cholesterol in epidermal sebum

[0418] The following table outlines the (sebaceous gland) cholesterol peak areas of the sebum samples. Intradermal administration of BoNT / A induced an increase in the excretion of cholesterol to the epidermis (at levels immediately following dosing). This data is shown in the bar graph of FIG. 4. [Table 8] group: group, vehicle: solvent, Adapalene: adapalene, Animal ID: animal ID, Peak area (μV*sec), Mean: mean, SD: standard deviation

[0419] Example 4

[0420] BoNT / A induces increased levels of squalene in epidermal sebum

[0421] The table below outlines the squalene peak areas (sebaceous gland) of sebum samples. Intradermal administration of BoNT / A induced an increase in excretion of squalene into the epidermis (at levels immediately following dosing). This is particularly surprising, since mouse sebum generally does not contain squalene (or contains only insignificant amounts). The data are shown as a bar graph in Figure 3. [Table 9] group: group, vehicle: solvent, Adapalene: adapalene, Animal ID: animal ID, Peak area (μV*sec), Mean: mean, SD: standard deviation

[0422] Example 5

[0423] BoNT / A induces increased excretion of squalene into the epidermis

[0424] The following table outlines the peak areas of wax esters (from the sebaceous gland) in sebum samples. Intradermal administration of BoNT / A induced an increase in the excretion of wax esters into the epidermis (at levels immediately following dosing). Three wax esters (designated wax 2, 3, and 4) were analyzed. The data are shown in the bar graph in Figure 5.

[0425] Wax 2 [Table 10] group: group, vehicle: solvent, Adapalene: adapalene, Animal ID: animal ID, Peak area (μV*sec), Mean: mean, SD: standard deviation

[0426] Wax 3 [Table 11] group: group, vehicle: solvent, Adapalene: adapalene, Animal ID: animal ID, Peak area (μV*sec), Mean: mean, SD: standard deviation

[0427] Wax 4 [Table 12] group: group, vehicle: solvent, Adapalene: adapalene, Animal ID: animal ID, Peak area (μV*sec), Mean: mean, SD: standard deviation

[0428] Example 6

[0429] BoNT / A induces increased levels of cholesteryl esters in epidermal sebum

[0430] The following table outlines the peak areas of cholesteryl esters (from the sebaceous gland) in sebum samples. Intradermal administration of BoNT / A induced an increase in the excretion of cholesteryl esters to the epidermis (at levels immediately following dosing). This data is shown in the bar graph of FIG. 6. [Table 13] group: group, vehicle: solvent, Adapalene: adapalene, Animal ID: animal ID, Peak area (μV*sec), Mean: mean, SD: standard deviation

[0431] Example 7

[0432] BoNT / A induces retention of substance P in the dermis

[0433] Skin tissues were homogenized (1 mg tissue: 0.01 ml buffer) in DPBS containing protease inhibitor cocktail by mechanical grinding using ceramic beads (3 cycles, 6,800 rpm, 30 s) followed by centrifugation at 12,000 × g for 10 min at 4 °C. The supernatant was collected and the levels of substance P were estimated using an ELISA kit (as described in Materials and Methods). Substance P is expressed in pg / g tissue.

[0434] Intradermal administration of BoNT / A induced retention of substance P in the epidermis (at levels immediately following dosing). The table below shows the levels of substance P in the back samples. This data is presented in the bar graph of FIG. 7. [Table 14] [Table 15] group: group, vehicle: solvent, Adapalene: adapalene, Animal ID: animal ID, Concentration (pg / g tissue): concentration (pg / g tissue), Mean: mean, SD: standard deviation

[0435] Example 8

[0436] Evolution of clinical scores of erythema and scaling

[0437] Clinical scores (erythema and scaling of the back skin) were followed and recorded on days 1, 5, 10, and 15. No erythema or scaling was observed in groups 1, 3, and 4 (vehicle, Dysport 0.1 U, and Dysport 0.3 U, respectively) in this study.

[0438] Higher scores of erythema and scaling were observed in the reference group 2 compared to the group treated with Dysport. Only two mice in group 5 treated with 1 U of Dysport showed very slight scaling marks (score=1) on day 5, which disappeared on day 10.

[0439] The scores are outlined in the table below, and the data is presented in a bar graph in Figure 8. Evolution of clinical scores (erythema) of the skin on the back in Group 2 (mean + / - standard deviation): [Table 16] Group: Group, Adapalene: Adapalene, Parameter: Parameter, Day: Day, Aminal ID: Animal ID, Mean: Mean, SD: Standard deviation, Erythema score: Erythema score

[0440] Evolution of clinical scores (scaling) of the skin on the back in Group 2 (adapalene) and Group 5 (Dysport 1U) (mean + / - standard deviation) [Table 17] Group: group, Adapalene: adapalene, Dysport 1U / mouse: Dysport 1U / mouse, Parameter: parameter, Day: day, Aminal ID: animal ID, Mean: mean, SD: standard deviation, Scaling score: scaling score

[0441] Example 9

[0442] Sebaceous surface following treatment

[0443] On day 15, samples of skin harvested around the upper two injection sites were collected and fixed for HES (hematoxylin / eosin / saffron) and Ki67 staining.

