WO2018082034A1

WO2018082034A1 - Uses of triterpenoid mixture for treating multiple sclerosis

Info

Publication number: WO2018082034A1
Application number: PCT/CN2016/104681
Authority: WO
Inventors: Teng-Hai Chen; Cheng-Po HUNAG; Kuang-Dee Chen; Mon-Tarng Chen; I-Shuan CHAO; Chien-Yuan Wang; Shin Huei HUANG; Cheng Chiu TSAO
Original assignee: Trineo Biotechnology Co., Ltd
Priority date: 2016-11-04
Filing date: 2016-11-04
Publication date: 2018-05-11
Also published as: EP3509602A4; EP3509602A1

Abstract

Disclosed herein is a novel use of a triterpenoid mixture for the treatment of multiple sclerosis (MS). The triterpenoid mixture consists of, ganoderic acid A (GAA), ganoderic acid B (GAB), ganoderic acid C (GAC), ganoderic acid C₅ (GAC₅), ganoderic acid C₆ (GAC₆), ganoderic acid D (GAD), ganoderic acid E (GAE), ganoderic acid G (GAG), and ganoderenic acid D. The triterpenoid mixture may be administered to a subject suffering from MS in a dose of about 1 to 10 mg/Kg to ameliorate or alleviate symptoms associated with MS.

Description

[Title established by the ISA under Rule 37.2] USES OF TRITERPENOID MIXTURE FOR TREATING MULTIPLE SCLEROSIS

BACKGROUND OF THE INVENTION

1. FIELD OF THE INVENTION

The present disclosure in general relates to a novel use of a triterpenoid mixture for the treatment of Multiple sclerosis (MS) .

2. DESCRIPTION OF RELATED ART

Multiple sclerosis (MS) is a disease involving one’s immune system attacking the central nervous system, such as brain, spinal cord, and optic nerves. As part of the immune attack on the central nervous system, diffuse patches of demyelination in the brain and spinal cord are often observed that result in multiple neurologic symptoms and signs, usually with repeated relapse and remission. Currently, there is no cure for MS, however, therapeutics that relieve symptoms of MS are available, such as oral administration of adrenocortical steroid or vitamin B₁₂, and subcutaneous injection of interferon beta. However, the use of adrenocortical steroid is limited to acute attack, for long term use of steroid may cause unwanted side effects. As to interferon beta, it is expensive, thus such treatment would become costly for long-term usage； moreover, subcutaneous injection of interferon beta would require hospital attendance, which further lowers the patient’s degree of compliance.

Accordingly, there is a high demand of an improved medicine for ameliorating and/or relieving symptoms of MS, in which the medicine is suitable for oral administration and is effective for the treatment of MS without generating any unwanted side effects.

SUMMARY

The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the present invention or delineate the scope of the present invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

In general, the present disclosure relates to the unexpected discovery of the novel use of a triterpenoid mixture in treating a subject suffering from multiple sclerosis (MS) . Thus, the triterpenoid mixture is useful for developing medicaments for treating diseases and/or conditions related to MS.

Accordingly, the first aspect of the present disclosure aims at providing a use of the triterpenoid mixture for the manufacture of a medicament suitable for treating MS.

According to embodiments of the present disclosure, the medicament comprises 10 to 1, 000 mg of the triterpenoid mixture； preferably, 50 to 500 mg of the triterpenoid mixture； and more preferably, 100 to 300 mg of the triterpenoid mixture.

According to preferred embodiments of the present disclosure, the triterpenoid mixture consists of, ganoderic acid A (GAA) , ganoderic acid B (GAB) , ganoderic acid C (GAC) , ganoderic acid C₅ (GAC₅) , ganoderic acid C₆ (GAC₆) , ganoderic acid D (GAD) , ganoderic acid E (GAE) , ganoderic acid G (GAG) , and ganoderenic acid D, in which GAA is present in about 30-40％by weight in the mixture, the GAB, GAC, GAD and GAG are respectively present in about 10-15％by weight in the mixture, and the GAC₅, GAC₆, GAE and ganoderenic acid D are respectively present in about 3-6％by weight in the mixture, in which the total weight of the triterpenoid mixture is taken as 100％.

According to preferred embodiments of the present disclosure, the medicament is suitable for oral administration.

