Treatment of hyperpigmentation conditions by botulinum toxins

Abstract

The present invention relates to the use of botulinum toxins for the treatment of hyperpigmentation conditions. The use includes intracutaneously injecting botulinum toxin into the skin of a patient with hyperpigmentation at a specific depth, volume and dose to specifically target the epidermal layer and the upper dermal layer. For example, an intradermal injection of botulinum toxin according to the invention may be used to treat chloasma.

Description

Technical Field

[0001] This invention relates to the use of botulinum toxin in the treatment of hyperpigmentation. This use includes intradermal injection of botulinum toxin at a specific depth, volume, and dose into the skin of a patient suffering from hyperpigmentation, thereby specifically targeting the epidermis and upper dermis. For example, intradermal injection of botulinum toxin according to the invention can be used to treat melasma. Background Technology

[0002] Hyperpigmentation is a common condition that occurs when a localized area of ​​skin becomes darker than the surrounding skin due to the production of excessive melanin. Melanin is produced by specialized cells called melanocytes. These cells are located in the basal layer of the epidermis, the deepest layer of the epidermis, which forms the interface (dermal-epidermal junction (DEJ)) with the underlying dermis. Melanin synthesis begins with the amino acid tyrosine, which is converted to dihydroxyphenylalanine (DOPA) by tyrosinase. DOPA is then converted to dopaquinone, a precursor to eumelanin and pheomelanin, the two types of melanin that influence skin color. Eumelanin is black, resulting in a darker skin tone, while pheomelanin is reddish-brown, resulting in a lighter skin tone. The relative amounts of eumelanin and pheomelanin produced by melanocytes determine skin color. Once produced, melanin is transferred from melanocytes to neighboring skin cells (called epidermal keratinocytes). Melanin is then evenly distributed throughout the skin, absorbing and scattering UV radiation from the sun, helping to protect the skin from damage.

[0003] The prevalence of hyperpigmentation varies considerably depending on underlying causes, geographic location, and other factors. For example, studies suggest that up to 90% of people may experience some form of hyperpigmentation in their lifetime, varying in severity. This can include age spots, melasma, post-inflammatory hyperpigmentation, and other types of hyperpigmentation. For instance, in the United States, melasma is estimated to affect up to 5–10% of people, with a higher incidence in women, especially those who are pregnant or taking oral contraceptives.

[0004] While hyperpigmentation is harmless to health, it can be a significant cosmetic concern for many, impacting self-esteem and quality of life. Currently, various treatment options exist for hyperpigmentation, including topical creams containing ingredients such as hydroquinone, kojic acid, and retinoids, chemical peels, laser therapy, cryotherapy, and other procedures. However, these treatments may have side effects such as skin irritation, hypopigmentation, allergic reactions, and photosensitivity, and their effectiveness is limited, leading to unsatisfactory results.

[0005] In addition, botulinum toxin (botulinum neurotoxin or BoNT) has been suggested as an alternative treatment option for hyperpigmentation. Botulinum toxin is a neurotoxin derived from Clostridium botulinum that blocks the release of acetylcholine into the synaptic cleft, thereby inhibiting cholinergic neuromuscular innervation. For example, WO 2018 / 222652 A1 discloses a method for treating hyperpigmentation by administering a composition comprising botulinum neurotoxin BoNT / DC. ​​According to its embodiments, BoNT / DC reduces melanin content in human skin equivalent models and human explant skin. These embodiments also describe intradermal treatment of solar lentigines by injecting 50 μl of BoNT / DC toxin at each injection point using a 30 G needle, and intradermal treatment of melasma by injecting 2 μl of BoNT / DC toxin at each injection point using a multi-needle dermal syringe system.

[0006] Typically, BoNT is administered topically via subcutaneous or intramuscular injection. However, treatment of hyperpigmentation requires shallower injections, usually intradermal injections (IVIs). IVIs have seen increasing use in recent years. However, IVIs are more challenging to perform than subcutaneous and intramuscular injections when using a standard syringe for injecting botulinum toxin. This is especially true for targeting the active substance to both the epidermis and upper dermis. Using a syringe with a standard needle for IVIs will produce different results in terms of application area and / or injection depth. Furthermore, a relatively high volume is usually used to ensure that BoNT reaches the target cells through diffusion and dispersion of the toxin into the target skin layer. This, in turn, increases costs due to the use of large volumes of BoNT. It also leads to a higher risk of overdose / side effects. To date, no established administration regimen for BoNT has been found to reliably and effectively treat hyperpigmentation at a limited cost. The purpose of this invention

[0007] In view of the above, the object of the present invention is to provide an improved botulinum toxin treatment for hyperpigmentation that is effective, reliable and cost-effective. Summary of the Invention

[0008] This invention is based on the discovery that deep-controlled injection of low-volume BoNTs into the dermis can specifically target the viable epidermis and upper dermis, more specifically the basal layer and adjacent layers, in a safe, accurate, and reliable manner. Since the basal layer contains target cells such as melanocytes, the BoNT injection of this invention can be used to treat various melanin-related conditions, particularly hyperpigmentation due to excessive melanin production, such as melasma. Furthermore, the specific delivery of low-volume BoNTs to the viable epidermis and upper dermis allows for the use of reduced amounts of BoNT, thereby significantly saving costs.

[0009] In a first aspect, the present invention relates to the use of botulinum toxin for the treatment of hyperpigmentation, comprising injecting botulinum toxin into the skin of a patient affected by hyperpigmentation, wherein the botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site.

[0010] In a preferred embodiment, the distance between injection sites is at least 5 mm, more preferably 5 mm to 20 mm, even more preferably 5 mm to 15 mm, even more preferably 10 mm to 20 mm, and most preferably 10 mm to 15 mm.

[0011] In the context of this invention, there is no particular limitation on the hyperpigmentation condition to be treated, and it can be a condition selected from the group consisting of melasma, freckles, solar lentigines, café-au-lait spots, post-inflammatory hyperpigmentation (PIH), nevi, and hyperpigmented nevi. Melasma is a particularly preferred condition to be treated. The mentioned conditions are generally cosmetic in nature, as they involve aesthetic features of the body that may cause dissatisfaction with one's appearance but are not threatening or do not lead to substantial health problems requiring therapeutic intervention.