[0444] Several parameters were evaluated on HES sections: epidermal thickness, dermal inflammation, utricle and sebaceous gland surface.

[0445] The surface of 10 sebaceous glands was calculated for each mouse on HES sections. Adapalene significantly increased the surface of the sebaceous glands, whereas Dysport (BoNT / A) did not. The results (average of 10 measurements per mouse) are shown in the table below, together with the standard deviation. The data are shown in a bar graph in Figure 9. [Table 18] [Table 19] group: group, vehicle: solvent, Adapalene: adapalene, Animal ID: animal ID, Sebaceous gland surface: sebaceous gland surface, mean: mean, SD: standard deviation

[0446] Example 10

[0447] Utricular surface following treatment

[0448] The surface of 10 utricles was calculated for each mouse on HES sections. Adapalene significantly reduced the surface of the utricle, whereas Dysport(BoNT / A) did not. The results (average of 10 measurements per mouse) are shown in the table below, together with the standard deviation. The data are shown in a bar graph in FIG. 10. [Table 20] [Table 21] group: group, vehicle: solvent, Adapalene: adapalene, Animal ID: animal ID, utriculi surface: sebaceous gland surface, mean: mean, SD: standard deviation

[0449] Example 11

[0450] Epidermal thickness following treatment

[0451] The thickness of the epidermis (back skin area) was calculated for each mouse on the HES sections. Adapalene significantly increased the thickness of the epidermis, whereas Dysport (BoNT / A) did not. The results (average of 6 measurements per mouse) are shown in the table below with standard deviation. The data are shown in a bar graph in Figure 11. [Table 22] [Table 23] group: group, vehicle: solvent, Adapalene: adapalene, Animal ID: animal ID, Epidermis thickness: epidermal thickness, mean: mean, SD: standard deviation

[0452] Example 12

[0453] Dermal inflammation following treatment

[0454] Inflammation in the dermis (skin area on the back) was measured as the number of nuclei / 1,000 μm 2 Each mouse was assessed on HES sections by measuring

[0455] Adapalene significantly increased inflammation, whereas Dysport (BoNT / A) significantly decreased inflammation (immediately following dosing). The results are shown in the table below with standard deviations. This data is shown in the bar graph in Figure 12. [Table 24] [Table 25] group: group, vehicle: vehicle, Adapalene: adapalene, Animal ID: animal ID, Dermis inflammation (nuclei1,000μm 2) / : Inflammation of the dermis (nucleus / 1,000μm 2 ), mean: average, SD: standard deviation

[0456] Example 13

[0457] Keratinocyte proliferation following treatment

[0458] Keratinocyte proliferation was determined by automated counting of intraepidermal positive Ki67 cells from Ki67 immunostained sections. Adapalene caused a significant increase in keratinocyte proliferation, whereas Dysport (BoNT / A) did not. The results (average of 6 fields per section) are shown in the table below. The data are shown in a bar graph in Figure 13. [Table 26] [Table 27] group: group, vehicle: vehicle, Adapalene: adapalene, Animal ID: animal ID, Ki67 positive cells per mm 2 : Ki67 positive cells per square millimeter, mean: average, SD: standard deviation

[0459] Example 14

[0460] Fibroblast proliferation following treatment

[0461] Assessment of fibroblast proliferation was performed by automated counting of positive Ki67 cells in the dermis from Ki67 immunostained sections, together with morphological analysis for differentiation of fibroblasts (thus allowing quantification of the proliferating fibroblast population). Adapalene, but not Dysport (BoNT / A), caused a significant increase in fibroblast proliferation. The results (average of 6 fields per section) are shown in the table below. The data are presented as a bar graph in Figure 14. [Table 28] [Table 29] group: group, vehicle: vehicle, Adapalene: adapalene, Animal ID: animal ID, Ki67 positive cells per mm 2 : Ki67 positive cells per square millimeter, mean: average, SD: standard deviation

[0462] Example 15

[0463] Sebocyte proliferation following treatment

[0464] Sebocyte proliferation was assessed by automated counting of positive Ki67 cells within sebaceous glands from Ki67 immunostained sections, together with morphological analysis for differentiation of sebocytes (thus allowing quantification of the proliferating sebocyte population). No changes in fibroblast proliferation were observed in any of the treatment groups. The results (average of 6 fields per section) are shown in the table below. The data are presented as a bar graph in Figure 15. [Table 30] group: group, vehicle: vehicle, Adapalene: adapalene, Animal ID: animal ID, Ki67 positive cells per mm 2 : Ki67 positive cells per square millimeter, mean: average, SD: standard deviation

[0465] Example 16

[0466] Levels of IL-1α cytokine following treatment

[0467] Skin tissues were homogenized (1 mg tissue: 0.01 ml buffer) in DPBS containing protease inhibitor cocktail by mechanical grinding using ceramic beads (3 cycles, 6,800 rpm, 30 s) followed by centrifugation at 12,000 × g for 10 min at 4 °C. The supernatant was collected and the levels of IL-1α were estimated using an ELISA kit (as described in Materials and Methods). The levels of IL-1α are shown in ng / g tissue.

