A NOVEL COMBINATION OF EPIMEDIUM-DERIVED-FLAVONOIDS- FRACTIONS FOR PREVENTION OF STEROID-INDUCED OSTEONECROSIS

FIELD OF THE INVENTION

The present invention relates to Epimedium wushanense extracts, pharmaceutical composition comprising Epimedium wushanense extracts, methods of preparing the pharmaceutical composition having the Epimedium wushanense extracts and their use, in particular for inhibiting thrombosis and lipid deposition, and more particularly, for the prevention or treatment of steroid- associated osteonecrosis.

BACKGROUND OF THE INVENTION

Steroid is commonly prescribed for treatment of medical conditions. Pulsed steroids are frequently prescribed as life-saving agent for serious infectious diseases such as Severe Acute Respiratory Syndrome (SARS) and Acquired Immune Deficiency Syndrome (AIDS), or chronic autoimmune disease such as Systemic Lupus Erythematosus (SLE) and Rheumatoid Arthritis (RA) (1- 4). Inevitably, steroid-associated osteonecrosis is frequently reported (5-9), which has been recently highlighted after life-saving treatment for Severe Acute Respiratory Syndrome (SARS) patients (10-12). Total joint replacement is the last option for treatment of osteonecrosis, yet the post-surgical prognosis is poor

in osteonecrosis patients treated with steroid (7, 13-14). Therefore, prevention of development of steroid-associated osteonecrosis before formation of osteonecrosis lesions is highly desirable (8).

A consensus etiopathogenesis of steroid-associated osteonecrosis has been recently unified on both intravascular thrombosis-induced occlusion and extravascular lipid-deposit-induced compression, leading to an impaired structure-function of intra-osseous blood supply system (8,9,13,15). For intravascular thrombosis, imbalance between coagulation and fibrinolysis, which predisposes to both hypercoagulation and hypofibrinolysis, has consistently presented itself in the intravascular events (16-18). For extravascular lipid deposition, abnormal lipid transportation to the peripheral tissue (19) and excessive PPAR γ2-mediated adipogenesis (20) has been involved in extravascular events.

Outbreak of Severe Acute Respiratory Syndrome (SARS) in China in 2003 provides a unique evidence-based medicine for studying steroid-associated osteonecrosis. There is a remarkable difference in prevalence of osteonecrosis recovered from SARS patients between North China 32.7% (14) and South China 5-6% (10,11 ). Interestingly, the frequency of herb preparation used for SARS patients during rehabilitation after pulsed steroid-treatment was much higher in South China (conventional herbal intervention with World Health Organization (WHO) recommended procedure) than in North China (WHO recommended procedure without conventional herbal intervention) (3, 20-22).

The lower osteonecrosis prevalence in South China was suggested to be associated with their potential effects on prevention of steroid-associated osteonecrosis. Antiviral herbal Epimedium with immunomodulation was one of the medicinal herbs (21 ), which has been also used as a broad-spectrum "bone strengthening" herb in traditional Chinese medicine to treat many musculoskeletal diseases (23).

Since inhibition of thrombosis and lipid deposition may help prevent steroid-associated osteonecrosis, understanding of the mechanism of inhibition is crucial. However, there is a need for developing potent and specific chemical inhibitors targeting both thrombosis and lipid deposition for preventing or treating steroid-associated osteonecrosis. Further, there is a need for developing a pharmaceutical composition which can inhibit both thrombosis and lipid deposition and is easy to prepare and handle for use in routine and industrial chemical synthesis.

SUMMARY OF THE INVENTION

It has been found that compounds of the present invention inhibit both thrombosis and lipid deposition for preventing or treating steroid-assoicated osteonecrosis. The present invention therefore provides a novel pharmaceutical composition comprising an extract of Epimedium wushanense Ying for inhibiting thrombosis and lipid deposition.

Accordingly, the present invention includes a pharmaceutical composition wherein the extract is a combination of prenylated flavonol glycoside compounds of the Formula (I):

in which

Ri is selected from the group consisting of glucose, rhamnose, xylose and combinations thereof;

R ₂ is selected from the group consisting of glucose, rhamnose, xylose and hydrogen; and

R ₃ is hydrogen or Ci _-6 alkyl.

Also included in the scope of the present invention is a pharmaceutical composition comprising an extract of Epimedium wushanense Ying, wherein the extract is selected from the group consisting of Epimedoside A, Hexandraside F, Epimedin A, Epimedin B ₁ Epimedin C, lcariin, Baohuoside-I and combinations thereof.

The present invention also includes a pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers or excipients. Still

further, the present invention includes a pharmaceutical composition which is formulated into various preparations in different dosage forms.

In another aspect of the present invention, there is provided a method of inhibiting thrombosis and lipid deposition comprising administering a pharmaceutical composition having an extract of Epimedium wushanense Ying to a subject in need thereof. In particular, the thrombosis is intravascular thrombosis and the lipid deposition is extravascular lipid deposition.

In yet another aspect of the invention, there is provided a method of preventing or treating steroid-associated osteonecrosis comprising administering a pharmaceutical composition having an extract of Epimedium wushanense Ying to a subject in need thereof.

In yet another further aspect of the present invention, there is provided a use of a pharmaceutical composition having an extract of Epimedium wushanense Ying for the manufacture of a medicament for inhibiting thrombosis and lipid deposition. In particular, the thrombosis is intravascular thrombosis and the lipid deposition is extravascular lipid deposition.

