WO2016162489A1

WO2016162489A1 - Treatment of nail disorder with an indigo naturalis or indigo-producing plant extract

Info

Publication number: WO2016162489A1
Application number: PCT/EP2016/057773
Authority: WO
Inventors: Philippe Andres; Laurent Chantalat; Yin-Ku Lin
Original assignee: Galderma Sa
Priority date: 2015-04-09
Filing date: 2016-04-08
Publication date: 2016-10-13

Abstract

A pharmaceutical or cosmetic composition for use in the treatment of a nail disorder comprising an Indigo Naturalis or Indigo-producing plant extract, and a method of treating a nail disorder comprising administering a therapeutically effective amount of an Indigo Naturalis or Indigo-producing plant extract to subjects in need thereof are described.

Description

TREATMENT OF NAIL DISORDER WITH AN INDIGO NATURALIS OR INDIGO-PRODUCING PLANT EXTRACT

TECHNICAL FIELD

The present invention relates to a use of an Indigo Naturalis or Indigo-producing plant extract, particular in a use for treating a nail disorder, including nail psoriasis or nail dystrophy, such as brittle nails.

BACKGROUND

Nail Dystrophy is a condition that impedes the healthy, normal growth of the fingernail plate. It refers to poor nail formation, usually as the result of external factors (trauma, excessive moisture or chemicals) or specific diseases (including but not limited to infections, genodermatoses...), and may cause nail brittleness or fragility, nail splitting, or peeling nails, or partial destruction of the nail plate. When a trauma or an infection involves the distal part of the nail, it may result in the nail plate splitting from the nail bed, i.e., a process called onycholysis.

Nail psoriasis is common, occurring in up to half of patients with psoriasis and in 90% of patients with psoriatic arthritis. Left untreated, it may progress to debilitating nail disease, which leads to significant functional impairment. The most common clinical signs of nail psoriasis are nail plate pitting and onycholysis. Other classical signs include oil drop discoloration, subungual hyperkeratosis, and splinter hemorrhages. Severe psoriatic nail disease can lead to functional and social impairments if left untreated.

It is always desirable to develop medications for treating nail dystrophy, onycholysis, or nail psoriasis. SUMMARY

The present invention relates to an Indigo Naturalis or Indigo-producing plant extract that is effective for treating a nail disorder selected in the group consisting of nail dystrophy, onycholysis, and nail psoriasis.

In one aspect, the present invention provides a pharmaceutical or cosmetic composition for use in the treatment of nail dystrophy or nail psoriasis, said composition comprising an Indigo Naturalis or Indigo-producing plant extract.

In another aspect, the present invention provides pharmaceutical or cosmetic composition for use in the treatment of onycholysis, said composition comprising Indigo Naturalis or Indigo-producing plant extract.

In another aspect, the present invention provides use of an Indigo Naturalis or Indigo-producing plant extract in the preparation of a medicament for treating a nail disorder selected in the group consisting of nail dystrophy, onycholysis, and nail psoriasis.

In another aspect, the present invention provides an Indigo Naturalis or Indigo-producing plant extract for treating a nail disorder selected in the group consisting of nail dystrophy, onycholysis, and nail psoriasis.

In further aspect, the present invention provides a method of treating a nail disorder selected in the group consisting of nail dystrophy, onycholysis, and nail psoriasis, comprising administering a therapeutically effective amount of an Indigo Naturalis or Indigo-producing plant extract to a subject (e.g., human) in need thereof.

In an aspect, the present invention provides a method of the treatment of a nail disorder selected in the group consisting of nail dystrophy, onycholysis, and nail psoriasis, comprising administering topically or orally an effective amount of an Indigo Naturalis or Indigo-producing plant extract to a subject in need thereof.

In some embodiments of the invention, Indigo Naturalis or Indigo-producing plant extract is administered topically at a dose of 0.001-0.1 mg to a nail.

In some embodiments of the invention, Indigo Naturalis or Indigo-producing plant extract is administered topically at a dose of 0.005-0.05mg to a nail. In some embodiments of the invention, Indigo Naturalis or Indigo-producing plant extract is administered once or twice daily.

The Indigo Naturalis extract or Indigo-producing plant extract includes any extract obtained from an Indigo Naturalis or Indigo-producing or Indigo-bearing plant as starting material.

DESCRIPTION OF DRAWINGS

Figure 1(a): Clinical appearance of the nails of a patient having Candida albicans infection and chronic onycholysis with disappearing nail bed prior to treatment.

Figure 1(b): Marked improvement in Candida albicans infection and chronic onycholysis with disappearing nail bed after four (4) months of treatment with Lindioil.

Figure 2: Trial profile. Lindioil= Indigo Naturalis extract in oil; R = right hand; L = left hand

Figure 3: Clinical improvement on the appearance of nail psoriasis before (top) and after (bottom) the Lindioil treatment

Figure 4: Fig.4a SHNAPSI scores in the Lindioil group and the Calcipotriol group - Fig. 4b MTNAPSI scores in the Lindioil group and the Calcipotriol group

DETAILED DESCRIPTION

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art.

Indigo Naturalis, a traditional Chinese medicine (TCM) also known as Qingdai, is obtained from one or more plants including Indigofera tinctoria L., Baphicacanthus cusia (Nees) Bremek (syn. Strobilanthes cusia (Nees), Persicaria tinctoria (Alton) Spach. (syn. Polygonum tinctorium Alton, P. tinctorlum Lour.) and Isatls tinctoria L. (syn. Isatls Indlgotlca Fort.), such as from the plant leaves or stems, after harvest and collection, may be processed by, for example, fermentation. Qingdai is the current name for Indigo Naturalis. Indigo Naturalis is usually a dark-blue powder. It is obtained from Indigo-bearing or Indigo-producing plants with a NaOH or KOH aqueous solution and corresponds to a mixture of around 5-15% organic compounds including alkaloids among which indigo and indirubin are present, and 85-95 % inorganic compounds such as calcium carbonate and calcium hydroxide.