The second aspect of the present disclosure aims to provide a method of treating a subject suffering from MS. The method comprises the step of, administering to the subject the present triterpenoid mixture described above in a dose of about 1 to 10 mg/Kg to ameliorate or alleviate symptoms associated with MS.

According to preferred embodiment of the present disclosure, the GAA is present in about 30-40％by weight in the mixture, the GAB, GAC, GAD and GAG are respectively present in about 10-15％by weight in the mixture, and the GAC₅, GAC₆, GAE and ganoderenic acid D are respectively present in about 3-6％by weight in the mixture, in which the total weight of the mixture is taken as 100％.

According to preferred embodiment of the present disclosure, the present triterpenoid mixture is administered to the subject orally.

According to preferred embodiment of the present disclosure, the subject is a mammal, preferably a human.

The details of one or more embodiments of this disclosure are set forth in the accompanying description below. Other features and advantages of the invention will be apparent from the detail descriptions, and from claims.

It is to be understood that both the foregoing general description and the following detailed description are by examples, and are intended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate various example systems, methods and other exemplified embodiments of various aspects of the invention. The present description will be better understood from the following detailed description read in light of the accompanying drawings, where,

FIG 1 illustrates the clinical scores of EAE mice in accordance with one embodiment of the present disclosure；

FIG 2 are photographs illustrating the spinal cord tissues taken from EAE mice stained with H&E in accordance with one embodiment of the present disclosure, in which A: the vehicle control, and B: the present triterpenoid mixture-treated, 100x magnification, the inflammatory cells, demyelination and/or apoptotic cells were indicated by boxes； and

FIG 3 is a bar graph depicting the effect of the present triterpenoid mixture on blood IL-17 level in accordance with one embodiment of the present disclosure.

DETAILED DESCRIPTIONOF THE PREFERRED EMBODIMENTS

The detailed description provided below in connection with the appended drawings is intended as a description of the present disclosure and is not intended to represent the only forms in which the present disclosure may be constructed or utilized.

1. Definitions

For convenience, certain terms employed in the context of the present disclosure are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skill in the art to which this invention belongs.

The term “treatment” as used herein are intended to mean obtaining a desired pharmacological and/or physiologic effect, e.g., inhibiting or suppressing the progression of MS. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment” as used herein includes preventative (e.g., prophylactic) , curative or palliative treatment of a disease in a mammal, particularly human； and includes: (1) preventative (e.g., prophylactic) , curative or palliative treatment of a disease or condition (e.g., MS) from occurring in an individual who may be pre-disposed to the disease but has not yet been diagnosed as having it； (2) inhibiting a disease (e.g., by arresting its development) ； or (3) relieving a disease (e.g., reducing symptoms associated with the disease) .

The term “administered” , “administering” or “administration” are used interchangeably herein to refer a mode of delivery, including, without limitation, intraveneously, intramuscularly, intraperitoneally, intraarterially, intracranially, or subcutaneously administering an agent of the present invention (e.g., the present triterpenoid mixture) . In some embodiments, the triterpenoid mixture of the present disclosure are formulated into powders for mixed with suitable carrier (e.g., buffer solution) before use, such as intraveneous injection. In other embodiments, the triterpenoid mixture of the present disclosure is formulated into powders with suitable excipients for direct oral ingestion.

The term “an effective amount” as used herein refers to an amount effective, at dosages, and for periods of time necessary, to achieve the desired result with respect to the treatment of a disease resulted from MS. For example, in the treatment of MS, the present triterpenoid mixture which decrease, prevents, delays or suppresses or arrests any clinical signs of the MS would be effective. An effective amount of an agent is not required to cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered or prevented, or the disease or condition symptoms are ameliorated. The specific effective or sufficient amount will vary with such factors as the particular condition being treated, the physical condition of the patient (e.g., the patient's body mass, age, or gender) , the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any) , and the specific formulations employed and the like. Effective amount may be expressed, for example, as the total mass of the active agent (e.g., in grams, milligrams or micrograms) or a ratio of mass of the active agent to body mass, e.g., as milligrams per kilogram (mg/kg) . The effective amount may be divided into one, two or more doses in a suitable form to be administered at one, two or more times throughout a designated time period.