[0012] In a second aspect, the present invention relates to a method for treating hyperpigmentation, comprising injecting botulinum toxin into the skin of a patient affected by hyperpigmentation, wherein the botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site.

[0013] In the third and fourth aspects, the present invention relates to the use of botulinum toxin in the treatment of rosacea and methods for treating rosacea, respectively. The use and methods involve injecting botulinum toxin into the skin of a patient affected by rosacea, wherein the botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site.

[0014] In this invention, the botulinum toxin is preferably type A. Furthermore, the botulinum toxin may be in a form without complexing proteins or in the form of a complex containing complexing proteins. Preferably, the botulinum toxin is type A and is in a form without complexing proteins, or in an alternative preferred embodiment, the botulinum toxin is type A and is in the form of a complex containing complexing proteins.

[0015] Preferred embodiments are set forth in the appended dependent claims and in the following detailed description in conjunction with the embodiments and drawings provided therein. Attached Figure Description

[0016] Figure 1 The distribution of fluorescently labeled inactive BoNTs in an ex vivo human skin model after injection using a MicronJet 600 microneedle device is shown (E = epidermis, D = dermis). All images were taken at the same magnification. Scale bar = 0.1 cm. (A): Overlay of fluorescence microscopy (concentric structures in light to dark gray shading around the injection site) and bright-field microscopy (gray). (B): Graph showing the BoNT distribution, expressed as BoNT signal (relative fluorescence intensity) as a function of skin depth.

[0017] Figure 2 The distribution of fluorescently labeled inactive BoNTs in an ex vivo human skin model after injection using a 1.4 mm U-needle is shown (E = epidermis, D = dermis). Scale bar = 0.1 cm. (A): Overlay of fluorescence microscopy (concentric structures in light to dark gray shading around the injection site) and bright-field microscopy (gray). (B): Graph showing the BoNT distribution, expressed as BoNT signal (relative fluorescence intensity) as a function of skin depth.

[0018] Figure 3The distribution of fluorescently labeled inactive BoNTs in an ex vivo human skin model after injection using a conventional 30G x ½” needle is shown (E = epidermis, D = dermis). All images were taken at the same magnification. Scale bar = 0.1 cm. (A): Overlay of fluorescence microscopy (concentric structures in light to dark gray shading around the injection site) and bright-field microscopy (gray). (B): Graph showing the BoNT distribution, expressed as BoNT signal (relative fluorescence intensity) as a function of skin depth. Detailed Implementation

[0019] The discovery leading to this invention is that depth-controlled injection of relatively low-volume BoNTs into specific layers of the dermis enables reliable targeting of BoNTs in the epidermis and upper dermis, particularly the basal layer, thus enabling applications across a wide range of conditions. Surprisingly, it was found that the injection bolus in the upper dermis was redirected to the upper skin layers, namely the upper dermis and the basal layer (the deepest layer of the epidermis). This allows for specific targeting of cells located in these upper skin layers, cells involved in a variety of diseases, conditions, and ailments. For example, it is possible to specifically target melanocytes contained in the basal layer to treat general hyperpigmentation, particularly melasma.

[0020] Precise injection of low-volume BoNTs at accurate depths not only delivers BoNTs specifically to the skin layer containing target cells (such as melanocytes), but also allows for the use of reduced amounts of BoNTs, resulting in significant cost savings. Furthermore, optimal dosing leads to a reduced risk of overdose, thereby minimizing the risk of side effects. Therefore, overall, treatment of diseases, conditions, or illnesses using this injection method is more accurate, reliable, and cost-effective compared to conventional intradermal injections using standard needles with high variability in injection depth and volume.

[0021] As used herein, the term “hyperpigmentation” refers to a skin condition caused by increased melanin production, characterized by localized areas of skin that are darker in color than the surrounding skin.

[0022] As used herein, the term "epidermis" has its common meaning in the art unless otherwise stated. It is the outermost of the three layers of skin, the middle layer being the dermis, and the innermost layer being the subcutaneous tissue. The epidermis consists of four or five layers, which, from the outermost to the innermost, are: the stratum corneum, the stratum lucidum (only in the palms and soles), the stratum granulosum, the stratum spinosum, and the stratum basale. The stratum basale contains keratinocytes, melanocytes, and Merkel cells. The thickness of the human epidermis varies by region. In the context of this invention, the thickness of the stratum corneum is typically from about 10 μm to about 40 μm, typically about 20 μm, and the thickness of the active epidermal layer can be from about 30 μm to about 150 μm, typically from about 40 μm to about 100 μm. As used herein, the term "active epidermis" refers to all epidermal layers except the stratum corneum, including the stratum basale.

[0023] As used herein, the term "dermis" generally refers to the layer of skin below the epidermis and above the subcutaneous tissue (which contains fat and connective tissue). The thickness of the dermis varies depending on its location on the body. In the context of this invention, the thickness is typically from 1500 μm to 4000 μm. The dermis can be further divided into papillary dermis and reticular dermis. The papillary dermis is a thin upper layer containing capillaries. The thickness of the papillary dermis is typically from about 300 μm to about 400 μm. The reticular dermis is a much thicker lower layer than the papillary dermis, containing connective tissue and dense collagen bundles.

[0024] The term "dermal-epidermal junction," "DEJ," or simply "dermal-epidermal junction" refers to the structure at the interface between the epidermis and dermis. It comprises a dense network of collagen that creates the undulations of the dermal-epidermal junction (DEJ). The DEJ is approximately 80 nm in size and ensures several functions within the skin, namely ensuring a good connection between the epidermis and dermis to maintain skin integrity and providing a physical boundary layer between fibroblasts and keratinocytes.

[0025] As used herein, the term "upper dermis" or "upper dermal layer" refers to the dermal layer that is closer to the epidermis than the subcutaneous tissue. These layers typically include limited portions of the papillary and reticular layers of the dermis.