An Indigo Naturalis or Indigo-producing plant extract, as used herein, refers to an extract from Indigo Naturalis or from the leaves and/or stems (or a part thereof) of one or more Indigo-bearing plant or Indigo-producing plant, where the extraction may be performed by using organic solvent(s) and/or non-organic solvent(s). The extract may include enriched ingredients (having a higher w/w percentage than that existing in Indigo Naturalis) such as tryptanthrin, isatin, indirubin, indigo, and qingdainone. The extract may be a solid, liquid, or any form in-between (e.g., semi-solid).

In a particular embodiment, the Indigo Naturalis or Indigo-producing plant extract is enriched in indirubin, for example the extract may contain indirubin in an amount of at least 65% w/w of the extract, for example, 65%-90% w/w of the extract. The extract may further contain indigo in an amount of 0.1%- 15% w/w of the extract. The extract may also contain tryptanthrin and/or qingdainone each in an amount of 0.1-5% w/w.

One example of the Indigo Naturalis or Indigo-producing plant extract is an ethyl acetate extract (EA-extract), which may be prepared as illustrated by Example 2 in this application. The content of each ingredient in the extract may vary. As an example, the extract may contain indirubin in an amount of at least 65% w/w of the extract, for example, 65%-90% w/w of the extract. The extract may further contain indigo in an amount of 0.1%- 15% w/w of the extract. The extract may also contain tryptanthrin and/or qingdainone each in an amount of 0.1-5% w/w.

A further example of the Indigo Naturalis extract is an oil extract, particularly an olive oil extract. An oil extract can be prepared by the method disclosed in the patent US 8,784,905. More specifically, the oil extract of Indigo Naturalis is an oil-extracted product of Indigo Naturalis which is obtained by a process comprising extracting Indigo Natural is powder with an oil under heating, optionally followed by a refining treatment by filtration. More preferably, in said process, the oil-extracted product is obtained after the refining treatment has a decreased indigo content. In said process, extracting Indigo Natural is powder is more particularly conducted at an elevated temperature not higher than 155° C, and preferably conducted at a temperature ranging from 100° C. to 155° C. The oil used in said process is preferably selected from the group consisting of vegetable oils, animal oils, mineral oils, and combinations thereof. More preferably, the oil is a vegetable oil and can be selected from the group consisting of olive oil. cottonseed oil, sesame oil, sunflower seed oil, peanut oil, wheat germ oil, soybean oil, jojoba oil, evening primrose oil, coconut oil. palm oil. sweet almond oil, aloe oil, apricot kernel oil. avocado oil, borage oil, hemp seed oil, macadamia nut oil, rose hip oil, pecan oil, hazelnut oil. sasanqua oil, rice bran oil, shea butter, corn oil, camellia oil, grape seed oil, canola oil, castor oil, and combinations thereof. The content of each ingredient in the extract may vary. As an example, the extract may contain indirubin in an amount of at least 65% w/w of the total amount of extracted alkaloids, for example, 65%-90% w/w of the total amount of extracted alkaloids. The extract may further contain indigo in an amount of 0.1%- 15% w/w of the total amount of extracted alkaloids. The extract may also contain tryptanthrin and/or qingdainone each in an amount of 0.1-5% w/w of the total amount of extracted alkaloids.

Another example of Indigo Naturalis or Indigo-producing plant extract is an extract prepared by a process comprising the following steps:

a) an extraction step: extracting Indigo Naturalis or the leaves and/or stems of one or more Indigo-bearing plants or Indigo-producing plants, preferably selected from the group consisting of Indigofera tinctoria L, Baphicacanthus cusia (Nees) Bremek (syn. Strobilanthes cusia (Nees)), Persicaria tinctoria (Alton) Spach. (syn. Polygonum tlnctorium Alton, P. tlnctorlum Lour.) and Isatls tinctoria L. (syn. Isatls indigotica Fort.) and/or Strobilanthes Formosanus, with a first polar solvent or moderately polar solvent to obtain a mixture of extraction; b) a filtration step: filtering the mixture of extraction to obtain a filtrate;

c) a concentration step: concentrating the filtrate to obtain a crude extract;

d) a washing step: washing the crude extract with a non-polar solvent, and optionally a second polar solvent, to obtain a washing mixture; and

e) a filtration step: filtering the washing mixture to obtain a refined extract optionally after a drying step, for example, according to a conventional method for drying.

In a particular embodiment, a crude extract obtained from the concentration step c) is subjected to the following procedure for at least one cycle till obtaining a refined extract: the crude extract is washed by a solvent (step d)), and filtered (step (e)) to yield a refined extract, optionally followed by a drying step. According to a specific embodiment, the washing step d) and filtration step e) are performed by only one cycle to obtain the refined extract. When more than one cycle is applied, the same or different solvents for washing can be used. Further, the crude extract can be washed with a solvent under reflux, the mixture can be cooled to room temperature and then filtered to yield a refined extract, optionally followed by a drying step.

In a preferred embodiment, two cycles are performed. Particularly, the crude extract obtained by the concentration step c) is washed in a non- polar solvent, preferably hexane (step d) and filtered (step e), optionally followed by a drying step. The hexane extract is then washed by an organic polar solvent, preferably ethanol (step d) and then filtered (step e) to obtain a refined extract, optionally followed by a drying step.

Optionally, a micronization step is performed after step e), providing thereby a refined extract having a particle size between 25 and 35 μιη, preferably of about 30 μιη.

In another preferred embodiment, when the refined extract is micronized in the last step, 99% of the obtained particles are less or equal to 30 μιη. In a preferred embodiment, a refined extract may be prepared by a process comprising the following steps consisting of: a) (i) adding an extracting solvent, a polar or moderately polar solvent (such as an alcohol or ethyl acetate), to Indigo Naturalis powder to yield a mixture; (ii) heating and stirring the mixture for a period of time (e.g., 30 min, 1 hour, 2 hours); b) (iii) filtering the heated mixture while hot to remove insoluble by-products to yield a filtrate; c) (iv) concentrating the filtrate to yield a crude extract; d) (v) adding a washing solvent (for example, water a non-polar and/or a polar solvent or a mixture thereof) to the crude extract to yield a washing mixture; (vi) heating and stirring the washing mixture for a period of time (e.g. , 30 min, 1 hour, 2 hours); e) (vii) filtering the washing mixture, for example at room temperature (e.g. 18-35°C) to collect a refined extract; optionally (viii) repeating steps (v) to (vii) until the amount of indirubin (% w/w) in the refined extract is more than 55% (w/w), preferably more than 65%> (w/w) as measured by HPLC method, and optionally (ix) drying the residue according to a conventional method (e.g., air-drying, lyophilization) to obtain a dried extract. The washing solvent in steps (v) and (viii) can be the same or different.