In a still further aspect of the present invention, there is provided a use of a pharmaceutical composition having an extract of Epimedium wushanense Ying for the manufacture of a medicament for preventing or treating steroid-associated osteonecrosis.

Moreover, in a further aspect of the present invention, there is provided a method for preparing a pharmaceutical composition, in which the method comprises the steps of:

(a) extracting the plant Epimedium wushanense or a part thereof with a solvent to obtain an extract; and

(b) purifying the extract to obtain prenylated flavonol glycoside compounds of the Formula (I):

in which

Ri is selected from the group consisting of glucose, rhamnose, xylose and combinations thereof;

R ₂ is selected from the group consisting of glucose, rhamnose, xylose and hydrogen; and R ₃ is hydrogen or Ci _-6 alkyl.

Further, within this aspect of the invention, the prenylated flavonol glycoside compounds of the formula I is selected from the group consisting of

Epimedoside A, Hexandraside F, Epimedin A, Epimedin B, Epimedin C, lcariin,

Baohuoside-I and combinations thereof.

By using the above-described method, it has been found that flavonoids fraction extractions from E. wushanense can be achieved with stability and with reproducible results, with both small scale batch and large scale batch. Another advantage of the present invention is that high yields can be achieved from both small scale batch and large scale batch extractions. The extracts can then be purified to afford the pharmaceutical composition of the present invention with high flavonoids content.

For purposes of summarizing the invention and the advantages achieved over the prior art, certain objects and advantages of the invention have been described above. Of course, it is to be understood that not necessarily all such objects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example, those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objects or advantages as may be taught or suggested herein.

Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the

invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described in relation to the drawings.

Figure 1A shows the general structure of the compounds of the Formula (I). Figure 1B shows the chemical information of the major seven compounds in the flavonoids. The common structure is 8-prenylkaempferol. Ri and R2 are substituted by glucose, rhamnose, or xylose, and R ₃ is replaced by methyl. Figure 2 shows the 2-D HPLC profile of Epimedium-derived Flavonoids

(EF).

Figure 3 shows the 3-D HPLC profile of Epimedium-derived Flavonoids (EF).

Figure 4 shows the high similarity in HPLC fingerprints of Epimedium- derived flavonoids from different extraction scales. Upper line is the small scale batch and the lower one is the large scale batch. Three mobile phases were used:

(A) water, (B) methanol, (C) acetonitrile. The elution profile was: 0-25min, A:B:C

(65:12:23), 25-30min, linear gradient to A:B:C (40:35:25), 30-60min, linear gradient to A:B:C (35:40:25), and 61-65min, linear gradient to A:B:C (20:55:25); flow rate: 1.0 mL/min; wavelength: 270nm; oven temperature: 3O ⁰ C.

Figure 5 shows the similarities between the preparations of the small scale batch and the large scale batch.

Figure 6 shows the key characteristics during histopathological identification. Figure 6A shows that Osteonecrosis (ON) lesion was found with trabecular bone containing considerable empty lacunae. Figure 6B shows that in ON ⁺ rabbits, thrombi were predominantly found in small marrow vessels in ON ⁺ rabbits, and marrow was predominantly occupied by numerous fat cells. Hematoxylin-Eosin staining, x 200.

Figure 7 shows the Histopathological data analysis. Figure 7A shows the incidence of ON in each group: CON (13/14, 93%), L-EF (9/16, 56%), M-EF (2/16, 13%), H-EF (1/16, 6%). Figure 7B shows that there was no significant difference in the ON Extent among all the groups. Figure 7C and Figure 7D show that in both Thrombotic Vessel Counts, and Fat Cell Area Fraction, there were similarities in changing pattern over time, i.e. either attenuated in L-EF group or prevented in both M-EF and H-EF groups when compared to that in CON group. • for CON group; ♦ for L-EF group; ■ for M-EF group; ▲ for H-EF group; * for P<0.05.

Figure 8 shows the Hematology / cytology data analysis. Significantly decreased APTT (Figure 8B) and t-PA/PAI-l (Figure 8C) from baseline in CON group were attenuated in L-EF group or prevented in both M-EF and H-EF groups at week 1 post induction, whereas significantly increased TM (Figure 8A), LDL/HDL (Figure 8C) and Adipocyte positive colonies (Figure 8D) in CON group were attenuated in L-EF group or prevented in both M-EF and H-EF groups after induction. P<0.05 for comparison with CON; # P<0.05 for comparison with

baseline. • for CON group; ♦ for L-EF group; ■ for M-EF group; ▲ for H-EF group.

Figure 9 shows the dynamic MRI data during longitudinal study. In Figure 9A, at week 1 post induction, the significantly decreased PEP in CON group was attenuated in L-EF group or prevented in both M-EF and H-EF groups. In Figure 9B, throughout the experimental period, the significantly increased PSp in CON group was attenuated in L-EF group or almost prevented in both M-EF and H-EF groups. Figure 9C shows the comparison in representative Dynamic MRI derived Time-Intensity Curve at week 1 post induction between CON group and M-EF group. * for P<0.05. • for CON group; ♦ for L-EF group; ■ for M-EF group; ▲ for H-EF group. Black curve for M-EF group; Grey curve for CON group.