[0468] Intradermal administration of BoNT / A did not alter IL-1α secretion into the epidermis, whereas adapalene treatment significantly reduced the levels of IL-1α. The table below shows the levels of IL-1α in the back samples. The data are presented in a bar graph in FIG. 16. [Table 31] [Table 32] group: group, vehicle: vehicle, Adapalene: adapalene, Animal ID: animal ID, Concentration (ng / g tissue): concentration (ng / g tissue), mean: mean, SD: standard deviation

[0469] Consider

[0470] The purpose of this study was to evaluate the efficacy of Dysport in a rhino mouse model of sebaceous gland function.

[0471] Briefly, animals were injected under anesthesia via the intradermal route (single injection) with a neurotoxin (Dysport) in three increasing doses (0.1, 0.3, and 1 Dysport unit per mouse). An additional group of mice received adapalene gel 0.1% by topical application daily for 15 days as a reference. The "vehicle only" group was injected via the intradermal route with saline alone as a control. During this period, animals were observed daily for clinical signs of toxin injection. Body weight and parameters of the back skin were measured on days 1, 5, 10, and 15. The occurrence of erythema and scaling at the application sites was also examined on the same days. The experiment was terminated by ethical euthanasia on day 15. Sebum was collected from the treated areas of the skin for lipidomic assays, followed by skin samples for histological analysis and for determining the levels of IL-1α cytokine and substance P.

[0472] No major observations or adverse effects were noted in groups 1, 2, 3, and 4 during the study. No significant changes were evident in the thickness of the back skin in the groups treated with Dysport (<10% between days 1 and 15), and a slight and non-significant increase in skin thickness (+14%) was observed in the adapalene group compared to the vehicle group. The treatment group of mice treated daily with adapalene also showed more erythema (mean score between 0.3 and 1.1) on the back skin from days 5 to 15 than the other groups, and scaling (mean score between 0.5 and 1.3) also increased here during the same period. No signs of erythema or scaling were observed in mice from groups 1, 3, and 4. Overall, macroscopic skin observations did not reveal any noticeable events after Dysport treatment.

[0473] Regarding histopathological observations on HES sections, Dysport had no significant effect on the surface area of ​​the utricle at any of the doses tested (12% increase in the surface area of ​​the utricle in group 3 (Dysport 0.1 U), 11% decrease in group 4 (Dysport 0.3 U) and 9.5% decrease in group 5 (Dysport (1 U)), whereas adapalene dramatically inhibited this parameter (94% decrease), a major effect of retinoid activity in the Rhino acne model. Regarding the utricle parameters, the surface area of ​​the sebaceous gland was not altered by Dysport at any of the three doses tested. Mice treated daily with adapalene showed a significant increase in the sebaceous gland surface (+82% vs. the vehicle group). This result is consistent with the results of the ineffective sebum production that depletes the adjacent sebaceous glands and leads to swelling. This could be explained by the opening of the utricle, which became extremely tight after adapalene treatment, which may have been caused by the endothelial cell wall. HES staining revealed significant epidermal hyperplasia (increase in epidermal thickness by +188%) associated with parakeratosis in mice treated with adapalene. This effect is widely described during repeated treatment with retinoids as a side effect, and is often accompanied by increased epidermal inflammation (increase of +23% in the adapalene group in this study). Dysport did not induce any associated hyperplasia of the epidermis at any of the three doses (change in epidermal thickness between 8% and 11% in groups 3, 4, and 5). Moreover, Dysport dose-dependently reduced immune cell infiltration in the dermis, evidence of a significant anti-inflammatory effect (reduction of 10%, 19%, and 26% vs. vehicle, groups 3, 4, and 5, respectively).