In a more preferred embodiment, a refined extract is prepared by a process comprising the steps of:

a) extracting Indigo Naturalis with ethanol at reflux between 2 and 8 hours,

b) filtering the mixture at a temperature not less than 65 °C to obtain a filtrate,

c) concentrating the filtrate, to obtain a crude extract, said crude extract is optionally filtered (with addition of water) in order to remove completely the solvent and the last components still present in the solvent and dried,

d) (i) washing the crude extract with hexane at a temperature not less than 50°C between 15 and 60 min,

(ii) filtering at room temperature the mixture obtained at step d) (i) to obtain a product, optionally rinsing it with ethanol and water (iii) washing the product obtained at step d) (ii) with ethanol at reflux, and

e) filtering at room temperature the washing mixture obtained at step d) and drying the resulting product at a temperature less than 80°C to obtain an extract which is optionally micronized.

In another preferred embodiment, when the refined extract is micronized in the last step, the particle size is around 99% in the range 25 to 35 μιη, preferably of about 30 μιη.

As used herein, "about" or "around" will be understood by a person of ordinary skill in the art and will vary to some extent on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, "about" or "around" will mean up to plus or minus 20%>, preferably 10% of the particular term.

The term "refined extract", as used herein, refers to a solid, semi-solid or oily extract which contains less than 10%> (w/w) of water and/or solvents used in the process for preparing the said refined extract. A refined extract is more preferably characterized by an increase amount of active ingredients, including alkaloids among which indigo, indirubin, tryptanthrin, and/or qingdainone are present, preferably enriched in indirubin, compared to Qingdai or Indigo Naturalis. More specifically, the refined extract according to the invention comprises at least 60%, or more preferably more than 65 %, (w/w) of active ingredients, including indigo, indirubin, tryptanthrin, and/or qingdainone.

The term "crude extract", as used herein, refers to a solid, semi-solid or oily extract which contains less than 15% (w/w) (e.g., 5-15%, 5-10%) of water and/or solvents used in the process for preparing the refined extract. The crude extract is less enriched in indirubin, than the refined extract as compared to Qingdai or Indigo Naturalis. The crude extract is obtained by the concentration step c) according to the invention. The concentration step is more particularly carried out by sending the filtrate to a concentrator (for instance at reduced pressure), as to remove water and/or solvents used in the process and concentrating thereby the active ingredients present in the extract, including indigo, indirubin, tryptanthrin, and/or qingdainone.

"one cycle", as used herein, refers to the two steps of the washing step d) and filtration step e) which are performed sequentially once, "two cycles", as used herein, refers to the two steps of the washing step d) and filtration step e) which are performed sequentially twice.

According to a specific embodiment, the Indigo Naturalis extract or Indigo-producing plant extract according to the invention is an oil extract as defined above or an extract of Indigo-producing plant obtained by the process as above detailed comprising steps (a)-(e), optionally including one of the above described specific embodiments.

Nail dystrophy, as used herein, should be understood a nail disease or disorder not caused or associated with nail psoriasis.

Onycholysis, as used herein, is a nail disease or disorder as separation of the nail plate from the underlying nail bed and/or lateral nail folds. It generally starts at the distal free margin of the nail plate and progresses proximally. The distal nail bed may appear to shrink, the nail bed becomes cornified and leads to the condition known as the disappearing nail bed if onycholysis is present for a long period of time. Once this happens, it is unlikely that the nail plate will reattach. The most common cause of onycholysis is trauma. Even slight trauma can cause onycholysis when it happens repetitively— for example, the daily tapping of long fingernails on a keyboard or counter. Onycholysis also can be caused by manicure tools that are pushed beneath the nail to clear dirt or smooth the nail. Too much moisture also can cause the problem.

Nail psoriasis is common, occurring in up to half of patients with psoriasis. The most common clinical signs of nail psoriasis are nail plate pitting and onycholysis. Other classical signs include oil drop discoloration, subungual hyperkeratosis, and splinter hemorrhages.

A therapeutically effective amount refers to a dose level of an Indigo Naturalis or Indigo-producing plant extract that yields a therapeutic benefit (for example, amelioration, reduction or cure of the diseases, disorder or symptoms of nail psoriasis, nail dystrophy, or onycholysis) to a patient on average.

The present invention provides a pharmaceutical composition comprising an Indigo Naturalis or Indigo-producing plant extract for treating a nail disorder selected in the group consisting of nail dystrophy, onycholysis, and nail psoriasis, preferably for treating topically said nail disorder.

The extract may be used directly without further formulation or included in a pharmaceutical or cosmetic composition that comprises the extract.

The extract may comprise indirubin in an amount of at least 65% w/w of the extract, for example, 65%-90% w/w of the extract. It may further comprise indigo in an amount of 0.1%- 15% w/w of the extract, and in another further embodiment, the extract may also comprise indigo in an amount of 0.1%- 15% w/w of the extract and tryptanthrin and/or qingdainone each in an amount of 0.1-5% w/w of the extract.

The compositions, methods or uses of the invention may be used alone (i.e., in replacement of current treatments) or in combination with current treatments to improve their efficacy.

In an embodiment, Indigo Naturalis or Indigo-producing plant extract is used as the sole active ingredient (e.g. as a single therapy). According to this embodiment, the composition preferably comprises an Indigo Naturalis or Indigo-producing plant extract as the sole active ingredient.