Figure 10 shows representative three-dimensional angiograms and histograms from MicroCT-based angiography. Figure 10A shows pattern of angiographic structural units in both baseline before induction and M-EF or H-EF group at week 2 post induction. Figure 10B shows pattern of angiographic structural units in CON group at week 1 post induction. Figure 10C shows pattern of angiographic structural units in CON group at week 2 post induction. Figure 10D shows that in CON group, angiographic structural units ranged from 400- 600μm or 36-200μm were less at week 1 post induction than at baseline, whereas angiographic structural units ranged from 200-400μm were more at week 1 post induction than at baseline. Figure 10E shows that angiographic structural units ranged from 36-200μm or 400-600μm were slightly lower in L-EF

group at week 1 post induction than that at baseline. Figure 1OF shows no difference in pattern of angiographic structural units between baseline before induction and M-EF group or H-EF group at week 1 post induction. Figure 10G shows angiographic structural units ranged from 400-600μm were less in CON group at week 2 post induction than that at baseline, whereas angiographic structural units ranged from 36-200μm were more in CON group at week 2 post induction than that at baseline. Figure 10H shows that angiographic structural units ranged from 400-600μm were slightly lower in L-EF group at week 2 post induction than that at baseline, whereas angiographic structural units ranged from 36-200μm were slightly higher than that at baseline. Figure 101 shows that there is no difference in pattern of angiographic structural units between baseline and M-EF group or H-EF group at week 2 post induction. Red circle for CON group at week 1 post induction. Blue square for Baseline before induction. Brown rhombus for L-EF group. Green rhombus for either M-EF or H-EF group. Figure 11 shows that there are extravascular leakage particles during perfusion for MicroCT-based angiography. Figures 11A and 11B show representative histology images of particles (indicated by arrow) were extravascularly distributed in CON group at week 2 post induction, whereas particles were intravascular^ distributed in M-EF group at week 2 post induction. Figures 11 C and 11D show representative histograms of extravascular leakage particles mainly ranged from 200 to 400 μm. Extravascular leakage particles found more in CON group since week 1post induction, whereas those rarely

found in both M-EF and H-EF groups but few in L-EF after induction. • for CON group; ♦ for L-EF group; ■ for M-EF group; A for H-EF group.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It has been demonstrated for the first time that a pharmaceutical composition comprising an extract of Epimedium wushanense Ying can be used for inhibiting thrombosis and lipid deposition, and that these compositions can prevent or treat steroid-associated osteonecrosis.

While not wishing to be limited by theory, these effects are likely due to a unique mechanism which can target the two pathways of steroid-induced osteonecrosis. That is, by inhibiting both intravascular thrombosis and extravascular lipid deposition.

Thus, the pharmaceutical composition of the present invention represents a new class of drugs that can act through both pathways. The pharmaceutical composition of the present invention can simultaneously target both intravascular thrombosis and extravascular lipid deposition. Subsequently, the pharmaceutical composition of the present invention is useful for preventing or treating steroid- associated osteonecrosis.

Accordingly, in one of its aspect, the present invention includes a pharmaceutical composition in which the extract is a combination of prenylated flavonol glycoside compounds of the Formula (I):

(I) in which

Ri is selected from the group consisting of glucose, rhamnose, xylose and combinations thereof;

R ₂ is selected from the group consisting of glucose, rhamnose, xylose and hydrogen; and

R ₃ is hydrogen or d-β alkyl.

The compounds of Formula (I) include those in which Ri is selected from the group consisting of rhamnose, glucose-rhamnose, xylose-rhamnose and rhamnose-rhamnose. The compounds of Formula (I) include those in which R ₂ is glucose or hydrogen. The compounds of Formula (I) include those in which R ₃ is hydrogen or C _h alky!. In embodiments of the invention, R ₃ is methyl.

More particularly, in embodiments of the invention, the compounds are selected from the group consisting of Epimedoside A, Hexandraside F, Epimedin A, Epimedin B, Epimedin C, lcariin, Baohuoside-I and combinations thereof.

The term "C _h alky!" as used herein means straight and/or branched chain, saturated alkyl radicals containing from one to n carbon atoms and includes (depending on the identity of n) methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl,

isobutyl, t-butyl, 2,2-dimethylbutyl, n-pentyl, 2-methylpentyl, 3-methylpentyl, 4- methylpentyl, n-hexyl and the like.

In embodiments of the pharmaceutical composition of the present invention, Epimedoside A is present in an amount of about 0.5 to 3.5% by weight, Hexandraside F is present in an amount of about 0.2 to 2.5% by weight, Epimedin A is present in an amount of about 0.5 to 3.0% by weight, Epimedin B is present in an amount of about 0.5 to 3.5% by weight, Epimedin C is present in an amount of about 2.0 to 6.0% by weight, lcariin is present in an amount of about 70.0 to about 95.0% by weight and Baohuoside-I is present an amount of about 0.5 to about 4.0% by weight.

In more particular embodiments of the pharmaceutical composition of the present invention, Epimedoside A is present in an amount of about 1.3 to 2.3% by weight, Hexandraside F is present in an amount of about 0.5 to 1.5% by weight, Epimedin A is present in an amount of about 0.7 to 1.7% by weight, Epimedin B is present in an amount of about 1.0 to 2.0% by weight, Epimedin C is present in an amount of about 3.2 to 5.2% by weight, lcariin is present in an amount of about 75.0 to about 85.0% by weight and Baohuoside-I is present an amount of about 1.5 to about 2.5% by weight.