[0026] As used herein, the term "injection site" refers to the location on the skin where an injection device is placed and botulinum toxin is injected into the dermis through one or more needles. If the injection device used to inject botulinum toxin is a syringe with a single needle, the injection site is the point on the skin where that single needle enters the skin, which may also be referred to herein as the "injection point". If the injection device has multiple needles, i.e., two, three, four or more needles, the injection site is the point on the skin where the multiple needles enter the skin.

[0027] As used herein, the term "distance between injection sites" refers to the distance between one injection site and another adjacent injection site. If the injection device used to inject botulinum toxin is a syringe with a single needle, the distance between injection sites is the distance between the site on the skin where the single needle enters the skin in the first injection and the site on the skin where the single needle enters the skin in the second adjacent injection. If the injection device has multiple needles, i.e., two, three, four, or more needles, the distance between injection sites is the shortest distance between any of the multiple needles of the injection device that enter the skin in the first injection and any of the multiple needles of the injection device that enter the skin in the second adjacent injection.

[0028] As used in this article, the term "injection depth" refers to the depth of injection measured from the skin surface to the tip of the needle penetrating the skin and reaching the dermis.

[0029] The terms "volume per injection site" and "dose per injection site" refer to the volume and dose (in units) of botulinum toxin injected at one injection site. If an injection device with multiple needles (i.e., two, three, four, or more needles) is used, the terms "volume per injection site" and "dose per injection site" refer to the volume and dose of botulinum toxin injected at one site using all the needles in the multi-needle injection device, not the volume and dose of botulinum toxin injected at one site using only a single needle in the multi-needle injection device.

[0030] As used herein, the term "injection device" refers to any device suitable for injecting botulinum toxin into the dermis, such as a syringe. An injection device can be a conventional syringe equipped with a single hollow needle, or preferably a microneedle device. As used herein, a "microneedle device" refers to a device having multiple needles (also called "microneedles"), such as two, three, four, five, or more needles. The needles are typically hollow and in fluid contact with a reservoir within the device filled with the botulinum toxin solution to be injected. The needles can be made of various materials, such as stainless steel or silicone. A suitable microneedle device for this invention is, for example, the MicronJet™ 600 silicone microneedle device available from NanoPass Technologies Ltd.

[0031] As used herein, the term "subject" is not particularly limited, but generally refers to a person receiving therapeutic or cosmetic treatment. In this context, a subject is preferably a person receiving cosmetic treatment. The term "subject" may be used interchangeably with the term "patient" in this document.

[0032] As used herein, the term "effective amount" refers to an amount of botulinum toxin that is generally sufficient to cause the desired change in a subject. For example, when the desired effect is a reduction in melanin, an effective amount of botulinum toxin is an amount that causes at least a significant reduction in melanin without causing significant toxicity.

[0033] The terms “a”, “an”, and “the” as used in the context of this invention, as well as similar references, should be interpreted to cover both singular and plural forms, and thus may also refer to “at least one” or “more than one”, unless otherwise stated herein or clearly contradicted by the context.

[0034] The term "comprising," and the terms "including," "containing," and any variations thereof, are intended to indicate a non-exclusive inclusion, such that a process, method, product obtained by a process, composition, or formulation that comprises, includes, or contains one or more elements may include not only those elements but also other elements not expressly listed for use in such a process, method, product obtained by a process, composition, or formulation. Furthermore, within the framework of this invention, it is intended that each of the terms "comprising," "including," "containing," and any variations thereof may be replaced by the term "composed of," or any variation thereof, which will be understood to refer to an exclusive inclusion of the indicated element.

[0035] In the context of this invention, a value without decimal places should be understood to include all values ​​with one or more decimal places, given by applying general rounding rules. For example, the value 3 includes 2.5 or 2.50 (rounded up to 3) and 3.4 or 3.49 (rounded down to 3), as well as all values ​​in between. The same applies to the endpoints of the range, i.e., the range 3 to 5 can mean, for example, 2.5-3.4 to 4.5-5.4.

[0036] In a first aspect, the present invention relates to the use of botulinum toxin for the treatment of hyperpigmentation, comprising injecting botulinum toxin into the skin of a patient affected by hyperpigmentation, wherein the botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site.

[0037] Preferably, the injection depth is 0.45 mm to 0.90 mm, more preferably 0.50 mm to 0.80 mm, even more preferably 0.55 mm to 0.70 mm, and most preferably 0.60 mm. Because it is administered by injection, no botulinum toxin is applied to the skin, and the selected injection depth results in no or minimal backflow of botulinum toxin from the injection channel. Furthermore, according to the invention, the injection depth is achieved in a controlled and reliable manner. For example, at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% of injections according to the invention have an injection depth of X ± 50 µm, where X is from 450 µm to 950 µm. Preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and even more preferably at least 95% of injections according to the invention have an injection depth of X ± 50 µm, where X is from 550 µm to 750 µm. Furthermore, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and even more preferably at least 95% of the injections according to the invention have an injection depth of X ± 50 µm, wherein X is 600 µm to 650 µm.

[0038] The volume of each injection site is preferably 15 μl to 30 μl or 10 μl to 25 μl, more preferably 15 μl to 25 μl, and most preferably 20 μl to 25 μl. The dose at each injection site is preferably 0.50 U to 10.0 U, more preferably 1.0 U to 8.0 U, even more preferably 1.5 U to 6 U, and even more preferably 2.0 U to 4.0 U.

[0039] According to the present invention, the distance between injection sites is preferably at least 5 mm, more preferably 5 mm to 20 mm, for example 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, and 20 mm. Even more preferably, the distance between injection sites is 5 mm to 15 mm, even more preferably 10 mm to 20 mm, and most preferably 10 mm to 15 mm. Furthermore, the injection density, i.e., the number of injections per treated area, is typically per 4 cm². 2 1 to 16 injections. Preferably, the number of injections per 4 cm² of treated area. 2 1.5 to 8 injections, preferably every 4 cm 2 Two to four injections.