In another embodiment, the Indigo Naturalis or Indigo-producing plant extract can be used in combination with at least one other therapy. The pharmaceutical composition may be formulated into a suitable dosage form for topical or oral administration using technology well known to those skilled in the art. The pharmaceutical composition can additionally comprise a pharmaceutically acceptable carrier such as those widely employed in the art of drug-manufacturing. For instance, the pharmaceutically acceptable carrier may include one or more of the following agents: solvents such as olive oil, olive oil refined, cottonseed oil, sesame oil, sunflower seed oil, peanut oil, wheat germ oil, soybean oil, jojoba oil, evening primrose oil, coconut oil, palm oil, sweet almond oil, aloe oil, apricot kernel oil, avocado oil, borage oil, hemp seed oil, macadamia nut oil, rose hip oil, pecan oil, hazelnut oil, sasanqua oil, rice bran oil, shea butter, corn oil, camellia oil, grape seed oil, canola oil, castor oil, and combinations thereof, preferably olive oil refined, emulsifiers, suspending agents, decomposers, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, thickening agent such as beeswax and/or petroleum jelly, preservatives, lubricants, absorption delaying agents, liposomes, antioxidants such as butylhydroxytoluene or butylhydroxyanisole and the like. A topical formulation suitable for the pharmaceutical composition according to the present invention may be an emulsion, a gel, an ointment, a cream, a patch, an embrocation, an aerosol, a spray, a lotion, a serum, a paste, a foam, or a drop. In some embodiments, the pharmaceutical composition is formulated into an external preparation by admixing the extract according to the present invention with a base such as those that are well known and commonly used in the art.

According to a specific embodiment, the compositions, methods or uses of the invention are suitable for a topical treatment of candidiasis.

In some other embodiments, the dosage and the frequency of administration of the pharmaceutical composition may vary depending on the following factors: the severity of the disease to be treated, the route of administration, and the weight, age, physical condition and response of the subject to be treated. Additionally, the present invention also provides a cosmetic composition comprising the Indigo Naturalis or Indigo-producing plant extract. The composition according to the present invention may contain 0.001-10 mg, for example 0.01-1 mg of one or more active ingredients per lg composition.

In further or additional embodiments, the amount of the extract is in the range of about 0.001 to about 1000 mg/kg body weight/day, for example, about 0.01 to about 500, 300, or lOOmg/kg body weight/day. In further or additional embodiments, administration can be performed daily or even several times per day, if necessary. By way of examples, the extract of the invention can be administered once, twice, three, four, five or six times a week or more, or once, twice, three or four times a day or more. Duration of the treatment may vary and depends on the severity of the disease. It may last for instance from one week to several months (such as from 2, 3, 4, 5, 6 or 7 weeks to 12, 18, 24, 30, or 36 weeks).

The present invention also provides a cosmetic composition comprising the Indigo Naturalis or Indigo-producing plant extract. The composition according to the present invention may be present in a form adapted for topical application comprising a cosmetically or dermato logically acceptable carrier or medium. "Cosmetically or dermatologically acceptable" means media which are suitable for a use in which they come into contact with the skin or human skin appendages without posing a risk of toxicity, intolerance, instability, allergic reaction, etc. The extract may be present in the cosmetic composition at a concentration from approximately 0.0005 to 500 ppm, lOOOppm, or 10,000ppm. In the cosmetic composition, the Indigo Naturalis or Indigo-producing plant extract may be previously solubilized in one or more cosmetically or dermatologically acceptable solvents, such as water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, petroleum jelly, a vegetable oil or any mixture of these solvents.

The present invention provides a method of treating a nail disorder selected in the group consisting of nail dystrophy, onycholysis, and nail psoriasis, comprising administering a therapeutically effective amount of an Indigo Naturalis or Indigo-producing plant extract to a subject in need thereof. The Indigo Naturalis or Indigo-producing plant extract and compositions above can be used in the treatment or alleviation of a disease or condition. By treatment it is meant at least an alleviation of the symptoms associated with the pathological condition afflicting the subject, where alleviation is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the pathological condition being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the host no longer suffers from the pathological condition, or at least the symptoms that characterize the pathological condition. As such, treatment includes both curing and managing a disease condition. Accordingly, the extract and compositions above can be used in the treatment or alleviation of a nail disorder selected in the group consisting of nail dystrophy, onycholysis, and nail psoriasis.

The efficacy of the extract and compositions can be evaluated by in vivo models with respect to their activities in treating diseases or disorders, for example, clinically trails on humans.

The subject in need of candidiasis treatment according to the invention is any mammal, including a human or a non-human mammal, preferably a human mammal.

Within the context of the invention, the term treatment denotes curative, symptomatic, and preventive treatment. The compositions of the invention will not necessarily cure the patient who has the nail disease but will diminish in a satisfactory manner delaying or slowing thereby the progression or preventing thereby complications of the nail disease. This will ameliorate consequently the patients' nail condition. The compositions of the invention can also be administered to those who do not have the nail disease yet but who would normally develop such nail disease or be at increased risk for said disease, they will not develop said disease. Treatment also includes delaying the development of nail disease in an individual who will ultimately develop said disease or would be at risk for the disease due to age, personal or familial history, or genetic abnormalities. By delaying the onset of the nail disease, compositions of the invention have prevented the individual from getting the nail disease during the period in which the individual would normally have gotten said disease or reduce the rate of development of the nail disease or some of its symptoms.

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present application will be obtained by reference to the following detailed description that sets forth illustrative embodiments.

While embodiments of the present invention have been shown and described herein such embodiments are provided by way of example only. It should be understood that the above described embodiments may be combined if compatible and various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. Those ordinary skilled in the art will appreciate that numerous variations, changes, and substitutions are possible without departing from the invention. It is intended that the following claims define the scope of aspects of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art. All documents, or portions of documents, cited in the invention including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

The percentage herein is expressed by weight relative to the weight of the extract, unless otherwise specified.

Further aspects and advantages of the invention will be disclosed in the following illustrative experimental section. EXAMPLES

1. Preparation of refined Indigo Naturalis or Indigo-producing plant extracts and analytical methods for analysis

Example 1: Preparation of a refined Indigo Naturalis extract

Qingdai as used in the following preparation is obtained from Delong Pharmaceutical (Indigo 2.62% and Indirubin 0.284% (HPLC method depicted in Example 7 A) and tryptanthrin 0.0046%.