[0474] Finally, Ki67 immunostaining, a biomarker for cell proliferation, was used to highlight the proliferative effect of treatment with Dysport or adapalene on sebocytes (cells of the sebaceous gland), fibroblasts (cells of the dermis), and keratinocytes (cells of the epidermis). While Dysport did not induce any proliferative effect on sebaceous, dermal, or epidermal cells, a significant increase in Ki67 positive cells was observed in both the epidermis and dermis of the adapalene group (30% and 43% increase in keratinocytes and fibroblasts, respectively). This proliferative effect was due to the epidermal hyperplasia and immune cell infiltration observed in this group. Substance P and IL-1α were detected by ELISA in tissue extracts from the different groups. In control and adapalene mice, the levels of substance P were similar (390.2 ± 106.3 and 418.0 ± 52.4 pg / g tissue, respectively). Dysport treatment increased the levels of substance P in a dose-dependent manner (575.3±263.8, 969.2±233.2, and 1,101.8±254.7 pg / g tissue in groups 3, 4, and 5, respectively). Mice treated with Dysport at two doses, 0.3 U and 1 U, showed significant changes in the content of substance P compared to control mice (+148% and +182%, respectively, p<0.001). This result could be explained by a mechanism of action of a neurotoxin that inhibits exocytosis at the cellular level, thus leading to the accumulation of the neuropeptide as substance P in the nerve endings of the skin. Thus, this result confirms the in vivo activity of Dysport.

[0475] Concerning the levels of IL-1α, repeated application of adapalene dramatically reduced the levels of IL-1α (47.7±11.5 vs. 880.6±200.3 ng / g tissue, p<0.001, 96% reduction). IL-1α is the major cytokine in the epidermis, which is constitutively secreted by keratinocytes upon activation under perturbations of the skin environment, such as skin lesions, bacterial infections, or chemicals, and is predominantly increased in the proinflammatory flow. The inhibition of IL-1α in the adapalene group is due to the anti-inflammatory properties of retinoids. Dysport did not alter the levels of IL-1α at any of the three doses tested (939.5±175.0, 738.5±140.0, and 892.0±259.7 ng / g tissue in groups 3, 4, and 5, respectively).

[0476] Finally, the lipid content of sebum from back skin did not change under Dysport treatment, and the levels of fatty acids increased (+83%) in mice treated with 1 U of Dysport, as noted in each result, by changes in the peak areas of lipid compounds on the chromatogram. Cholesterol and squalene were also significantly increased in sebum from treatment with 0.3 U and 1 U of Dysport (cholesterol, increased +99% and +154% in mice treated with 0.3 U and 1 U, respectively; squalene, increased +110% and +175% in mice treated with 0.3 U and 1 U, respectively), and the levels of wax esters of interest were altered under Dysport. The levels of wax 2 increased by +126%, +128%, and +195% in mice treated with 0.1 U, 0.3 U, and 1 U of Dysport, respectively. Treatment with Dysport also increased compounds wax 3 and wax 4 in sebum in the same manner as wax 2. Subsequently, cholesteryl esters in sebum were significantly increased by 440% at the highest dose of Dysport. Conversely, adapalene did not significantly change the lipid content of sebum. Taken together, these results suggest that treatment with Dysport may act on sebum content by increasing the levels of some lipids that exhibit protective properties for the skin.

[0477] In conclusion, adapalene demonstrated a dramatic reduction in the surface of the utricle in the present study, a major effect usually observed with retinoids in the rhino model of acne, suggesting a beneficial effect of this reference compound. Although Dysport did not change the surface area of ​​the utricle at any of the doses tested; it did not induce any of the adverse effects usually observed with retinoid treatment, such as epidermal hyperplasia and dermal inflammation. Dysport did not enhance cell proliferation in the epidermis and dermis, and showed an anti-inflammatory effect on the dermis from a dose of 0.3 U, presenting a beneficial effect in this model. The proinflammatory cytokine IL-1α was not affected by Dysport. Conversely, Dysport dose-dependently increased the levels of substance P in the skin, a neuropeptide present in nerve endings and supporting the in vivo activity of the neurotoxin in the present study. Finally, the lipid content of sebum was altered under Dysport treatment, and an increase in the amount of some lipids with skin-protecting properties could be a beneficial effect of treatment with Dysport in skin conditions.