[0040] In the context of this invention, the hyperpigmentation condition to be treated is preferably selected from melasma, freckles, solar lentigines, café-au-lait spots, post-inflammatory hyperpigmentation (PIH), nevi, and hyperpigmented nevi. Melasma is the preferred hyperpigmentation condition to be treated herein.

[0041] These hyperpigmentation conditions are cosmetic conditions, meaning they affect a subject's appearance rather than their health and do not require any therapeutic treatment. However, because cosmetic conditions refer to aesthetic features of the body, they can lead to dissatisfaction with one's appearance and affect self-esteem and quality of life. Therefore, individuals may choose to seek cosmetic treatments to improve their appearance and enhance their self-confidence. Therefore, the use of botulinum toxin in treating hyperpigmentation conditions according to this invention is purely for cosmetic purposes, i.e., for improving or enhancing physical appearance, and not for therapeutic reasons, i.e., for treating or preventing medical conditions.

[0042] As used in this article, the term "melasma" refers to acquired hyperpigmentation occurring in sun-exposed areas. It is a type of hyperpigmentation characterized by dark patches on the face, particularly the forehead, cheeks, and upper lip, occasionally on the neck, and rarely on the forearms. It is more common in women and is often associated with hormonal changes, such as those occurring during pregnancy or when using birth control pills.

[0043] As used in this article, the term "freckles" (also known as "spots") refers to small, flat, dark (usually brown) spots that are often hereditary and typically appear on the face, chest, and arms. Freckles are more common in people with fair skin. They are caused by increased melanin production due to sun exposure.

[0044] As used in this article, the term "solar nevus" (also known as "age spots" or "liver spots") refers to flat, brown spots that appear on the skin due to aging and sun exposure. They are most common on areas of skin that are frequently exposed to sunlight, such as the face, hands, and arms.

[0045] As used herein, the term "café-au-lait spots" refers to a skin pigmentation condition characterized by flat, light to dark brown spots of oval or irregular shape on the skin. These spots may be present at birth or develop later in childhood.

[0046] As used herein, the term "post-inflammatory hyperpigmentation" (PIH) refers to a condition that occurs after skin irritation or injury. It is characterized by the appearance of dark brown, black, or reddish-brown patches or spots on the skin. In response to skin irritation or injury, the body produces excessive melanin, causing the affected area to darken. Common causes of PIH include acne, eczema, psoriasis, insect bites, burns, cuts, and surgical scars. While PIH is not a serious medical condition and does not pose any health risk, it is often a significant cosmetic concern for affected individuals.

[0047] As used in this article, the term "mole" refers to a pigmented area of ​​skin that is typically round or oval, and can vary in size, color, and texture. Moles are usually brown or black and can be flat or raised. Moles can appear at any age, but most develop during childhood or adolescence. Specific examples of moles include congenital nevi and acquired nevi (which are usually caused by sun exposure).

[0048] As used herein, the term "hyperpigmented nevus" refers to an area of ​​skin pigmentation that may be present at birth or acquired later in life. Examples include congenital melanocytic nevus, acquired melanocytic nevus, halo nevus, Spitz nevus, blue nevus, Becker nevus, and brown-blue nevus.

[0049] As used herein, the term "therapeutic condition" refers to a condition or disease that affects the health of a subject and is treated through a therapy. In the context of this invention, the term "therapy" refers to the treatment of a condition or disease that restores health by curing the disease, or to preventative treatment aimed at maintaining health by preventing adverse reactions that would otherwise occur, or to relieving symptoms of pain and suffering. In other words, the terms "therapeutic" or "treatment" refer to restoring the human body from a pathological state to its original (healthy) state, or preventing a pathological state, while cosmetic treatments begin with a general state of normalcy or a state of health in a narrow sense.

[0050] The botulinum toxin used in this invention is not particularly limited and includes any serotype of botulinum toxin (BoNT / AH) in a form without complexing proteins or in a complex containing complexing proteins. Preferably, the botulinum toxin is serotype A or B (BoNT / A, BoNT / B), particularly preferably serotype A (BoNTA). More preferably, the botulinum toxin is serotype A, even more preferably serotype A1 (BoNT / A1), and most preferably BoNT / A1 produced by the Clostridium Hall strain. Preferably, the botulinum toxin is type A and in a form without complexing proteins or in a complex containing complexing proteins, more preferably, type A botulinum toxin in a form without complexing proteins.

[0051] As used herein, the term “botulinum toxin” (“BT”) is used synonymously with the term “botulinum neurotoxin” (“BoNT”). These terms are intended to refer to the toxin form without chelating proteins, i.e., the (active) neurotoxic polypeptide that ultimately inhibits the release of acetylcholine (also referred to herein as “pure botulinum neurotoxin,” “neurotoxic component,” “150 kDa neurotoxin,” or “botulinum neurotoxin (150 kD)”) and the toxin form containing chelating proteins (i.e., a complex of the neurotoxic component and the chelating protein). A botulinum toxin complex refers to a high-molecular-weight complex of the neurotoxic component and a group of chelating proteins (NAPs), including 900 kDa, 500 kDa, and 300 kDa type A botulinum toxin complexes. The chelating proteins are nontoxic non-hemagglutinin (NTNHA), which are distinct hemagglutinins (HAs) in AD serotype strains. For example, the 900 kDa complex is contained in onabotulinumtoxin A (Botox ® / Vistabel ® , Allergan, Inc., Irvine, CA, USA), and abobotulinumtoxin A (Dysport ® Azzalure ® Ipsen, Paris, France) Alluzience ® (lpsen / Galderma) and Innotox ® (Medytox) contains a toxin complex as an active agent. Preferably, the botulinum toxin is contained in Xeomin. ® Pure botulinum neurotoxin or Xeomin ® Or included in Botox ® Or Dysport ® The toxin complex in it, or Botox ® Or Dysport ® .