500g of Qingdai were suspended in 10L ethyl acetate. The mixture was stirred in reflux for two hours, and then filtered at 75°C. The filtrate was concentrated at reduced pressure to yield a dark solid. The crude extract was stirred in 250mL hexane and heated to reflux for one hour. After cooling to room temperature, the suspension was filtered to give a dark residue.

0.50g of the dark residue were refiuxed in 25mL hexane again for one hour, and cooled to room temperature, followed by filtration to give a refined extract as a dark red solid 452mg. HPLC: 62.9% indirubin, 12.9% indigo, and 0.53% tryptanthrin.

Example 2: Preparation of a refined Indigo Naturalis extract

500g of Qingdai as used in Example 1 were suspended in 10L alcohol (ethanol).

The mixture was stirred in reflux for two hours, and then filtered at 75°C. The filtrate was concentrated at reduced pressure to yield a dark solid, which was stirred in 260mL hexane and heated to reflux for one hour. Upon cooling to room temperature, the suspension was filtered to give a dark residue.

0.80g of the dark residue were refiuxed in 24mL alcohol (ethanol) for an additional hour, and then cooled to room temperature, followed by filtration to give a refined extract as a dark red solid (538mg). HPLC: 83.6%> indirubin, 6.35%> indigo, and 0.75% tryptanthrin. Example 3: Preparation of a refined Indigo Naturalis extract

500g of Qingdai as used in Example 1 were suspended in 10L ethyl acetate. The mixture was stirred in reflux for two hours, and then filtered while hot. The filtrate was concentrated at reduced pressure to yield a dark solid. The crude extract was stirred in 250mL hexane and heated to reflux for one hour. After cooling to room temperature, the suspension was filtered to give a dark residue.

0.75g of the dark residue were refluxed in 22.5mL ethanol for one hour, and cooled to room temperature, followed by filtration to give a refined extract as a dark red solid (538mg). HPLC: 77.9% indirubin, 15.9% indigo, and 0.56% tryptanthrin.

Example 4: Preparation of a refined Indigo Naturalis extract

500g of Qingdai as used in Example 1 were suspended in 2.1L DMF. The mixture was stirred at 50°C for 40 minutes. Upon cooling to 20°C, the suspension was filtered. The filtrate was concentrated at reduced pressure to yield a dark solid, which was stirred in 130mL hexane and heated to reflux for one hour. Upon cooling to 20°C, the suspension was filtered to give a dark residue.

1.56g of the dark residue was washed with 46.8 ml ethanol, and heated to reflux for one hour, and then cooled to 20°C, followed by filtered to yield a refined extract (766mg). HPLC: 66.3%, indirubin, 9.76% indigo.

Example 5: Preparation of a refined Indigo Naturalis extract

500g of Qingdai as used in Example 1 were suspended in 3L DMF. The mixture was stirred at 30°C for 1 hour, and then filtered. The filtrate was concentrated at reduced pressure to yield a dark solid, which was stirred in 230mL hexane and heated to reflux for one hour. Upon cooling to 20°C, the suspension was filtered to give a dark residue.

1.96g of the dark residue was washed with 59mL 85% ethanol (85% aq. alcohol), and heated to reflux for one hour followed by filtration while hot to yield a refined extract (1.02g). HPLC: 69.4% indirubin, 18.7% indigo, and 0.62% tryptanthrin. Example 6: Preparation of a refined Indigo Naturalis extract lOOg of Qingdai was extracted with 2L of ethanol 92% (92% aqueous ethanol) for 2 hours under reflux conditions. Upon completion, the mixture was filtered while hot on AF6 filter (Buchner) to obtain a dark blue-red solution as a filtrate. This filtrate was reduced under vacuum to dryness to give 2.4 g of dry residue. This residue was washed with 120 mL of hexane for lh under reflux. Upon completion, the mixture was cooled to room temperature for 2h then filtered under vacuum to yield 312.9mg of a dark red refined extract.

280mg of this refined extract were washed with 15 mL of ethanol 92% (92% aqueous ethanol) for lh under reflux. Upon completion the solution was cooled to room temperature, and then filtered to yield 159 mg of a dark red/burgundy refined extract after drying in oven (80°C) for lh30. (0.18%); HPLC: 82.31% indirubin, 8.99% indigo, and 0.81% tryptanthrin. Example 7: Micronization step

The micronization step of refined Indigo Naturalis or Indigo-producing plant extract obtained in the previous examples is performed with the following equipment:

- Micronizer: spiral jet Mill Diameter 200

- Feeder: this equipment is used for the dosage of powder to feed the micronizer. The dosage is made thanks to two screws.

This system allows a regularity of the flow.

Micronization consists to project grains of powder with jet of air. The contact of grains permits their explosion.

Following parameters of micronization are recorded during the micronization:

- Ring pressure: 6 bar

- Injector pressure: 3 bar

- The flow of powder feed: 25 kg/h

The micronizer allows a cylindrical enclosure - holes around the enclosure for the injection of air.

Powder is introduced in the micronizer; grains are propelled thanks to jet of air. When grains have the good size, they are concentrated in the center of the micronizer and they are breathed. To avoid any contamination by foreign particles or broken pieces of the equipment, an additional sieving (sieve: 700 μιη) is performed.

The step is done manually after the micronization and before the packaging. A granulomere analysis of the homogeneous product obtained was carried out according to the particular size distribution (PSD) method [Analytical specifications: D99 < 30 urn].

Example 8

Here a patient is presented with recalcitrant chronic onycholysis and disappearing nail bed that responded dramatically to topical treatment of Indigo Naturalis extract in oil (i.e. Lindioil, the refined olive oil extract prepared in example 2 of US 8,784,905). In addition, there has been no remission for over four years.