The pharmaceutical compositions of the present invention are suitably formulated for administration to human subjects in a biologically compatible form suitable for administration in vivo. Accordingly, in another aspect, the present

invention includes a pharmaceutical composition of the present invention further comprising one or more pharmaceutically acceptable carriers or excipients.

The pharmaceutical composition of the invention can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (2003 - 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999. On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.

The term "pharmaceutically acceptable" means compatible with the treatment of animals, in particular, humans.

In accordance with the methods of the invention, the described pharmaceutical composition thereof may be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The compositions of the invention may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump or transdermal (topical) administration. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical modes of

administration. Parenteral administration may be by continuous infusion over a selected period of time.

A pharmaceutical composition of the invention may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the composition of the invention may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. A composition of the invention may also be administered parenterally.

Solutions of a composition of the invention can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.

Ampoules are convenient unit dosages.

Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device. Alternatively, the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as fluorochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer.

Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.

Compositions for topical administration may include, for example, propylene glycol, isopropyl alcohol, mineral oil and glycerin. Preparations suitable for topical administration include liquid or semi-liquid preparations such as liniments, lotions, applicants, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes; or solutions or suspensions such as drops. In

addition to the aforementioned ingredients, the topical preparations may include one or more additional ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives, e.g. methyl hydroxybenzoate (including anti-oxidants), emulsifying agents and the like. Sustained or direct release compositions can be formulated, e.g. liposomes or those wherein the active compound is protected with differentially degradable coatings, such as by microencapsulation, multiple coatings, etc. It is also possible to freeze-dry the compounds of the invention and use the lypolizates obtained, for example, for the preparation of products for injection. The pharmaceutical composition of the invention may be administered to a subject alone or in combination with other pharmaceutically acceptable carriers, as noted above, and/or with other pharmaceutically active agents for the prevention or treatment of steroid-associated osteonecrosis, the proportion of which is determined by the solubility and chemical nature of the composition, chosen route of administration and standard pharmaceutical practice.

In embodiments of the present invention, the pharmaceutical composition is formulated into various preparations in different dosage forms. The dosage of the compositions of the invention can vary depending on many factors such as the pharmacodynamic properties of the composition, the mode of administration, the age, health and weight of the recipient, the nature and extent of the symptoms, the frequency of the treatment and the type of concurrent treatment, if any, and the clearance rate of the composition in the animal to be treated. One

of skill in the art can determine the appropriate dosage based on the above factors. The pharmaceutical composition of the present invention may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. For ex vivo treatment of cells over a short period, for example for 30 minutes to 1 hour or longer, higher doses of compound may be used than for long term in vivo therapy.

The pharmaceutical composition of the present invention can be used alone or in combination with other agents or therapies. In more particular embodiments of the present invention, the pharmaceutical composition can be used for inhibiting intravascular thrombosis. In another more particular embodiment of the present invention, the pharmaceutical composition can be used for inhibiting extravascular lipid deposition. In yet another embodiment of the present invention, the pharmaceutical composition of the present invention can be used for the prevention or treatment of steroid-associated osteonecrosis. Still further, also within the scope of the present invention is a pharmaceutical composition comprising an extract of Epimedium wυshanense Ying, wherein the extract is selected from the group consisting of Epimedoside A, Hexandraside F, Epimedin A ₁ Epimedin B, Epimedin C, lcariin, Baohuoside-I and combinations thereof for inhibiting thrombosis and lipid deposition. Particularly, in embodiments of the invention, the Epimedoside A is present in an amount of about 0.5 to 3.5% by weight, Hexandraside F is present in an amount of about 0.2 to 2.5% by weight, Epimedin A is present in an amount of about 0.5 to 3.0%

by weight, Epimedin B is present in an amount of about 0.5 to 3.5% by weight, Epimedin C is present in an amount of about 2.0 to 6.0% by weight, icariin is present in an amount of about 70.0 to about 95.0% by weight and Baohuoside-I is present an amount of about 0.5 to about 4.0% by weight. In more particular embodiments of the invention, Epimedoside A is present in an amount of about 1.3 to 2.3% by weight, Hexandraside F is present in an amount of about 0.5 to 1.5% by weight, Epimedin A is present in an amount of about 0.7 to 1.7% by weight, Epimedin B is present in an amount of about 1.0 to 2.0% by weight, Epimedin C is present in an amount of about 3.2 to 5.2% by weight, Icariin is present in an amount of about 75.0 to about 85.0% by weight and Baohuoside-I is present an amount of about 1.5 to about 2.5% by weight.

In embodiments of the invention, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers or excipients. Further, in embodiments of the invention, the composition is formulated into various preparations in different dosage forms.

The pharmaceutical composition of the present invention can be used for inhibiting intravascular thrombosis. In other embodiments of the invention, the pharmaceutical composition can be used for inhibiting extravascular lipid deposition. Moreover, in embodiments of the invention, the pharmaceutical composition can be used for the prevention or treatment of steroid-associated osteonecrosis.

The present invention further includes a method of inhibiting thrombosis and lipid deposition which comprises administering an effective amount of a pharmaceutical composition of the invention to a subject in need thereof. The present invention also includes a use of a pharmaceutical composition of the invention to inhibit thrombosis and lipid deposition and a use of a pharmaceutical composition of the invention to prepare a medicament to inhibit thrombosis and lipid deposition.

More particularly, the method of inhibiting thrombosis and lipid deposition comprises administering a pharmaceutical composition having an extract of Epimedium wushanense Ying to a subject in need thereof.