[0052] In this invention, the botulinum toxin can be a natural neurotoxin obtainable from the bacterium *Clostridium botulinum* or any other botulinum toxin, such as botulinum toxins obtainable from alternative sources, including recombinant technologies and genetic or chemical modifications. Chimeric or genetically modified botulinum toxins, i.e., botulinum toxins containing mutations including substitutions, deletions, and insertions, are also included in the terms "botulinum toxin," "neurotoxic component," etc. Preferably, the mutation does not impair any biological activity of the botulinum toxin. However, it is also contemplated to use mutations to modulate the biological activity of the botulinum toxin. Botulinum toxins containing chemically modified amino acids, such as one or more glycosylated, acetylated, or otherwise modified amino acids, may also be beneficial for toxin uptake or stability. Particularly preferred is the esterification of the neurotoxic component.

[0053] In the context of this invention, dosage is expressed in biological units because the botulinum toxin used may contain, for example, varying percentages of inactive toxin that contribute to the total protein load but not to efficacy. In the context of this invention, the biological potency of the botulinum toxin is determined using a mouse bioassay (MBA). The MBA determines the mean lethal dose (LD50) of the toxin / neurotoxin after intraperitoneal injection in mice. 50 The LD50 is defined as the dose of toxin / neurotoxin that kills 50% of a group of mice. Based on this, as used herein, one unit (U) of toxin / neurotoxin is defined as a mouse LD50 (1.0 LD50 = 1.0 U). The LD50 mouse bioassay is the gold standard among various biological, chemical, or immunological assays and activity determinations of botulinum toxin, and is known to those skilled in the art (see, e.g., Pearce, LB; Borodic, GE; First, ER; MacCallum, RD Measurement of botulinum toxin activity: Evaluation of the lethality assay. Toxicol. Appl. Pharmacol. 1994, 128, 69-77).

[0054] Another useful method for determining the biological activity (biological potency) of botulinum neurotoxin is cell-based potency assays, as disclosed, for example, in WO2009 / 114748, WO 2013 / 049508, or WO 2014 / 207109. The activity results obtained using such cell-based assays correspond to the activity values ​​obtained from mouse intraperitoneal (ip) LD50 assays, since these values ​​are calibrated using an LD50 reference standard.

[0055] Because LD50 testing varies among manufacturers of commercial botulinum toxin formulations, the unit potency declared by manufacturers for their commercial botulinum toxin formulations is proprietary and not easily comparable. Therefore, within the framework of this invention, the conversion rates provided below are used to establish incobotulinumtoxin A (“INCO”; Xeomin ® Bocouture ® Serum type A botulinum toxin, without complexing proteins; Merz Pharmaceuticals GmbH), onabotulinumtoxin A ("ONA"; Botox) ® Vistabel®; botulinum toxin complex of serotype A; Allergan Inc., abobotulinumtoxin A (“ABO”; Dysport) ® Azzalure ® ; serotype A botulinum toxin complex; Medicis Pharmaceutical Corp., Galderma Lab.), rimabotulinumtoxin B ("RIM"; Myobloc ® NeuroBloc ® ; serum type B botulinum toxin; Solstice Neurosciences Inc.) and PurTox ® The relative potency of (“TBD”; serotype A botulinum toxin; Mentor Worldwide LLC). In this document, the conversion ratio of ONA to INCO is 1:1. The conversion ratio of ONA / INCO:ABO is 1:2.5. The conversion ratio of ONA / INCO:RIM is 1:50, and the conversion ratio of ONA / INCO:TBD is 1:1.5. Furthermore, preferably, in the context of this invention, 1 U of INCO (Xeomin) ® ) and 1U of onabotulinumtoxinA ("ONA"; Botox ® This should be considered as corresponding to a mouse LD50 (1.0 LD50) or 1 U as measured by the method described above.

[0056] Typically, the botulinum toxin used in this invention is in the form of a liquid composition. As is known in the art, the liquid composition can be formulated using various techniques depending on the desired application. It can be provided as a ready-to-use liquid formulation or as a lyophilized powder typically reconstituted in physiological saline prior to use. Preferably, the botulinum toxin used in this invention is in the form of an aqueous solution, more preferably a saline solution or physiological saline solution, and most preferably a phosphate-buffered saline solution. The aqueous solution may additionally contain one or more pharmaceutically acceptable substances. Suitable pharmaceutically acceptable substances include those well known in the art, see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

[0057] Specifically, aqueous botulinum toxin solutions or compositions may contain other carriers or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc. Therefore, aqueous botulinum toxin compositions may contain glycerol, protein stabilizers (such as human serum albumin (HSA)) or non-protein stabilizers (such as polyvinylpyrrolidone (PVP)), hyaluronic acid, or free amino acids (such as methionine or histidine). Suitable non-protein stabilizers are disclosed in WO 2005 / 007185 or WO 2006 / 020208. Furthermore, it may be free of amino acids and / or free of stabilizing peptides (e.g., composed of 5 to 50 amino acids, 10 to 40 amino acids, or 15 to 30 amino acids). Botulinum toxin compositions may also contain nonionic or ionic surfactants, such as polysorbate or poloxamer. Suitable formulations for use in formulations containing HSA-stabilized botulinum toxin according to the invention are disclosed, for example, in US 8,398,998 B2.

[0058] Preferably, the botulinum toxin used in this invention is present in the form of an aqueous solution containing sodium chloride (NaCl), preferably in the form of a physiological saline solution (i.e., a solution containing a physiological concentration of sodium chloride, such as about 9 g / L NaCl), which also contains one or more of the following (i) to (ix): (i) no other excipients (except NaCl), (ii) human serum albumin (HSA) and sugars, especially monosaccharides or disaccharides, (iii) human serum albumin (HSA) and lactose, (iv) human serum albumin (HSA) and sucrose, (v) monosaccharides and / or disaccharides (e.g., lactose and / or sucrose), (vi) no buffer solution, (vii) no single amino acid, (viii) no human serum albumin (HSA), sodium chloride and lactose or no HSA, sodium chloride and sucrose, or (ix) no HSA and sodium chloride, or any combination of (i) to (ix).