The term “subject” or “patient” is used interchangeably herein and is intended to mean a mammal including the human species that is treatable by the compound of the present invention. The term “mammal” refers to all members of the class Mammalia, including humans, primates, domestic and farm animals, such as rabbit, pig, sheep, and cattle； as well as zoo, sports or pet animals； and rodents, such as mouse and rat. Further, the term “subject” or “patient” intended to refer to both the male and female gender unless one gender is specifically indicated. Accordingly, the term “subject” or “patient” comprises any mammal which may benefit from the treatment method of the present disclosure. Examples of a “subject” or “patient” include, but are not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl. In a preferred embodiment, the subject is a human.

The term “pharmaceutically acceptable” refers to molecules and compositions that do not produce an adverse or undesirable reaction (e.g., toxicity, or allergic reaction) when administered to a subject, such as a human.

The term “excipient” as used herein means any inert substance (such as a powder or liquid) that forms a vehicle/carrier for the active agent. The excipient is generally safe, non-toxic, and in a broad sense, may also include any known substance in the pharmaceutical industry useful for preparing pharmaceutical compositions such as, fillers, diluents, agglutinants, binders, lubricating agents, glidants, stabilizer, colorants, wetting agents, disintegrants, and etc.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in the respective testing measurements. Also, as used herein, the term “about” generally means within 10％, 5％, 1％, or 0.5％of a given value or range. Alternatively, the term “about” means within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Other than in the operating/working examples, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, durations of times, temperatures, operating conditions, ratios of amounts, and the likes thereof disclosed herein should be understood as modified in all instances by the term “about. ” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that can vary as desired. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

The singular forms “a” , “and” , and “the” are used herein to include plural referents unless the context clearly dictates otherwise.

2. DETAIL DESCRIPTION OF PREFERRED EMBODIMENTS

The present disclosure is based, at least in part, on the unexpected discovery that a triterpenoid mixture prepared in accordance with examples of the present disclosure may suppress or inhibit the progression of multiple sclerosis. Accordingly, the present triterpenoid mixture is useful for the development of a medicament for treating diseases, disorders and/or conditions related to MS.

The practices of this invention are hereinafter described in detail with respect to use of a triterpenoid mixture, which consists of, ganoderic acid A (GAA) , ganoderic acid B (GAB) , ganoderic acid C (GAC) , ganoderic acid C₅ (GAC₅) , ganoderic acid C₆ (GAC₆) , ganoderic acid D (GAD) , ganoderic acid E (GAE) , ganoderic acid G (GAG) , and ganoderenic acid D； the preparation of a medicament for preventing or treating MS, or disease caused thereby, in a subject or patient. Results of the present studies, as described herein below, show that the present triterpenoid mixture possess minimum or no cytotoxicity toward normal cells, and reduces the level of the inflammatory cytokine IL-17 in the blood, thereby suppresses the progression of MS in vivo.

The first aspect of the present application is therefore directed to a method of treating a subject having or suffering from MS. The method comprises the step of, administering to the subject in need thereof, an effective amount of the present triterpenoid mixture, which consists of: ganoderic acid A (GAA) , ganoderic acid B (GAB) , ganoderic acid C (GAC) , ganoderic acid C₅ (GAC₅) , ganoderic acid C₆ (GAC₆) , ganoderic acid D (GAD) , ganoderic acid E (GAE) , ganoderic acid G (GAG) , and ganoderenic acid D； so as to alleviate or ameliorate the symptoms associated with MS.

According to embodiments of the present disclosure, GAA is present in about 30-40％by weight in the triterpenoid mixture, the GAB, GAC, GAD and GAG are respectively present in about 10-15％by weight in the triterpenoid mixture, and the GAC₅, GAC₆, GAE and ganoderenic acid D are respectively present in about 3-6％by weight in the triterpenoid mixture, in which the total weight of the triterpenoid mixture is taken as 100％. According to one specific embodiment of the present disclosure, GAA is present in about 35％by weight in the triterpenoid mixture, the GAB, GAC, GAD and GAG are respectively present in about 12％by weight in the triterpenoid mixture, and the GAC₅, GAC₆, GAE and ganoderenic acid D are respectively present in about 3, 6, and 4.5％by weight in the triterpenoid mixture, in which the total weight of the triterpenoid mixture is taken as 100％.

According to preferred embodiments of the present disclosure, the present triterpenoid mixture is administered to the subject orally, intravenously, or subcutaneously in the amount of 0.1-100 mg/Kg body weight of the subject, preferably in the amount of 0.5-50 mg/Kg body weight of the subject； more preferably in the amount of 1-10 mg/Kg body weight of the subject.