[0478] [Item 1] 1. A method for treating a skin condition comprising intradermally administering to a patient a Clostridial neurotoxin: Here, following administration, said Clostridial neurotoxin induces secretion of one or more sebaceous gland lipids into the epidermal layer of the skin. [Item 2] 1. A Clostridial neurotoxin for use in a method of treating a skin condition comprising administering the Clostridial neurotoxin intradermally to a patient: Here, following administration, said Clostridial neurotoxin induces secretion of one or more sebaceous gland lipids into the epidermal layer of the skin. [Item 3] Non-therapeutic uses of clostridial neurotoxins for cosmetic treatment of the skin: Here, the Clostridial neurotoxin induces the secretion of one or more sebaceous gland lipids into the epidermal layer of the skin. [Item 4] Non-therapeutic uses of Clostridial neurotoxins to promote skin rejuvenation: Here, the Clostridial neurotoxin induces the secretion of one or more sebaceous gland lipids into the epidermal layer of the skin. [Item 5] 1. A method for treating a skin condition comprising intradermally administering to a patient a Clostridial neurotoxin: wherein, following administration, said Clostridial neurotoxin induces: - secretion of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin. [Item 6] 1. A Clostridial neurotoxin for use in a method of treating a skin condition comprising administering the Clostridial neurotoxin intradermally to a patient: wherein, following administration, said Clostridial neurotoxin induces: - secretion of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin. [Item 7] Non-therapeutic uses of clostridial neurotoxins for cosmetic treatment of the skin: wherein said Clostridial neurotoxin induces: - secretion of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin. [Item 8] Non-therapeutic uses of Clostridial neurotoxins to promote skin rejuvenation: wherein said Clostridial neurotoxin induces: - secretion of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin. [Item 9] 9. The non-therapeutic use according to any one of items 3-4 or 7-8, wherein the Clostridial neurotoxin is administered by intradermal administration. [Item 10] A Clostridial neurotoxin for the method or use according to any one of items 1-2 or 5-6: The Clostridial neurotoxin is administered to a patient having a level of the one or more sebaceous lipids in an epidermal layer that is lower than the level of the one or more sebaceous lipids in an epidermal layer of a subject not having the skin condition. [Item 11] A Clostridial neurotoxin for the method or use according to any one of items 1 to 2, 5 to 6 or 10: wherein the skin condition is caused by a reduced level of the one or more sebaceous gland lipids in the epidermal layer of the patient compared to the level of the one or more sebaceous gland lipids in the epidermal layer of a subject not having the skin condition. [Item 12] A method according to any one of the preceding items, a Clostridial neurotoxin for use, or a non-therapeutic use: Here, the Clostridial neurotoxin is administered to a patient whose skin has been exposed to the agent, causing a decrease in the level of the one or more sebaceous lipids in the patient's epidermal layer compared to the level of the one or more sebaceous lipids in the epidermal layer of a subject not exposed to the agent. [Item 13] A method according to any one of the preceding items, a Clostridial neurotoxin for use, or a non-therapeutic use: Here, the agent is a chemical selected from metals, soap chemicals, fragrances, preservatives, plant-derived chemicals, and paraphenylenediamines. [Item 14] A method according to any one of the preceding items, a Clostridial neurotoxin for use, or a non-therapeutic use: wherein said Clostridial neurotoxin is administered to a patient having sebum comprising: - fatty acids at a concentration of <50% (v / v); - wax esters at a concentration of <20% (v / v); - Squalene at a concentration of <10% (v / v); and / or - Cholesterol at a concentration of <4% (v / v). [Item 15] A Clostridial neurotoxin for the method or use according to any one of items 1-2, 5-6 or 10-14: wherein the skin condition is a condition associated with abnormal sebaceous gland lipid levels; The sebaceous lipids are present in the epidermal layer at a lower level than in the epidermal layer of patients not having the skin condition. [Item 16] A Clostridial neurotoxin for the method or use according to any one of items 1 to 2, 5 to 6 or 10 to 15: Here, the skin condition is one or more conditions selected from acne, atopic dermatitis, Netherton syndrome, psoriasis, dehydrated skin (e.g., dry or cracked skin), actinic keratosis, rosacea, carbuncle, eczema, cellulitis, dermatitis, skin cancer, and lichen pilaris. [Item 17] A Clostridial neurotoxin for the method or use according to any one of items 1 to 2, 5 to 6 or 10 to 16, wherein the skin condition is one or more conditions selected from psoriasis, eczema, and dermatitis. [Item 18] A Clostridial neurotoxin for the method or use according to any one of items 1 to 2, 5 to 6 or 10 to 17: wherein the skin condition is one or more selected from atopic dermatitis, psoriasis, xerosis, dehydrated skin (e.g., dry or cracked skin), eczema (e.g., atopic eczema), type 2 Gaucher disease, Sjogren-Larsson syndrome, ichthyosis (e.g., lamellar ichthyosis), X-linked ichthyosis, bullous congenital ichthyosiform erythroderma, essential FFA deficiency, aging dry skin, and anhidrotic ectodermal dysplasia. [Item 19] 19. A clostridial neurotoxin for the method or use according to item 18: wherein the skin condition is one or more selected from xerosis, psoriasis, atopic dermatitis, and ichthyosis, for example xerosis, psoriasis, or ichthyosis. [Item 20] A method according to any one of the preceding items, a Clostridial neurotoxin for use, or a non-therapeutic use: Here, the Clostridial neurotoxin is administered to a non-facial area of ​​the patient's skin. [Item 21] A method according to any one of the preceding items, a Clostridial neurotoxin for use, or a non-therapeutic use: Here, the Clostridial neurotoxin is administered to the patient's hands (both), feet (eg, both feet), neck, scalp, and / or back (eg, upper back). [Item 22] A method according to any one of the preceding items, a Clostridial neurotoxin for use, or a non-therapeutic use: wherein following administration of said Clostridial neurotoxin: - no increase is induced in the level of sebum on the skin of the patient relative to a reference standard; preferably no decrease is induced in the level of sebum on the skin of the patient relative to a reference standard; - the reference standard corresponds to the level of sebum on the skin of a subject who has not been administered the Clostridial neurotoxin, or the reference standard corresponds to the level of sebum on the skin of the patient prior to administration of the Clostridial neurotoxin. [Item 23] A method according to any one of the preceding items, a Clostridial neurotoxin for use, or a non-therapeutic use: wherein said patient does not have oily skin before and / or after administration of said Clostridial neurotoxin. [Item 24] A method according to any one of the preceding items, a Clostridial neurotoxin for use, or a non-therapeutic use: wherein the patient has a mass of ≦180 mg / cm 2 of skin on the forehead, nasolabial folds, and / or nose. 2 have a level of sebum. [Item 25] A method according to any one of the preceding items, a Clostridial neurotoxin for use, or a non-therapeutic use: Here, following administration to the patient, the Clostridial neurotoxin induces retention of a tachykinin peptide within the dermis of the skin. [Item 26] 26. A method according to claim 25, a Clostridial neurotoxin for use, or a non-therapeutic use: wherein said tachykinin peptide is substance P (eg, SEQ ID NO: 17). [Item 27] A method according to any one of the preceding items, a Clostridial neurotoxin for use, or a non-therapeutic use: wherein said Clostridial neurotoxin is a BoNT / A neurotoxin. [Item 28] A method according to any one of the preceding items, a Clostridial neurotoxin for use, or a non-therapeutic use: wherein said Clostridial neurotoxin is a chimeric neurotoxin. [Item 29] 29. A method according to claim 28, a Clostridial neurotoxin for use, or a non-therapeutic use: wherein said chimeric neurotoxin is selected from the group consisting of BoNT / DC and BoNT / X. [Item 30] 29. A method according to claim 28, a Clostridial neurotoxin for use, or a non-therapeutic use: wherein the chimeric neurotoxin is a H from a second neurotoxin. C LH from the first neurotoxin covalently bound to the domain N domain; preferably said first and second neurotoxins are different; The above LH N The C-terminal amino acid residue of the domain is LH N Domain and H C Splitting the Domain 3 10 corresponding to the first amino acid residue of the helix; and The above H C The N-terminal amino acid residue of the domain is LH in the second neurotoxin. N Domain and H C Splitting the Domain 3 10It corresponds to the second amino acid residue in the helix. [Item 31] 31. A method according to claim 30, a Clostridial neurotoxin for use, or a non-therapeutic use: Here, the first neurotoxin is BoNT / A and the second neurotoxin is BoNT / B. [Item 32] A method according to any one of the preceding items, a Clostridial neurotoxin for use, or a non-therapeutic use: wherein said intradermal administration comprises administration of said Clostridial neurotoxin at a dose ranging from about 0.00025 ng to 3 ng at the administration site. [Item 33] A method according to any one of the preceding items, a Clostridial neurotoxin for use, or a non-therapeutic use: wherein said intradermal administration comprises intradermal injection using a 30 gauge needle; preferably The 30 gauge needle is inserted into the dermis of the skin at an angle of approximately 5° to 15° to the flat surface of the skin. [Item 34] A method according to any one of the preceding items, a Clostridial neurotoxin for use, or a non-therapeutic use: Here, the epidermal layer is one or more selected from the stratum basale, stratum spinosum, stratum granulosum, or stratum corneum. [Item 35] A method according to any one of the preceding items, a Clostridial neurotoxin for use, or a non-therapeutic use: Here, the epidermis layer is the stratum corneum. [Explanation of symbols]