[0059] Particularly preferred is the botulinum toxin in the form of an aqueous formulation comprising botulinum toxin, sodium chloride, and human serum albumin; or in the form of an aqueous formulation comprising botulinum toxin, sodium chloride, human serum albumin, and lactose; or in the form of an aqueous formulation comprising botulinum toxin, sodium chloride, human serum albumin, and sucrose. Another particularly preferred aqueous formulation comprises botulinum toxin, sodium chloride, human serum albumin, and histidine. The botulinum toxin may be as defined above.

[0060] In a second aspect, the present invention relates to a method for treating hyperpigmentation, comprising injecting botulinum toxin into the skin of a patient affected by hyperpigmentation, wherein the botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site.

[0061] There are no particular limitations on hyperpigmentation conditions, including those defined above.

[0062] The method according to the second aspect of the invention is closely related to the use according to the first aspect of the invention. Therefore, all definitions, interpretations, and advantages given herein with respect to the use according to the first aspect also apply to the method according to the second aspect. In particular, the injection and administration protocols used in conjunction with the method according to the second aspect of the invention are the same as those disclosed above with respect to the use according to the first aspect of the invention.

[0063] In this invention, botulinum toxin is preferably type A. Furthermore, botulinum toxin can be in a form without complexing proteins or in a complex containing complexing proteins. Preferably, the botulinum toxin is type A and is in a form without complexing proteins, or in an alternative preferred embodiment, the botulinum toxin is type A and is in a complex containing complexing proteins.

[0064] In the third and fourth aspects, the present invention relates to the use of botulinum toxin in the treatment of rosacea and methods for treating rosacea, respectively. The use and methods involve injecting botulinum toxin into the skin of a patient affected by rosacea, wherein the botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site.

[0065] As used in this article, the term "rosacea" refers to a chronic skin condition that causes redness, flushing, and visible blood vessels on the face, particularly the cheeks, chin, nose, and central forehead. This is not a life-threatening condition, but rather a cosmetic concern that can cause significant social and psychological distress. In other words, rosacea is considered a cosmetic condition or state, not a therapeutic one. The mechanism of action of botulinum toxin in treating rosacea is not fully understood. One hypothesis is the inhibition of the release of neuropeptides associated with vasodilation and inflammation, such as substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), and acetylcholine (ACh), from presynaptic vesicles. Rosacea is considered a cosmetic condition, meaning one that affects a subject's appearance rather than their health, as explained above regarding hyperpigmentation.

[0066] The uses and methods according to the third and fourth aspects of the present invention are closely related to the uses according to the first aspect of the present invention and the methods according to the second aspect of the present invention, and differ from them only in that the condition to be treated is rosacea rather than hyperpigmentation. Therefore, all definitions, interpretations, and advantages given herein regarding the uses according to the first and second aspects are equally applicable to the uses and methods according to the third and fourth aspects of the present invention. In particular, any disclosure of the above-described injection and administration protocols related to the first and second aspects of the present invention should be understood as also representing the disclosures of the third and fourth aspects of the present invention.

[0067] Preferred administration methods of the first to fourth aspects of the present invention are described below.

[0068] In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 10 μl to 30 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 30 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 30 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 25 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 25 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 20 μl to 25 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 20 μl to 25 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site.

[0069] In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, and a dose of 0.50 U to 10.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 10 μl to 30 μl per injection site, and a dose of 0.50 U to 10.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 30 μl per injection site, and a dose of 0.50 U to 10.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 30 μl per injection site, and a dose of 0.50 U to 10.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 25 μl per injection site, and a dose of 0.50 U to 10.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 25 μl per injection site, and a dose of 0.50 U to 10.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 20 μl to 25 μl per injection site, and a dose of 0.50 U to 10.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 20 μl to 25 μl per injection site, and a dose of 0.50 U to 10.0 U per injection site.

[0070] In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, and a dose of 1.0 U to 6.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 10 μl to 30 μl per injection site, and a dose of 1.0 U to 6.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 30 μl per injection site, and a dose of 1.0 U to 6.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 30 μl per injection site, and a dose of 1.0 U to 6.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 25 μl per injection site, and a dose of 1.0 U to 6.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 25 μl per injection site, and a dose of 1.0 U to 6.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 20 μl to 25 μl per injection site, and a dose of 1.0 U to 6.0 U per injection site. In a preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 20 μl to 25 μl per injection site, and a dose of 1.0 U to 6.0 U per injection site.

[0071] In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, a dose of 0.25 U to 12.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 10 μl to 30 μl per injection site, a dose of 0.25 U to 12.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 30 μl per injection site, a dose of 0.25 U to 12.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 30 μl per injection site, a dose of 0.25 U to 12.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 25 μl per injection site, a dose of 0.25 U to 12.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 25 μl per injection site, a dose of 0.25 U to 12.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 20 μl to 25 μl per injection site, a dose of 0.25 U to 12.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm.In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 20 μl to 25 μl per injection site, a dose of 0.25 U to 12.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm.

[0072] In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, a dose of 0.50 U to 10.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 10 μl to 30 μl per injection site, a dose of 0.50 U to 10.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 30 μl per injection site, a dose of 0.50 U to 10.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 30 μl per injection site, a dose of 0.50 U to 10.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 25 μl per injection site, a dose of 0.50 U to 10.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 25 μl per injection site, a dose of 0.50 U to 10.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 20 μl to 25 μl per injection site, a dose of 0.50 U to 10.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm.In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 20 μl to 25 μl per injection site, a dose of 0.50 U to 10.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm.

[0073] In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, a dose of 1.0 U to 6.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 10 μl to 30 μl per injection site, a dose of 1.0 U to 6.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 30 μl per injection site, a dose of 1.0 U to 6.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 30 μl per injection site, a dose of 1.0 U to 6.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 25 μl per injection site, a dose of 1.0 U to 6.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 25 μl per injection site, a dose of 1.0 U to 6.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm. In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 20 μl to 25 μl per injection site, a dose of 1.0 U to 6.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm.In another preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 20 μl to 25 μl per injection site, a dose of 1.0 U to 6.0 U per injection site, and an injection distance of 5 mm to 20 mm, preferably 10 mm to 20 mm, more preferably 15 mm to 20 mm.