In embodiments of the present invention, the method is used for inhibiting intravascular thrombosis. Also within the scope of the present invention, the method is used for inhibiting extravascular lipid deposition.

It is to be clear that, in the method as described above, the extract is a combination of prenylated flavonol glycoside compounds of the Formula (I):

in which

Ri is selected from the group consisting of glucose, rhamnose, xylose and combinations thereof;

R ₂ is selected from the group consisting of glucose, rhamnose, xylose and hydrogen; and R ₃ is hydrogen or C _h alky!.

In embodiments of the invention, the extract is selected from the group consisting of Epimedoside A, Hexandraside F, Epimedin A, Epimedin B, Epimedin C, lcariin, Baohuoside-I and combinations thereof.

In yet another embodiment of the method described above, the pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers or excipients. Still further, in embodiments of the method described above, the pharmaceutical composition is formulated into various preparations in different dosage forms.

Also within the scope of the present invention is a method of preventing or treating steroid-associated osteonecrosis comprising administering a pharmaceutical composition having an extract of Epimedium wυshanense Ying to a subject in need thereof. In embodiments of the invention, the extract is a combination of prenylated flavonol glycoside compounds of the Formula (I):

in which

R ₁ is selected from the group consisting of glucose, rhamnose, xylose and combinations thereof;

R ₂ is selected from the group consisting of glucose, rhamnose, xylose and hydrogen; and

R ₃ is hydrogen or Ci _-6 alkyl.

In more particular embodiments of the invention, the extract is selected from the group consisting of Epimedoside A, Hexandraside F, Epimedin A, Epimedin B, Epimedin C, lcariin, Baohuoside-I and combinations thereof.

Further, in embodiments of the method of the present invention as described above, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients. Moreover, in embodiments of the method of the present invention as described above, the pharmaceutical composition is formulated into various preparations in different dosage forms.

Administering a compound to a subject includes in vivo, ex vivo and in vitro treatment.

The term an "effective amount" or a "sufficient amount " of an agent as used herein is that amount sufficient to effect beneficial or desired results, including clinical results, and, as such, an "effective amount" depends upon the context in which it is being applied. For example, in the context of administering an agent that prevents or treats steroid-associated osteonecrosis, an effective amount of an agent is, for example, an amount sufficient to achieve such a treatment as compared to the response obtained without administration of the agent.

As used herein, and as well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

The term "subject" as used herein includes all members of the animal kingdom including human. The subject is suitably human. Moreover, the present invention includes a use of a pharmaceutical composition having an extract of Epimedium wushanense Ying in the manufacture of a medicament for inhibiting thrombosis and lipid deposition. In

embodiments of the present invention, the use of the pharmaceutical composition in the manufacture of the medicament is for inhibiting intravascular thrombosis. In other embodiments of the present invention, the use is for inhibiting extravascular lipid deposition. Particularly, in the use as described above, the extract is a combination of prenylated flavonol glycoside compounds of the Formula (I):

in which

Ri is selected from the group consisting of glucose, rhamnose, xylose and combinations thereof;

F? ₂ is selected from the group consisting of glucose, rhamnose, xylose and hydrogen; and R ₃ is hydrogen or Ci _-6 alkyl.

In embodiments of the invention, the extract is selected from the group consisting of Epimedoside A, Hexandraside F, Epimedin A, Epimedin B,

Epimedin C, lcariin, Baohuoside-I and combinations thereof.

In other embodiments of the invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.

In yet another embodiment of the invention, the pharmaceutical composition is formulated into various preparations in different dosage forms.

Also within the scope of the invention is a use of a pharmaceutical composition having an extract of Epimedium wushanense Ying for the manufacture of a medicament for preventing or treating steroid-associated osteonecrosis.

In particular embodiments of the invention, the extract is a combination of prenylated flavonol glycoside compounds of the Formula (I):

in which R ₁ is selected from the group consisting of glucose, rhamnose, xylose and combinations thereof;

R ₂ is selected from the group consisting of glucose, rhamnose, xylose and hydrogen; and R ₃ is hydrogen or C _h alky!.

In more particular embodiments of the invention, the extract is selected from the group consisting of Epimedoside A, Hexandraside F, Epimedin A, Epimedin B, Epimedin C, lcariin, Baohuoside-I and combinations thereof.

Moreover, in embodiments of the invention, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers or excipients. Still further, the pharmaceutical composition may be formulated into various preparations in different dosage forms.

The pharmaceutical composition of the present invention can be prepared using a method comprising the steps of: (a) extracting the plant Epimedium wushanense or a part thereof with a solvent to obtain an extract; and

(b) purifying the extract to obtain prenylated flavonol glycoside compounds of the Formula (I):

in which

Ri is selected from the group consisting of glucose, rhamnose, xylose and combinations thereof;

F?2 is selected from the group consisting of glucose, rhamnose, xylose and hydrogen; and

R ₃ is hydrogen or C _h alky! .

In embodiments of the invention, the plant is Epimedium wushanense leaves.

In particular embodiments of the invention, the solvent in step (a) is about 40 to 90% alcohol, suitably about 40 to 80% alcohol, more suitably about 60 to 75% alcohol. In more particular embodiments of the invention, the alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol and hexanol. In still more particular embodiments of the invention, the solvent in step (a) is about 60 to 80% ethanol, mores suitably about 70% ethanol.