[0479] [Figure 1] sebum: sebum vehicle: Solvent adapalene [Figures 2-17] Gp: Group vehicle: Solvent Excipient: adapalene mouse: mouse [Figures 2-6] Peak area: Peak area [Figure 2] Fatty Acid Level: Fatty acid level [Figure 3] Squalene Level: Squalene Level [Figure 4] Cholesterol Level: Cholesterol level [Figure 5] Wax ester: Wax ester [Figure 6] Cholesteryl: [Figure 7] Substance P: Substance P Substance P level (pg / g tissue): [Figures 8-15] Mean: Average SD: standard deviation [Figure 8] Erythema: erythema Scaling: Scaling score: score Day: [Figure 9] Sebaceous glands surface: sebaceous glands surface [Figure 10] Utriculi surface: Utriculi surface [Figure 11] Back skin epidermis thickness: Back skin epidermis thickness Thickness: [Figure 12] dermis inflammation: Inflammation of the dermis nucleus: nucleus [Figures 13-15] ki67 positive cells per mm 2: ki67 cells per square millimeter [Figure 13] Keratinocyte proliferation: proliferation of keratinocytes [Figure 14] Fibroblasts proliferation: Fibroblast proliferation [Figure 15] Sebaceous glands proliferation: Sebaceous gland proliferation [Figure 16] IL-1 alpha: IL-1α IL-1 alpha level (ng / g tissue): IL-1α level (ng / g tissue) [Figure 17] Fatty acids: Fatty acids Monoglycerides Squalene: Waxes: Diglycerides Triglycerides Cholesteryl Esters: Signal: Signal Time (min): Time (min)

Claims

1. 1. A method for treating a skin condition comprising intradermally administering to a patient a Clostridial neurotoxin: Here, following administration, said Clostridial neurotoxin induces secretion of one or more sebaceous gland lipids into the epidermal layer of the skin.