A 78-year-old man with a 10-year history of onyco lysis, which developed two years after starting gardening regularly, had a nail with painless discoloration. He was diagnosed with onycolysis and Candida albicans infection. Subsequent treatment with topical anti-fungal ointment mildly improved the discoloration but did not resolve the onycolysis. The affected nail plate was surgically removed five years later but redeveloped onycolysis as soon as the fresh nail grew back completely. Consequently, he self-administered various over-the-counter topical treatments, such as Loceryl nail lacquer 5%, and again with no success in resolving the onycolysis. In addition, the distal nail bed appeared to shrink.

The patient first visited the outpatient clinic on August 31, 2008. A physical examination revealed discoloration of the right thumb nail with nail bed hyponychium cornifies and dermatoglyphyics (also known as disappearing nail bed) (Fig. la). There were no other skin disease lesions noted but a medical history revealed hyperhidrosis palmaris. The patient began to apply Lindioil to the affected nail twice daily. After four months of treatment, the patient achieved a fully formed nail plate, with no onycholysis and no side-effects (Fig. lb). Follow-up after more than four years showed only minor recurrences of onycholysis which were quickly controlled by repeated application of Lindioil. Comment

Here it is also shown that the Indigo Naturalis or Indigo-producing plant extract produced excellent therapeutic outcomes in treating onycholysis with disappearing nail bed.

In conclusion, this novel finding indicates that an Indigo Naturalis or Indigo-producing plant extract is an effective alternative therapy for treating chronic onycholysis with disappearing nail bed. Treatment with an Indigo Naturalis or Indigo-producing plant extract is appealing to patients because it is easy to apply and there are no side effects.

Example 9

In this study, a randomized, observer-blind, intra- subject trial was conducted to compare the efficacy of Indigo Naturalis extract in oil (Lindioil, a refined olive oil extract as prepared in example 2 of U.S. Patent No. US 8,784,905) with Calcipotriol solution in treating nail psoriasis.

ABBREVIATIONS

AE, Adverse event; CI, Confidence interval; Lindioil, Indigo Naturalis extract in oil; NAPSI, Nail Psoriasis Severity Index; MTNAPSI, modified target Nail Psoriasis Severity Index; PASI, Psoriasis Area and Severity Index; PGA, Physician global assessment; SD, Standard deviation; SGA, Subject global assessment; SHNAPSI, single hand Nail Psoriasis Severity Index.

PATIENTS AND METHODS

This was a randomized, observer-blind, vehicle-controlled and intra- subject trial. The study was reviewed and approved by the Institutional Review Board of the Chang Gung Memorial Hospital (CGMH IRB No.:99-3316A3). All patients signed the informed consent to participate in the trial. Subjects

Subjects with nail psoriasis were recruited by Dr. Lin in the Department of Traditional Chinese Medicine and Dermatology at the Chang Gung Memorial Hospital from January 2011 to May 2012. Patients aged 20-65 years with symmetrically comparable psoriatic nails on each hand were eligible. Females of childbearing age were instructed to use birth control measures during the study period. Patients with a known history of allergy to Indigo Naturalis were excluded, as were patients with onychomycosis or any other fingernail infections, those who used concomitant topical or systemic treatments. Pregnant and breast-feeding women were also excluded.

Treatment

The patients were randomized in a ratio of 1 : 1 by block randomization method. Lindioil (Indigo Naturalis extract in oil) was applied to fingernails of one hand (experimental group) and Calcipotriol solution was applied to fingernails of the contralateral hand (control group) twice daily for 24 weeks.

The powdered form of Indigo Naturalis used in this study was prepared from the leaves of Baphicacanthus cusia (Nees) Bremek and purchased from Xianyou county, Fujian province, China. The fingerprints and quantity analysis of standard samples, indirubin and indigo, were established and a specimen (SF-1) was deposited in the herbarium of the Chang Gung University, Taoyuan, Taiwan. Lindioil was prepared using powdered Indigo Naturalis mixed with olive oil: Kirkland Signature Pure Olive Oil (Italian, item 71008) and filtered by Chuang Song Zong Pharmaceutical Co., LTD, Taiwan. The Lindioil contained 0.2 mg/ml of indirubin that was tested by high-performance liquid chromatography and was placed in 5-ml eye drop bottles. Calcipotriol solution (Daivonex® scalp solution, Calcipotriol 50ug/ml) was purchased from LEO Pharmaceutical Products Ltd. (Ballerupt, Demark) and placed in 5-ml eye drop bottles.

All patients were instructed to apply one drop (0.05ml) of Lindioil or Calcipotriol solution onto the nail plate folds and hyponychium of affected nails twice daily. They were also instructed to avoid washing hands during the first 30 minutes after application, to avoid activities that might injure the nails, and to avoid cross-contamination between the two hands. Treatment was performed for a maximum of 24 weeks or until complete clearing of nail psoriasis or the development of obvious local or systemic adverse events (AEs) possibly related to treatment. Subjects who did not comply with the study protocol were terminated from the study. In addition, subjects were free to drop out of the study for any reason at any time.

Assessments

The severity of nail psoriasis was recorded at each visit (week 0, 4, 8, 12, 16, 20, and 24) by using the Nail Psoriasis Severity Index (NAPSI) and Modified Target Nail Psoriasis Severity Index (MTNAPSI). Before evaluation, all patients were requested to thoroughly clean their fingernails so that no stain was left to interfere with the assessment.

The Nail Psoriasis Severity Index (NAPSI) score is reported as a reproducible, objective, and simple tool for clinical assessment of nail psoriasis and is the only clinical scale that has been validated. The nail is divided by imaginary horizontal and longitudinal lines into quadrants. Each nail is given a score for nail bed psoriasis (0-4) and nail matrix psoriasis (0-4) depending on the presence of any of the features of nail psoriasis in that quadrant. The NAPSI score evaluates presence of signs in the nail bed (of onycholysis, splinter hemorrhages, nail bed discoloration, and subungual hyperkeratosis) and on the nail matrix (pitting, leukonychia, red spots in the lunula and nail plate crumbling) in all 10 fingernails, providing a maximum score of 80. Because we conduct a side-to-side comparison in our study, we modified NAPSI score to SHNAPSI (Single Hand Nail Psoriasis Severity Index) score to measure disease severity of all five fingernails in one hand providing a maximum score of 40. The MTNAPSI score for the target nail severity of nail matrix and nail bed psoriasis is from 0 (no sign) to 3 (severe involvement) in each nail quadrant, providing a maximum score of 96. In comparison, the NAPSI has limitation in terms of sensitivity and exclusion of types of changes and degree of severity as well as not providing information on the severity of involvement of each single nail, but rather reflects the overall severity of nail psoriasis. In this study, the primary efficacy measures were the SHNAPSI and the MTNAPSI for the single most severely affected nail on each hand. Two dermatologists, who were blinded to the treatment modality, rated the SHNAPSI or MTNAPSI based on the photographs of the fingernails. Prior to the study, the two dermatologists underwent a special training course in the assessment of NAPSI and MTNAPSI scores to minimize inter and intra-rater discrepancies.