In embodiments of the invention, in the method as described above, following step (a), the extract was suspended in water to form a solution. In embodiments of the invention, the solution is filtered to obtain a filtrate. In more particular embodiments of the invention, the filtrate is partitioned between water and an alcohol to obtain a water portion and an alcohol portion, and wherein the extract is in the alcohol portion. Still further, in embodiments of the invention, the alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol and hexanol. Particularly, in embodiments of the invention, the butanol alcohol is n-butyl alcohol.

It is an embodiment in the method of the present invention that the extract at step (b) is purified by purification techniques selected from the group

consisting of chromatography, recystallization, filtration and combinations thereof. It is a more particular embodiment of the invention that the extract at step (b) is purified by column chromatography in which the extract is fractionated to obtain a plurality of fractions. Particularly, in embodiments of the invention, the extract is eluted with chloroform-methanol. In embodiments of the invention, the chloroform-methanol is in the range of about 9:1 to 6:4. Still more particularly, in embodiments of the invention, the chloroform-methanol is in the range of about 8:2 to 7:3.

In yet another embodiment of the method as described above, the plurality of fractions are assayed for the prenylated flavonol glycoside compounds of the Formula (I). Particularly, in embodiments of the invention, the prenylated flavonol glycoside compounds of the Formula (I) is selected from the group consisting of Epimedoside A, Hexandraside F, Epimedin A, Epimedin B, Epimedin C, lcariin, Baohuoside-I and combinations thereof. While the following Examples illustrate the invention in further detail, it will be appreciated that the invention is not limited to the specific Examples.

EXAMPLES Chemical Compounds

Seven prenylated flavonol glycosides were extracted from Epimedium wushanense Ying, and identified as Epimedoside A (1), Hexandraside F (2), Epimedin A (3), Epimedin B (4), Epimedin C (5), lcariin (6), and Baohuoside-I (7) which can be seen in Figure 1.

In the HPLC profile, at 270 nm, their percentages of area under the curve

(AUC) were 1.8%, 1.0%, 1.2%, 1.5%, 4.2%, 80.9%, and 2.1%, respectively. The present inventors have found that investigation of the HPLC-UV-MS/MS spectra of flavonoid fraction from E. wushanense allowed rapid identification of 92.7% constituents in it (Figures 1 to 3).

Synthesis of the Pharmaceutical Composition of the Present Invention Plant Material Leaves of E. wushanense were collected from Lei Shan, Gui Zhou province, China, a planting field with Good Agricultural Practice (GAP) certification for E. wushanense, and authenticated by the College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. A voucher specimen (EW 2006) was preserved in Shenzhen Research Center of Traditional Chinese Medicines and Natural Products, Shenzhen, China.

Extraction:

Small Scale Extraction

The leaves of E. wushanense (2 kg) were extracted with 70% ethanol (20 L, reflux, 2 hrs, and * 2). The ethanol was removed under reduced pressure to give a residue (21 g), which was suspended in water and stirred. After standing for about 12 hrs, the solution was filtered and the filtrate was partitioned between water and n-BuOH. The n-BuOH portion (6.4 g) was concentrated by vacuum- evaporation and then subjected to silica gel column chromatography. The column was washed with chloroform-methanol system. The flavonoids extract obtained was analyzed by thin layer chromatography (TLC). Flavonoids extraction (2.3 g) was then concentrated in the 8:2 CHCI ₃ -MeOH and 7:3 CHCI ₃ -MeOH eluants.

Large Scale Extraction

The leaves of E. wushanense (50 kg) were extracted with 70% ethanol (500 L, reflux, 2 hrs, and * 2). The ethanol was removed under reduced pressure to give a residue (490 g), which was suspended in water and stirred. After standing for about 12 hrs, the solution was filtered and the filtrate was partitioned between water and n-BuOH. The n-BuOH portion (168 g) was concentrated by vacuum- evaporation and then subjected to silica gel column chromatography. The column was washed with chloroform-methanol system. The flavonoids extract obtained was analyzed by thin layer chromatography (TLC). Flavonoids extraction (49 g) was then concentrated in the 8:2 CHCI ₃ -MeOH and 7:3 CHCI ₃ -MeOH eluants.

Comparison between Small Scale and Large Scale Extractions To yield reproducible results, the plant resource was guaranteed by Good Agriculture Practice (GAP) plant habitat in Guizhou. The stability of the process of making flavonoids fraction from E. wushanense was proved by quantifying the similarity between two batches (small scale and large scale preparations) of samples by using vectorial angle method. The similarity of HPLC chromatograms between small scale batch and large scale batch was 98.2% according to retention times and 99.9% according to peak areas (Figure 4, Figure 5). MA TERIALS AND METHODS ANIMAL MODEL

Rabbits were intravenously injected with 10 μg/kg body weight of Lippolysaccharide (LPS; Escherichia coli 0111 :B4, Sigma-Aldrich, Inc. USA) on day 0 (week 0). Twenty-four hours later, three injections of 20 mg/kg of Methylprednisolone (MPS; Pharmacia & Upjohn, USA) were given intramuscularly at a time interval of 24 hours.