2. 1. A Clostridial neurotoxin for use in a method of treating a skin condition comprising administering the Clostridial neurotoxin intradermally to a patient: Here, following administration, said Clostridial neurotoxin induces secretion of one or more sebaceous gland lipids into the epidermal layer of the skin.

3. Non-therapeutic uses of clostridial neurotoxins for cosmetic treatment of the skin: Here, the Clostridial neurotoxin induces the secretion of one or more sebaceous gland lipids into the epidermal layer of the skin.

4. Non-therapeutic uses of Clostridial neurotoxins to promote skin rejuvenation: Here, the Clostridial neurotoxin induces the secretion of one or more sebaceous gland lipids into the epidermal layer of the skin.

5. 1. A method for treating a skin condition comprising intradermally administering to a patient a Clostridial neurotoxin: wherein, following administration, said Clostridial neurotoxin induces: - secretion of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin.

6. 1. A Clostridial neurotoxin for use in a method of treating a skin condition comprising administering the Clostridial neurotoxin intradermally to a patient: wherein, following administration, said Clostridial neurotoxin induces: - secretion of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin.

7. Non-therapeutic uses of clostridial neurotoxins for cosmetic treatment of the skin: wherein said Clostridial neurotoxin induces: - secretion of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin.

8. Non-therapeutic uses of Clostridial neurotoxins to promote skin rejuvenation: wherein said Clostridial neurotoxin induces: - secretion of one or more sebaceous lipids selected from squalene, fatty acids, cholesterol, and wax esters into the epidermal layer of the skin.

9. 9. The non-therapeutic use of any one of claims 3-4 or 7-8, wherein the Clostridial neurotoxin is administered by intradermal administration.

10. A Clostridial neurotoxin for the method or use according to any one of claims 1-2 or 5-6: The Clostridial neurotoxin is administered to a patient having a level of the one or more sebaceous lipids in an epidermal layer that is lower than the level of the one or more sebaceous lipids in an epidermal layer of a subject not having the skin condition.

11. A Clostridial neurotoxin for the method or use according to any one of claims 1 to 2, 5 to 6 or 10: wherein the skin condition is caused by a reduced level of the one or more sebaceous gland lipids in the epidermal layer of the patient compared to the level of the one or more sebaceous gland lipids in the epidermal layer of a subject not having the skin condition.

12. A method, a Clostridial neurotoxin for use, or a non-therapeutic use according to any one of the preceding claims. Here, the Clostridial neurotoxin is administered to a patient whose skin has been exposed to the agent, causing a decrease in the level of the one or more sebaceous lipids in the patient's epidermal layer compared to the level of the one or more sebaceous lipids in the epidermal layer of a subject not exposed to the agent.

13. A method, a Clostridial neurotoxin for use, or a non-therapeutic use according to any one of the preceding claims. Here, the agent is a chemical selected from metals, soap chemicals, fragrances, preservatives, plant-derived chemicals, and paraphenylenediamines.

14. A method, a Clostridial neurotoxin for use, or a non-therapeutic use according to any one of the preceding claims. wherein said Clostridial neurotoxin is administered to a patient having sebum comprising: - fatty acids at a concentration of <50% (v / v); - wax esters at a concentration of <20% (v / v); - Squalene at a concentration of <10% (v / v); and / or - Cholesterol at a concentration of <4% (v / v).

15. A Clostridial neurotoxin for the method or use according to any one of claims 1 to 2, 5 to 6 or 10 to 14. wherein the skin condition is a condition associated with abnormal sebaceous gland lipid levels; The sebaceous lipids are present in the epidermal layer at a lower level than in the epidermal layer of patients not having the skin condition.

16. A Clostridial neurotoxin for the method or use according to any one of claims 1 to 2, 5 to 6 or 10 to 15. Here, the skin condition is one or more conditions selected from acne, atopic dermatitis, Netherton syndrome, psoriasis, dehydrated skin (e.g., dry or cracked skin), actinic keratosis, rosacea, carbuncle, eczema, cellulitis, dermatitis, skin cancer, and lichen pilaris.

17. A Clostridial neurotoxin for the method or use according to any one of claims 1 to 2, 5 to 6 or 10 to 16. wherein the skin condition is one or more conditions selected from psoriasis, eczema, and dermatitis.