Changes in single hand NAPSI and modified target NAPSI scores after treatment were computed as follows: (Score prior to treatment - Score after treatment) / (Score prior to treatment) x 100%. In addition, clinical feature scores for pitting, leukonychia, red spots in the lunula, nail plate crumbing, onycholysis, splinter hemorrhages, subungual hyperkeratosis, and oil drop discoloration were assessed independently.

The dynamic Subject Global Assessment (SGA) and Physician Global Assessment (PGA) at week 24 were also assessed using baseline photos for comparison. The global assessment is an overall assessment of a patient's nail psoriasis, scored separately for each hand, which takes into consideration the quality and extent of nail disorders relative to the baseline assessment on a 6-point scale (0 = worse, 5 = cleared). A positive comprised of 3 categories: good response (score 3, 50-74% clearing with moderate improvement), excellent response (score 4, 75-99%) clearing with striking improvement), and clearance (score 5, 100% clearing). Non-response was defined as a fair (score 2, 25-49%) clearing with slight improvement), poor (score 1, 0-24% clearing with little or no change), or worse (score 0) rating. Furthermore, at week 24, subjects were also asked which treatment they preferred.

At each visit, we assessed the occurrence of AEs such as pain, erythema, and irritation or itching that might have been caused by the medication.

Sample size and statistics

The sample size of 30 was calculated based on the following: (1) mixed effect model with two within-factors (group and time), (2) the detectable difference in single-hand Nail Psoriasis Severity Index (SHNAPSI) score or modified target Nail Psoriasis Severity Index (MTNAPSI) score between two groups is 3 units, (3) the significance level is .05, and (4) the power is 0.9.

Continuous variables such as subject age and duration of psoriasis are expressed as mean ± SD or as a percentage. Categorical variables, such as gender, are expressed as frequency or percentage. The mixed-effect model with two within-factors (group and time) was used to account for time dependency of the repeated measurements and differences between the two hands. Pearson correlation coefficient was used to test the correlation between SGA and PGA. The comparison of the efficacy of the different treatments was conducted by a paired-T test. The overall significance level of this study was 0.05; however, the Bonferroni method reduced the significance level. This was accomplished by dividing the numbers of multiple comparisons when comparing SHNASPI (or MTNAPSI) between the two hands over time. In other words, when comparing SHNAPSI (or MTNAPSI) between the two hands over time, the significance level was .007 (= .05/7).

RESULTS

Demographics

A total of 43 subjects agreed to participate in the study and 33 subjects met the eligibility criteria. Of the 33 subjects (22 men, 11 women), the mean age was 41.9 ± 9.4 years. The mean duration of skin and nail psoriasis was 11.8 ± 10.9 and 5.8 ± 6.4 years, respectively. The mean Psoriasis Area Severity Index (PASI) score was 8.9 ± 8.2 and the mean NAPSI score was 54.3 ± 15.4 (Table I.)

At baseline, 18 of 33 subjects used the Lindioil on the right hand while 15 used the Lindioil on the left hand. At week 24, 28 subjects had completed the trial (Fig 2). Therefore, data for 31 subjects at baseline and 28 subjects at week 24 were included in the "as-treated" analysis in this study.

In this study, 323 fingers (total fmgers=330) were involved with psoriasis among which pitting was presented in 74.% of fingers, leukonychia was 41.8%, red spots was 1%, crumbling was 4%, onycholysis was 91% , splinter hemorrhage was 25.4%), subungual hyperkeratosis was 62.2%, and nail plate discoloration was 58.2%.

Efficacy

At baseline, both SHNAPSI and MTNAPSI scores were not statistically different between the Lindioil group and calcipotriol group. At the end of treatment, both SHNAPSI and MTNAPSI scores were lower in the L group (P < 0.001) compared with calcipotriol group (P < 0.001 ). (Fig 4a & 4b).

After 12 week of treatment, the mean percentage change in SHNAPSI was greater in the Lindioil group (24.3%) than in the calcipotriol group (13/9%) (P< 0.001). At the end of treatment (week 24), The Lindioil group (49.5%) had a greater mean percentage reduction in SHNAPSI than calcipotriol group (24.9%, P<0.001). Similarly, the mean percentage change in MTNAPSI was greater in the Lindioil group (77.9%o) compared with calcipotriol group (65.4%>, P <0.001) .

In terms of the percentage change of scores in nail feature from baseline to the end of treatment, no statistically significant difference was observed in splinter hemorrhage, subungual hyperkeratosis, leukonychia, red spots, and nail plate crumbling between the Lindioil and calcipotriol. However, a significant difference of score reduction was seen in the onycholysis, nail bed discoloration, and pitting between the two groups. (Table 2)

Table 2. Scores by nail bed and matrix features in the Lindioil and calcipotriol group

Time Scores¹ (mean+SD)