EXPERIMENTAL DESIGN

One hundred and twelve male 28-week-old New-Zealand white rabbits with body weight of 4-5 kg were housed at the Laboratory Animal Service Center of Prince of Wales Hospital, the affiliated hospital of the Chinese University of

Hong Kong. The animals were given a standard laboratory diet and water ad

lititum. The experimental protocol was approved by Animal Experiment Ethics Committee in Hong Kong. All the rabbits were given the injection protocol as described above (Animal Model) for inducing steroid-associated osteonecrosis to evaluate the prevention effect of Epimedium-derived flavonoids (EF). The rabbits were divided into 4 groups for the following daily oral administration: low dose EF group (L-EF; n=28; 10mg/kg body weight/day), middle dose EF group (M-EF; n=28; 20mg/kg body weight/day), high dose EF group (H-EF; n=28; 40mg/kg body weight/day), and one control group was given the corresponding vehicle of EF daily (CON; n=28). The phytochemical profile of EF, in which the common structure of the EF is 8-prenylkaempferol, was identified by HPLC. The two-dimensional HPLC profile of EF is shown in Figure 2, while the three-dimensional HPLC profile of EF is shown in Figure 3. At week 0 (immediately before LPS injection), week 1 and week 2 post induction, blood samples were taken for systemic examination of coagulation index, fibrinolysis index and lipid-transportation index. Marrow sample from iliac crest was also obtained for local evaluation of adipogenic potential index of mesenchymal stem cell. Dynamic-MRI was locally performed on proximal femur of each animal for intra-osseous perfusion function index at week 0 (immediately before LPS injection), week 1 and week 2 post induction before sacrifice. Furthermore, 4, 8 and 16 rabbits in each group were sacrificed at week 0, 1 and 2 post induction, respectively. After sacrifice using overdose Sodium Pentobarbitone, bilateral proximal femora were dissected for MicroCT-

based angiography, histopathological examination on local ON lesion, intravascular thrombosis and extravascular lipid-deposition. The body weight of each rabbit before sacrifice was documented. Survival condition throughout the experiment period in each group was recorded.

Mechanistic Pathway of the Pharmaceutical Composition of the Present

Invention: Effect on Pathogenic Events Involving Intravascular/Extravascular

Abnormalities

Intravascular Histopatholoov and its Contributive Events Histopathologically, thrombi were predominantly found in small marrow vessels in CON group since week 1 post induction (Figure 6). The Thrombotic Vessel Counts in CON group increased to peak level at week 1 post induciton, and then maintained until week 2 post indcution. The patterns of the Thrombotic Vessel Counts changes over time were different between CON group and the three EF groups. The increased Thrombotic Vessel Counts in CON group were attenuated in either L-EF group or prevented in both M-EF and H-EF group throughout the experimental period. Fisher's exact probability test showed that the order of the Thrombotic Vessel Counts at week 1 post indctuion was similar to those at week 2 post indctuion, i.e. CON > L-EF > M-EF = H-EF (Figure 7). For initially contributive endothelium damage event to intravascular thrombosis, the TM in CON group presented a significant increase at week 1 post induction (P=0.000), then declined toward baseline. The patterns of the TM

changes over time were significantly different between CON group and the three EF groups (P<0.05 for L-EF vs CON, P<0.01 for both M-EF vs CON and H-EF vs CON). Especially, the significantly increased TM in CON group was either attenuated in L-EF group or almost prevented in both M-EF and H-EF group at week 1 post induction (Figure 8A).

For contributive hypercoagulation event to intravascular thrombosis, the APTT in CON group decreased significantly at week 1 post induction (P<0.01 ), then declined toward baseline. The patterns of the APTT changes over time were significantly different between CON group and the three EF groups by Repeated Measure ANOVA (P<0.05 for L-EF vs CON, P<0.01 for both M-EF vs CON and H-EF vs CON). Especially, the significant decrease in APTT in CON group was either attenuated in L-EF group or almost prevented in both M-EF and H-EF group at week 1 post induction (Figure 8B).

For contributive hypofibrinolysis event to intravascular thrombosis, the tPA/PAI-l in CON group presented a significant decrease at week 1 post induction (P<0.01), then returned toward baseline. The patterns of the tPA/PAI-l changes over time were significantly different between CON group and the three EF groups (P<0.05 for L-EF vs CON, P<0.01 for both M-EF vs CON and H-EF vs CON). Especially, the significantly decreased tPA/PAI-l in CON group was either attenuated in L-EF group or almost prevented in both M-EF and H-EF group at week 1 post induction (Figure 8C).

Extravascular Histopathology and its Contributive Events

Histopathologically, marrow was predominantly occupied by numerous fat cells in CON group after induction (Figure 6). The Fat Cell Area Fraction in CON group increased signficantly at week 1 post inducton, and then maintained until week 2 post induction (P<0.05 for both). The patterns of the Fat Cell Area Fraction changes over time were different between CON group and the three EF groups. The significantly increased Fat Cell Area Fraction in CON group was either attenuated in L-EF group or prevented in both M-EF and H-EF groups throughout the experimental period. Fisher's exact probability test showed that the order of the Fat Cell Area Fraction at week 1 post induction was similar to those at week 2 post indctuion, i.e. CON > L-EF > M-EF = H-EF (Figure 7).

For contributive adipogenesis event to extravascular lipid-deposition, the Adipocyte Positive Colonies in CON group increased significantly at week 1 post induction (P<0.01 ), then moderately restored toward baseline. The patterns of the Adipocyte Positive Colonies changes over time were significantly different between CON group and the three EF groups (P<0.05 for L-EF vs CON, P<0.01 for both M-EF vs CON and H-EF vs CON). Especially, the significantly increased Adipocyte Positive Colonies in CON group were either attenuated in L-EF group or almost prevented in both M-EF and H-EF groups at week 1 post induction (Figure 8D).