18. A Clostridial neurotoxin for the method or use according to any one of claims 1 to 2, 5 to 6 or 10 to 17: wherein the skin condition is one or more selected from atopic dermatitis, psoriasis, xerosis, dehydrated skin (e.g., dry or cracked skin), eczema (e.g., atopic eczema), type 2 Gaucher disease, Sjogren-Larsson syndrome, ichthyosis (e.g., lamellar ichthyosis), X-linked ichthyosis, bullous congenital ichthyosiform erythroderma, essential FFA deficiency, aging dry skin, and anhidrotic ectodermal dysplasia.

19. 19. A clostridial neurotoxin for the method or use according to claim 18, wherein the skin condition is one or more selected from xerosis, psoriasis, atopic dermatitis, and ichthyosis, for example xerosis, psoriasis, or ichthyosis.

20. A method, a Clostridial neurotoxin for use, or a non-therapeutic use according to any one of the preceding claims. Here, the Clostridial neurotoxin is administered to a non-facial area of ​​the patient's skin.

21. A method, a Clostridial neurotoxin for use, or a non-therapeutic use according to any one of the preceding claims. Here, the Clostridial neurotoxin is administered to the patient's hands (both), feet (eg, both feet), neck, scalp, and / or back (eg, upper back).

22. A method, a Clostridial neurotoxin for use, or a non-therapeutic use according to any one of the preceding claims. wherein following administration of said Clostridial neurotoxin: - no increase is induced in the level of sebum on the skin of the patient relative to a reference standard; preferably no decrease is induced in the level of sebum on the skin of the patient relative to a reference standard; - the reference standard corresponds to the level of sebum on the skin of a subject who has not been administered the Clostridial neurotoxin, or the reference standard corresponds to the level of sebum on the skin of the patient prior to administration of the Clostridial neurotoxin.

23. A method, a Clostridial neurotoxin for use, or a non-therapeutic use according to any one of the preceding claims. wherein said patient does not have oily skin before and / or after administration of said Clostridial neurotoxin.

24. A method, a Clostridial neurotoxin for use, or a non-therapeutic use according to any one of the preceding claims. wherein the patient has a mass of ≦180 mg / cm 2 of skin on the forehead, nasolabial folds, and / or nose. 2 have a level of sebum.

25. A method, a Clostridial neurotoxin for use, or a non-therapeutic use according to any one of the preceding claims. Here, following administration to the patient, the Clostridial neurotoxin induces retention of a tachykinin peptide within the dermis of the skin.

26. 26. The method of claim 25, a Clostridial neurotoxin for use, or a non-therapeutic use: wherein said tachykinin peptide is substance P (eg, SEQ ID NO: 17).

27. A method, a Clostridial neurotoxin for use, or a non-therapeutic use according to any one of the preceding claims. wherein said Clostridial neurotoxin is a BoNT / A neurotoxin.

28. A method, a Clostridial neurotoxin for use, or a non-therapeutic use according to any one of the preceding claims. wherein said Clostridial neurotoxin is a chimeric neurotoxin.

29. 29. The method of claim 28, a Clostridial neurotoxin for use, or a non-therapeutic use: wherein said chimeric neurotoxin is selected from the group consisting of BoNT / DC and BoNT / X.

30. 29. The method of claim 28, a Clostridial neurotoxin for use, or a non-therapeutic use: wherein the chimeric neurotoxin is a H from a second neurotoxin. C LH from the first neurotoxin covalently bound to the domain N domain; preferably said first and second neurotoxins are different; The above LH N The C-terminal amino acid residue of the domain is LH N Domain and H C Splitting the Domain 3 10 corresponding to the first amino acid residue of the helix; and The above H C The N-terminal amino acid residue of the domain is LH in the second neurotoxin. N Domain and H C Splitting the Domain 3 10 It corresponds to the second amino acid residue in the helix.

31. 31. The method of claim 30, a Clostridial neurotoxin for use, or a non-therapeutic use: Here, the first neurotoxin is BoNT / A and the second neurotoxin is BoNT / B.

32. A method, a Clostridial neurotoxin for use, or a non-therapeutic use according to any one of the preceding claims. wherein said intradermal administration comprises administration of said Clostridial neurotoxin at a dose ranging from about 0.00025 ng to 3 ng at the administration site.

33. A method, a Clostridial neurotoxin for use, or a non-therapeutic use according to any one of the preceding claims. wherein said intradermal administration comprises intradermal injection using a 30 gauge needle; preferably The 30 gauge needle is inserted into the dermis of the skin at an angle of approximately 5° to 15° to the flat surface of the skin.

34. A method, a Clostridial neurotoxin for use, or a non-therapeutic use according to any one of the preceding claims. Here, the epidermal layer is one or more selected from the stratum basale, stratum spinosum, stratum granulosum, or stratum corneum.

35. A method, a Clostridial neurotoxin for use, or a non-therapeutic use according to any one of the preceding claims. Here, the epidermis layer is the stratum corneum.

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