Features

(wk. #) Lindioil Calcipotriol

Nail bed

Onycholysis 0 2.01 ± 0.47 2.00 ± 0.44 < 0.001²

0.821³

24 1.14 ± 0.71 1.43 ± 0.67 < 0.001⁴

Splinter

0 0.81 ± 0.72 0.90 ± 0.75 0.598² hemorrhages 0.875³

24 0.60 ± 0.66 0.89 ± 0.69

0.064⁴

Subungual 0 2.29 ± 1.28 2.26 ± 1.28 < 0.001² hyperkeratosis 0.202³

24 0.84 ± 0.96 1.38 ± 1.07 0.005⁴

Nail bed 0 2.34 ± 1.18 2.22 ± 1.21 < 0.001² discoloration 0.177³

24 0.88 ± 0.91 1.36 ± 1.14 0.002⁴

Nail matrix

Pitting 0 2.43 ± 1.11 2.36 ± 0.95 0.728²

0.150³

24 1.81 ± 1.24 2.29 ± 1.04

< 0.001⁴

Leukonychia 0 1.20 ± 0.78 1.14 ± 0.74- < 0.001²

0.282³

24 0.33 ± 0.53 0.67 ± 0.79

0.104⁴

Red spots in the 0 0.05 ± 0.26 0.03 ± 0.17 0.974² lunula 0.282³

24 0.00 ± 0.00 0.03 ± 0.14

0.947⁴

Nail plate 0 0.27 ± 0.81 0.39 ± 1.02 0.024² crumbing 0.558³

24 0.20 ± 0.53 0.30 ± 0.61

0.590⁴

Lidioil, Indigo Naturalis extract

¹Range of scores for single nail psoriasis is 0-4.

^2"4Mixed effect model: the main effect of time, group and interactions between time and group, respectively.

In the end of treatment, the dynamic SGA and PGA scores were highly correlated (r =0.68). According to the physicians' or subjects' assessment, there was a higher percentage of responders who achieved 'clearance', 'excellent good ', and 'good response' in Lindioil group (physicians 78,6%, subjects 96.4%>) than in the calcipotril group (physicians 42.9%>, subjects 60.7%>). (Table 3) In addition, after 24 weeks of treatment, 82.1% (23 of 28) of subjects reported that they preferred Lindioil, 17.9% (5 of 28) preferred calcipotriol.

Table 3. Subject and physician global assessment of Lindioil vs. Calcipotriol treatment at week 24 (n=28).

No. ofSGA(%) No. ofPGA(%)

Scores*

Lindioil Calcipotriol Lindioil Calcipotriol

5 5(17.9%) 1(3.57%) 2(7.14%) 1(3.57%)

4 15(53.6%) 5(17.9%) 14(50.0%) 3(10.7%)

3 7 (25%) 11(39.2%) 6(21.4%) 8(28.6%)

2 1(3.57%) 6(21.4%) 5(17.9%) 9(32.1%)

1 0 3(10.7%) 1(3.57%) 4(14.3%)

0 0 2(7.14%) 0 3(10.7%) SGA, subject global assessment; PGA, physician global assessment; Lindioil, Indigo Naturalis extract in oil.

* Scores:

• 5: clear, 100% clearing;

· 4: excellent, 75-99% clearing with striking improvement;

• 3: good, 50-74%) clearing with moderate improvement;

• 2: fair, 25-49% clearing with slight improvement;

• 1: poor, 0-24% clearing with little or no change;

• 0: worse.

SAFETY

None of the subjects experienced serious adverse effects during the study period. In the experimental group, 10 of the 33 subjects had nail pain at baseline and 8 of 10 (80%) were relieved on the first visit (week 4). In comparison, the control group had 10 subjects with nail pain and 5 of 10 (50%) were relieved on the first visit. In addition, 10 of the 33 subjects (30.30%>) in the control group suffered from nail pain after application compared to 2 subjects (6.06%) in the experimental group.

RESULTS

Of 33 subjects, 28 completed at week 24. At the end, comparing the experimental group to the control group, the mean percentage of reduction from the baseline SHNAPSI was 49.5 ± 26.21% vs. 24.9 ± 27.67%, and MTNAPSI was 77.9 ± 16.41% vs. 65.4 ± 17.27%, indicating that Lindioil was statistically superior to calcipotriol (P < 0.001, P < 0.001, respectively). The subject and physician's global assessments also showed similar results. There were no adverse events during 24 weeks of treatment. DISCUSSION

In this study, we found that Lindioil is significantly more effective than calcipotriol solution at reducing the severity of nail psoriasis. The mean percentage of SHNAPSI reduction from baseline in the Lindioil group is 49.5% and 24.9% in the calcipotril group at the end of 24-weeks treatment. This result is similar to that obtained in our previous pilot study.⁵ Furthermore, both the SGA and PGA revealed that Lindioil was superior to the calcipotriol solution. This study also demonstrated various nail features responded differently to treatment. We found onycholysis (91%o) and pitting (74%) were the most common features of nail psoriasis in our cases, which showed the greatest improvement after being treated with Lindioil.

In consideration of safety and effect, Lindioil is a good treatment modality for nail psoriasis in patients with either limited or extensive involvement of skin lesions.

The present study showed pitting and onychoysis responded well to Lindioil. Nail psoriasis is usually refractory to treatment. It is important to establish a safe and effective therapy for long-term use. In this study, we found that Lindioil was safe and effective for treating nail psoriasis, which was superior to clacipotriol solution, acceptable to subjects, indicating that it has great potential to become and effective alternative therapy for nail psoriasis.

Claims

A pharmaceutical or cosmetic composition comprising an Indigo Naturalis or Indigo-producing plant extract for a use in the treatment of a nail disorder selected in the group consisting of nail dystrophy, onycholysis, and nail psoriasis.

A pharmaceutical or cosmetic composition comprising an Indigo Naturalis or Indigo-producing plant extract for a use in the treatment of onycholysis.

A pharmaceutical or cosmetic composition according to any one of claim 1 or 2, wherein the Indigo Naturalis or Indigo-producing plant extract comprises indirubin in an amount of at least 65% w/w of the extract, preferably 65%-90% w/w of the extract

An Indigo Naturalis or Indigo-producing plant extract for a use in the treatment of a nail disorder selected in the group consisting of nail dystrophy, onycholysis, and nail psoriasis.

An Indigo Naturalis or Indigo-producing plant extract for a use in the treatment of onycholysis.

An Indigo Naturalis or Indigo-producing plant extract according to any one of claim 4 or 5, which comprises indirubin in an amount of at least 65% w/w of the extract, preferably 65%-90% w/w of the extract.