[0074] In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, a dose of 0.25 U to 1.0 U per injection site, and an injection distance of 5 mm to 10 mm. In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 10 μl to 30 μl per injection site, a dose of 0.25 U to 1.0 U per injection site, and an injection distance of 5 mm to 10 mm. In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 30 μl per injection site, a dose of 0.25 U to 1.0 U per injection site, and an injection distance of 5 mm to 10 mm. In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 30 μl per injection site, a dose of 0.25 U to 1.0 U per injection site, and an injection distance of 5 mm to 10 mm. In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 25 μl per injection site, a dose of 0.25 U to 1.0 U per injection site, and an injection distance of 5 mm to 10 mm. In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 25 μl per injection site, a dose of 0.25 U to 1.0 U per injection site, and an injection distance of 5 mm to 10 mm. In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 20 μl to 25 μl per injection site, a dose of 0.25 U to 1.0 U per injection site, and an injection distance of 5 mm to 10 mm. In another particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 20 μl to 25 μl per injection site, a dose of 0.25 U to 1.0 U per injection site, and an injection distance of 5 mm to 10 mm.

[0075] In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, a dose of 1.0 U to 8.0 U per injection site, preferably 2.0 U to 7.0 U, more preferably 3.0 U to 6.0 U, and an injection distance of 10 mm to 20 mm or 10 mm to 15 mm. In another particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 10 μl to 30 μl per injection site, a dose of 1.0 U to 6.0 U per injection site, preferably 2.0 U to 7.0 U, more preferably 3.0 U to 6.0 U, and an injection distance of 10 mm to 20 mm or 10 mm to 15 mm. In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 30 μl per injection site, a dose of 1.0 U to 8.0 U per injection site, preferably 2.0 U to 7.0 U, more preferably 3.0 U to 6.0 U, and an injection distance of 10 mm to 20 mm or 10 mm to 15 mm. In another particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 30 μl per injection site, a dose of 1.0 U to 8.0 U per injection site, preferably 2.0 U to 7.0 U, more preferably 3.0 U to 6.0 U, and an injection distance of 10 mm to 20 mm or 10 mm to 15 mm. In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 25 μl per injection site, a dose of 1.0 U to 8.0 U per injection site, preferably 2.0 U to 7.0 U, more preferably 3.0 U to 6.0 U, and an injection distance of 10 mm to 20 mm or 10 mm to 15 mm. In another particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 25 μl per injection site, a dose of 1.0 U to 8.0 U per injection site, preferably 2.0 U to 7.0 U, more preferably 3.0 U to 6.0 U, and an injection distance of 10 mm to 20 mm or 10 mm to 15 mm.In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 20 μl to 25 μl per injection site, a dose of 1.0 U to 8.0 U per injection site, preferably 2.0 U to 7.0 U, more preferably 3.0 U to 6.0 U, and an injection distance of 10 mm to 20 mm or 10 mm to 15 mm. In another particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 20 μl to 25 μl per injection site, a dose of 1.0 U to 8.0 U per injection site, preferably 2.0 U to 7.0 U, more preferably 3.0 U to 6.0 U, and an injection distance of 10 mm to 20 mm or 10 mm to 15 mm.

[0076] In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, a dose of 8.0 U to 12.0 U per injection site, and an injection distance of 15 mm to 20 mm. In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 10 μl to 30 μl per injection site, a dose of 8.0 U to 12.0 U per injection site, and an injection distance of 15 mm to 20 mm. In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 30 μl per injection site, a dose of 8.0 U to 12.0 U per injection site, and an injection distance of 15 mm to 20 mm. In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 30 μl per injection site, a dose of 8.0 U to 12.0 U per injection site, and an injection distance of 15 mm to 20 mm. In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 15 μl to 25 μl per injection site, a dose of 8.0 U to 12.0 U per injection site, and an injection distance of 15 mm to 20 mm. In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 15 μl to 25 μl per injection site, a dose of 8.0 U to 12.0 U per injection site, and an injection distance of 15 mm to 20 mm. In a particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 20 μl to 25 μl per injection site, a dose of 8.0 U to 12.0 U per injection site, and an injection distance of 15 mm to 20 mm. In another particularly preferred embodiment, botulinum toxin is injected intradermally at an injection depth of 0.50 mm to 0.80 mm, a volume of 20 μl to 25 μl per injection site, a dose of 8.0 U to 12.0 U per injection site, and an injection distance of 15 mm to 20 mm. Example Example 1 Distribution of fluorescently labeled BoNT in ex vivo human skin models

[0077] The distribution of fluorescently labeled inactive BoNTs was determined using an ex vivo human skin model. Inactive BoNTs (triple mutations in the light chain amino acid sequence) were labeled with Alexa Fluor 647 and dissolved in PBS buffer (pH 7.4) at a concentration of 0.58 mg / mL. The skin model consisted of ex vivo human abdominal skin. Subcutaneous tissue was almost completely removed. The trimmed skin was clamped under tension and incubated at 37°C. The model was kept hydrated by a cloth soaked in PBS buffer (pH 7.4) in contact with the reticular dermis. A low volume (0.01 mL) of BoNT solution (~10,000 units) was intradermally injected into a human skin model using (b) a MicronJet™ 600 silicone microneedle device (NanoPass Technologies Ltd.; hereinafter “MicronJet600”), (c) a silicone microneedle for intradermal delivery, designated Bella-mu 1.4 mm (U-Needle BV; hereinafter “U-needle”), and (d) a standard steel 30G x ½” hypodermal injection needle (hereinafter “G30½ needle”). After 4 hours of incubation, the distribution (diffusion and diffusion) of fluorescently labeled BoNT was determined by measuring the fluorescence signal of frozen sections of the human skin model.