According to preferred embodiments, the present triterpenoid mixture is administered orally. In this regard, the triterpenoid mixture is preferably in the form of powders that are formulated into tablets or capsules suitable for oral ingestion. Alternatively, the triterpenoid mixture may exist in the form of a solution or a suspension with the triterpenoid mixture homogeneously dispersed therein.

According to optional embodiments, the present triterpenoid mixture may be used in conjugation with another anti-MS agent. Anti-MS agent suitable for use with the present triterpenoid mixture is, for example, adrenocortical steroid, vitamin B₁₂, interferon beta and etc.

The second aspect of the present application is directed to a medicament for treating MS. The medicament comprises an effective amount of the present triterpenoid mixture, and a pharmaceutically acceptable excipient.

Generally, the present triterpenoid mixture is present in the medicament at a level of about 0.01％to 99.9％by weight, based on the total weight of the medicament. In some embodiments, the present triterpenoid mixture is present at a level of at least 0.1％by weight, based on the total weight of the medicament. In certain embodiments, the present triterpenoid mixture is present at a level of at least 5％by weight, based on the total weight of the medicament. In still other embodiments, the present triterpenoid mixture is present at a level of at least 10％by weight, based on the total weight of the medicament. In still yet other embodiments, the present triterpenoid mixture is present at a level of at least 25％by weight, based on the total weight of the medicament.

According to embodiments of the present disclosure, each medicaments comprises 10 to 1,000 mg of the triterpenoid mixture； preferably, 50 to 500 mg of the triterpenoid mixture； and more preferably, 100 to 300 mg of the triterpenoid mixture.

In some embodiments, the medicament of this invention further includes an agent (e.g., anti-MS agent) known to alleviate or ameliorate the symptoms of MS. Examples of such agent include, and are not limited to, adrenocortical steroid, vitamin B₁₂, interferon beta and etc. In one example, interferon beta is administered concurrently with the present triterpenoid mixture.

Pharmaceutically acceptable excipients are those that are compatible with other ingredients in the formulation and biologically acceptable.

The medicament may comprise different types of excipients depending on the intended routes of administration. The present composition may be administered intraveneously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intranasally, intrapleurally, intratracheally, intrarectally, topically, intramuscularly, subcutaneoustly, intravesicularlly, intrapericardially, intraocularally, orally, topically, locally, injection, inhalation, infusion, localized perfusion, in any suitable forms such as powders, creams, liquids, aerosols and etc.

The actual dosage of the medicament may be determined by the attending physician based on the physical and physiological factors of the subject, these factors include, but are not limited to, age, gender, body weight, the disease to be treated, severity of the condition, previous history, the presence of other medications, the route of administration and etc. According to non-limiting examples of the present disclosure, each dosage will give rise to 0.1-100 mg the present triterpenoid mixture per Kg body weight per administration, such as 0.1, 0.2, 0.4, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58.59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100 mg the present triterpenoid mixture per Kg body weight per administration； preferably, 0.5-50 mg the present triterpenoid mixture per Kg body weight per administration, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 mg the present triterpenoid mixture per Kg body weight per administration； more preferably in the amount of 1-10 mg the present triterpenoid mixture per Kg body weight per administration, such as, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 mg the present triterpenoid mixture per Kg body weight per administration.

The medicament containing the present triterpenoid mixture may be in a form suitable for oral use, for example, as tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Medicaments intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the present triterpenoid mixture in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate； granulating and disintegrating agents, for example, corn starch, or alginic acid； binding agents, for example starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc.

The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated to form osmotic therapeutic tablets for controlled release.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredients is mixed with water-miscible solvents such as propylene glycol, PEGs and ethanol, or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

For example, a solid oral medicament such as a tablet or capsule may contain from 1 to 99％ (w/w) the present triterpenoid mixture； from 0 to 99％ (w/w) diluent or filler； from 0 to 20％ (w/w) of a disintegrant； from 0 to 5％ (w/w) of a lubricant； from 0 to 5％ (w/w) of a flow aid； from 0 to 50％ (w/w) of a granulating agent or binder； from 0 to 5％ (w/w) of an antioxidant； and from 0 to 5％ (w/w) of a pigment. A controlled release tablet may in addition contain from 0 to 90％(w/w) of a release-controlling polymer.

A parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50％ (w/w) the present triterpenoid mixture； and from 50％ (w/w) to 99％ (w/w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water) ； and 0-20％ (w/w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilizers, tonicity adjusting agents and preservatives.

The medicaments of the present invention may be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, for example olive oil or peanut oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a preservative, and flavouring and colouring agents. The medicaments may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringers solution and isotonic sodium chloride solution. Co-solvents such as ethanol, propylene glycol or polyethylene glycols may also be used. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The present triterpenoid mixture may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the triterpenoid mixture with a suitable non-irritating excipient which is solid at ambient temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

For topical use, creams, ointments, gels, solutions or suspensions, etc., containingthe present triterpenoid mixture are employed. (For purposes of this application, topical application shall include mouth washes and gargles. ) Topical formulations may generally be comprised of a pharmaceutical carrier, co-solvent, emulsifier, penetration enhancer, preservative system, and emollient.

The following Examples are provided to elucidate certain aspects of the present invention and to aid those of skilled in the art in practicing this invention. These Examples are in no way to be considered to limit the scope of the invention in any manner. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent.

EXAMPLES

Materials and Methods

Animals. C57BL/6J mice were housed in an animal facility with ad libitum access to food and water.

Experimental Autoimmune Encephalomyelitis (EAE) Model of Multiple Sclerosis (MS)

To induce EAE, 10 weeks old female C57BL/6J mice were injected subcutaneously (s. c. ) at two sites on the flanks with 200 μL of an immunization mixture of myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG_35-55) in Complete Freund's Adjuvant (CFA) . The immunization mixture was prepared by mixing MOG_35-55 (2mg/mL) and CFA in a 1: 1 ratio until an emulsified mixture was formed. Pertussis toxin (PT, 400 ng) was given intraperitoneally on day 2 post-immunization. Mice were evaluated daily for clinical signs of disease for up to 14 days after inoculation. Clinical severity was assessed on a scale of 0-5 as indicated in Table 1.

Table 1

Treatment. The triterpenoid mixture of the present invention was orally administered to the MOG/CFA immunized mice with a clinical score of at least 1 at a dose of 600 mg/Kg for 7 consecutive days. Mice were sacrificed at the end of the experiment.

Hematoxylin and Eosin (H&E) Staining. H&E staining was used to show cellular infiltration of spinal cord white matter in mice with acute EAE in comparison with other groups of CFA control and the present mixture treated EAE mice. All staining was performed on tissues that were collected at the end of the experiment in accordance with standard procedures.

IL-17 ELISA.

IL-17 was measured by use of a commercial ELISA kit (Invitrogen cat #KMC3021) in accordance with the manufacturer’s instructions. Standard curves were obtained, and IL-17 levels were determined from the standard curves using standard statistical techniques.

Example 1 Effects of the Present Triterpenoid Mixture on EAE Mice

EAE is a CD4+ T cell-mediated demyelinating disease of the central nervous system (CNS) that serves as a model for MS in humans. The pathogenic mechanisms of EAE development include antigen-specific T cell activation and Th1 differentiation followed by T cell and macrophage infiltration into the CNS. IL-17 contributes to the pathology of multiple sclerosis (MS) . Prior studies indicated that MS lesions of human patients exhibited an increase of IL-17. Accordingly, the efficacy of the present triterpenoid mixture on EAE mice model of MS was evaluated by use of H&E staining, and the determination of blood IL-17 level.

EAE mice were induced and treated in accordance with the steps described in the “Materials and Methods” section, and results are summarized in Table 2 and FIGs 1 to 3.

According to data in Table 2 and FIG 1, the clinical signs of MS in EAE mice appeared on day 2, if left untreated, the EAE conditions worsened with time as the scores gradually increases to above 3.5； by contrast, EAE mice receiving treatment of the triterpenoid mixture had a low score, which is a clear indication that the present triterpenoid mixture was effective in suppressing the development of MS in EAE mice.

Table 2

The afore-mentioned results were confirmed by histological analysis, in which 6-10 um tissue sections were prepared from spinal cords of all mice at the end of the experiment. These tissues were stained with H&E to show inflammatory infiltration, and results from one representative example are illustrated in FIG 2. IN FIG 2, significant amounts of inflammatory cells, demyelination and apoptotic cells were observed in the controlled animals (FIG 2, panel A) ； while tissues taken from animals receiving treatment of the GAs mixture remained healthy (FIG 2, panel B) .