For contributive lipid transportation event to extravascular lipid-deposition, the LDL / HDL in CON group increased significantly at week 1 post induction

(P<0.01), then returned to baseline. The patterns of the LDL / HDL changes over time were significantly different between CON group and the three EF groups (P<0.05 for L-EF vs CON, M-EF vs CON and H-EF vs CON, respectively). Especially, the significant increase in LDL / HDL in CON group was either attenuated in L-EF group or almost prevented in both M-EF and H-EF group at week 1 post induction (Figure 8E).

TESTRESULTS

Neither bleeding nor death was found in each group throughout the experimental period. Osteonecrosis lesion was not found until week 2 post induction in each group. Osteonecrosis lesion was histopathologically characterized with trabecular bone containing considerable empty lacunae (Figure 6). The Osteonecrosis Incidence was 93% (15/16) in CON group, 56% (9/16) in L-EF group, 13% (2/16) in M-EF group and 6% (1/16) in H-EF group, respectively. Fisher's exact probability test showed that the Osteonecrosis Incidence in L-EF, M-EF and H-EF group was significantly lower than that in CON group (P=0.039, P=0.000 and P=0.000, respectively). The Osteonecrosis Incidence in M-EF and H-EF group was significantly lower than that in L-EF group (P=0.023 and P=0.005 for both), whereas no difference in the Osteonecrosis Incidence was found between M-EF and H-EF group (P=LOOO for both) (Figure 7A). There was no significant difference in the Osteonecrosis

Extent among CON group (2.8±0.8), L-EF group (2.5±0.6), M-EF group (2.4+0.7) and H-EF group (2.6±0.5) (Figure 7B).

PHARMACOLOGICAL DATA Effect on Pathophysiological Pathway Involving Abnormal Structure-Function of Intra-osseous Vasculature Vascular Perfusion Function

For Dynamic MRI based vascularization index, the PEP in CON group decreased significantly at week 1 post induction (P<0.01 ), then significantly increased over baseline at week 2 post induction (P<0.01 ). The patterns of the PEP changes over time were significantly different between CON group and the three EF groups (P<0.01 for L-EF vs CON, M-EF vs CON and H-EF vs CON, respectively). Especially, the significant decrease in PEP in CON group was either attenuated in L-EF group or almost prevented in both M-EF and H-EF groups at week 1 post induction (Figure 9A, 9C).

For Dynamic MRI based permeability index, the PSp in CON group increased significantly at week 1 post induction (P<0.01 ), then further significantly increased at week 2 post induction (P<0.01 ). The patterns of the PSp changes over time were significantly different between CON group and the three EF groups (P<0.05 for CON group vs CON, P<0.01 for both M-EF vs CON and H-EF vs CON). Especially, the significant increase in PSp in CON group

were either attenuated in L-EF group or almost prevented in both M-EF and H-EF groups throughout the experimental period (Figure 9B ₁ 9C).

Intra-osseous Vascular Structure Representative three-dimensional angiograms are shown in Figure 10A to

1OC and representative histograms are shown in Figure 10D to 101.

The angiograms in CON group at week 1 post induction showed dilated vessel-like structural units (>600μm) surrounded by both numerous middle size disseminated leakage-particle-like structural units (200~400μm) and few vessel- like structural units (36~200μm or 400~600μm), when compared to those at baseline (Figure 10D). Relative to those in CON group at week 1 post induction, no typical dilated vessel-like structural units surrounded by numerous middle size , disseminated leakage-particle-like structural units (200~400μm) were found in both M-EF group and H-EF groups with the exception of a few in the L-EF group. Reduced perfusion to vessel-like structural units (36~200μm or 400~600μm) were rarely found in both M-EF group and H-EF group but moderate in L-EF group (Figure 10E and 10F).

At week 2 post induction, the angiograms in CON group demonstrated dilated vessel-like structural units (>600μm) surrounded by both numerous small size vessel-like structural units (36~200μm) and numerous middle size disseminated leakage-particle-like structural units (200~400μm) when compared to those at baseline (Figure 10G). Relative to those in CON group at week 2 post

induction, dilated vessel-like structural units surrounded by both many small size vessel-like structural units (36~200μm) and numerous middle size disseminated leakage-particle-like structural units (200~400μm) were not found in both M-EF group and H-EF group but moderate in the L-EF group, and reduced perfusion to vessel-like structural units (400~600μm) were rarely found in both M-EF group and H-EF group but moderate in L-EF group (Figure 1OH and 101).

Examination of Size Distribution of Extravascular Leakage Particles

More extravascular leakage particles were found during perfusion for MicroCT-based angiography in CON group after induction, whereas they were rarely found in both M-EF and H-EF groups with the exception of a few in the L-

EF group after induction. All histograms showed that the size of the leakage particles ranged between 200μm and 400μm (Figure 11A-11 D).

RESUL TS AND DISCUSSION

EF exerted dose-dependent effect on inhibition of both thrombosis and lipid- deposition for maintaining integrity of structure-function of intraosseous vasculature and accordingly reducing incidence of steroid-associated ON in a rabbit model. The underlying mechanism could be explained by their counteraction effects on hypercoagulation, hypofibrinolysis, excessive adipogenesis and prominent lipid transport to peripheral tissues.

While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.

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