[0078] like Figure 1 As shown, the MicronJet600 resulted in a BoNT concentration halo at a depth of approximately 1.0 mm, with the highest BoNT concentration at shallow injection depths. In contrast, the U-shaped needle exhibited the highest BoNT concentration at a depth of approximately 1.4 mm (see [link to data]). Figure 2 And therefore, it acts on the mid-dermis region. As expected, the standard G30½ needle results in low injection accuracy with random injection depths (see...). Figure 3 (Two exemplary injections are shown). In other words, injections using a standard G30½ needle are applied randomly throughout the dermis.

[0079] In addition, such as from Figure 1 As can be seen, surprisingly, MicronJet600 injections are redirected to the upper dermal layers, specifically the upper dermal region, which includes the papillary region (the thinner top layer of the dermis) and the basal layer (the deepest layer of the epidermis). This allows for specific targeting of target cells, such as melanocytes involved in pigmentation, located at the dermal-epidermal junction (the interface between the basal epidermis and the papillary dermis). Furthermore, unlike injections using a standard G30½ needle, the BoNT distribution is consistent across all injections using MicronJet600, providing high injection accuracy and reliability.

[0080] Further conclusions from dye experiments suggest that the specific targeting effect of Micronjet is limited to lower injection volumes, such as approximately 0.05 mL. More specifically, toluidine blue, a blue dye, was used as a substitute for the BoNT solution, and MicronJet 600 was injected into the dermis of the aforementioned ex vivo human skin model at different volumes. Vertical diffusion of the dye was determined immediately after injection by gross cryosectioning of the human skin model. Using an injection volume of 0.0125 ml, a maximum vertical diffusion of 1.5 mm was observed. With a volume of 0.025 ml, the maximum vertical diffusion increased to 1.9 mm. Using an even higher volume of 0.050 ml, a maximum vertical diffusion of 2.0 mm was observed, meaning that the entire dermis (with the maximum vertical dimension / length of the dermis approximately 2 mm in certain cases) was filled with the solution from the upper dermis to the lower dermis. Example 2 Dosing and injection regimens for cosmetic treatment of melasma

[0081] Botulinum toxin solution is administered using the exemplary administration regimens according to the present invention shown in Table 1. Table 1. Dosing regimen

[0082] Generally, the injection dose increases with the injection distance. For example, if the dose at each injection site is 0.25 U to 2 U, the injection distance can be, for example, 5 mm. If the dose at each injection site is 2 U to 8 U, the injection distance can be, for example, 10 mm. If the dose at each injection site is 4 U to 16 U, the injection distance can be, for example, 20 mm.

[0083] The injected botulinum toxin is typically a 0.9% saline solution. A microneedle device is used to deliver the botulinum toxin into the upper dermis. For example, a syringe equipped with a MicronJet600 microneedle device can be used. This device can be used for multiple injections without needing to be changed after each injection.

[0084] Regarding the injection protocol, a multi-injection protocol is preferred for botulinum toxin. The number of injections depends on the size of the area to be treated (e.g., melasma). Typically, the injections are arranged in a grid pattern, such as a square grid. Based on experimental results using fluorescently labeled BoNTs, the lateral distribution of BoNTs was approximately 5.4 ± 0.3 mm after 4 hours. Therefore, depending on the dosage at each injection site, the appropriate distance between injection sites is approximately 5 mm or greater.

Claims

1. Use of botulinum toxin for the treatment of hyperpigmentation, including injecting botulinum toxin into the skin of a patient affected by hyperpigmentation, wherein the botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site.

2. The use according to claim 1, wherein the injection depth is 0.45 mm to 0.90 mm or 0.50 mm to 0.80 mm.

3. The use according to claim 1 or 2, wherein the volume of each injection site is 15 μl to 30 μl or 20 μl to 25 μl.

4. The use according to any one of claims 1 to 3, wherein the dose at each injection site is 0.5 U to 8.0 U or 1.0 U to 4.0 U.

5. The use according to any one of claims 1 to 4, wherein the distance between injection sites is 5 mm to 20 mm, 5 mm to 15 mm, 10 mm to 20 mm, or 10 mm to 15 mm.

6. The use according to any one of claims 1 to 5, wherein the hyperpigmentation condition is selected from melasma, freckles, solar lentigines, café-au-lait spots, post-inflammatory hyperpigmentation (PIH), nevi, and hyperpigmented nevi.

7. The use according to any one of claims 1 to 6, wherein the hyperpigmentation condition is melasma.

8. The use according to any one of claims 1 to 7, wherein the botulinum toxin is (i) type A, (ii) a form without complexing protein or a complex containing complexing protein, (iii) type A and a form without complexing protein or a complex containing complexing protein, or (iv) type A and a form without complexing protein.

9. Use of botulinum toxin for the treatment of rosacea, comprising injecting botulinum toxin into the skin of a patient affected by rosacea, wherein the botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site, preferably the use described herein as defined in any one of claims 2 to 8.

10. A method for treating hyperpigmentation, comprising injecting botulinum toxin into the skin of a patient affected by hyperpigmentation, wherein the botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site.

11. The method of claim 10, wherein the injection depth is as defined in claim 2 and / or the volume of each injection is as defined in claim 3.

12. The method of claim 10 or 11, wherein the dose at each injection site is as defined in claim 4 and / or the distance between injection sites is as defined in claim 5.

13. The method according to any one of claims 10 to 12, wherein the hyperpigmentation condition is selected from melasma, freckles, solar lentigines, café-au-lait spots, post-inflammatory hyperpigmentation (PIH), nevi, and hyperpigmented nevi.

14. The method according to any one of claims 10 to 13, wherein the botulinum toxin is (i) type A, (ii) a form without complexing proteins or a complex containing complexing proteins, (iii) type A and a form without complexing proteins or a complex containing complexing proteins, or (iv) type A and a form without complexing proteins.

15. A method for treating rosacea, comprising injecting botulinum toxin into the skin of a patient affected by rosacea, wherein the botulinum toxin is injected intradermally at an injection depth of 0.40 mm to 1.00 mm, a volume of 10 μl to 30 μl per injection site, and a dose of 0.25 U to 12.0 U per injection site, preferably wherein the method is as defined in claim 11 or 12.

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