IL-17 is known as an inflammatory chemokine that actives T cells and other types of immune cells and are expressed in large amounts in various immune diseases such as rheumatoid arthritis, lupus, asthma, and multiple sclerosis. Previous findings have confirmed that significant amounts of IL-17 proteins were identified in the spinal cord of EAE mice, thus the level of IL-17 is a good indicator for evaluating a candidate therapeutic of MS. The IL-17 level in EAE mice treated with or without the present triterpenoid mixture is illustrated in FIG 3.

According to FIG 3, the present triterpenoid mixture was capable of reducing the IL-17 level in EAE mice, which is consistent with the findings in FIGs 1 and 2 that the present triterpenoid mixture may suppress or inhibit the progression of MS.

It will be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those with ordinary skill in the art. The above specification, examples and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with ordinary skill in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of the present disclosure.

Claims

Use of a mixture for the manufacture of a medicament for treating multiple sclerosis (MS) , wherein the mixture consists of, ganoderic acid A (GAA) , ganoderic acid B (GAB) , ganoderic acid C (GAC) , ganoderic acid C₅ (GAC₅) , ganoderic acid C₆ (GAC₆) , ganoderic acid D (GAD) , ganoderic acid E (GAE) , ganoderic acid G (GAG) , and ganoderenic acid D.
The use of claim 1, wherein the GAA is present in about 30-40％ by weight in the mixture, the GAB, GAC, GAD and GAG are respectively present in about 10-15％ by weight in the mixture, and the GAC₅, GAC₆, GAE and ganoderenic acid D are respectively present in about 3-6％ by weight in the mixture.
The use of claim 2, wherein the GAA is present in about 35％ by weight in the mixture, the GAB, GAC, GAD and GAG are respectively present in about 12％ by weight in the mixture, and the GAC₅, GAC₆, GAE and ganoderenic acid D are respectively present in about 3, 6 and 4.5％ by weight in the mixture.
A mixture for use in the treatment of multiple sclerosis (MS) , wherein the treatment comprises administering an effective amount of the mixture that consists of, ganoderic acid A (GAA) , ganoderic acid B (GAB) , ganoderic acid C (GAC) , ganoderic acid C₅ (GAC₅) , ganoderic acid C₆ (GAC₆) , ganoderic acid D (GAD) , ganoderic acid E (GAE) , ganoderic acid G (GAG) , and ganoderenic acid D, to a subject in need thereof, to alleviate or ameliorate the symptoms associated with MS.
The mixture for use in claim 3, wherein the GAA is present in about 30-40％ by weight in the mixture, the GAB, GAC, GAD and GAG are respectively present in about 10-15％ by weight in the mixture, and the GAC₅, GAC₆, GAE and ganoderenic acid D are respectively present in about 3-6％ by weight in the mixture.
The mixture for use in claim 5, wherein the GAA is present in about 35％ by weight in the mixture, the GAB, GAC, GAD and GAG are respectively present in about 12％ by weight in the mixture, and the GAC₅, GAC₆, GAE and ganoderenic acid D are respectively present in about 3, 6 and 4.5％ by weight in the mixture.
A method of treating multiple sclerosis (MS) in a subject comprising administering to the subject a mixture consisting of, ganoderic acid A (GAA) , ganoderic acid B (GAB) , ganoderic acid C (GAC) , ganoderic acid C₅ (GAC₅) , ganoderic acid C₆ (GAC₆) , ganoderic acid D (GAD) , ganoderic acid E (GAE) , ganoderic acid G (GAG) , and ganoderenic acid D, in a dose of about 1 to 10 mg/Kg to ameliorate or alleviate symptoms associated with MS.
The method of claim 7, wherein the GAA is present in about 30-40％ by weight in the mixture, the GAB, GAC, GAD and GAG are respectively present in about 10-15％ by weight in the mixture, and the GAC₅, GAC₆, GAE and ganoderenic acid D are respectively present in about 3-6％ by weight in the mixture.
The method of claim 9, wherein the GAA is present in about 35％ by weight in the mixture, the GAB, GAC, GAD and GAG are respectively present in about 12％ by weight in the mixture, and the GAC₅, GAC₆, GAE and ganoderenic acid D are respectively present in about 3, 6 and 4.5％ by weight in the mixture.
The method of claim 7, wherein the subject is a human.