WO2002066491A1 - Water soluble boswellic acids, their preparation and use for treating imflammatory conditions - Google Patents

Abstract

A new composition which can be formed through a method comprising: (a) dissolving mixtures of boswellic acids in a water and alcohol solution to form a mixture; (b) adding one or more alkali salts to the mixture to form a salt solution; (c) filtering the solution to separate un-reacted alkali salt from a filtrate; and (d) recovering the soluble boswellic acid mixture from the filtrate. Additionally, the new composition can be formed by using super critical carbon dioxide. The new composition can be used to alleviate numerous inflammatory conditions, including, but not limited to, rheumatoid arthritis and osteoarthritis, colon cancer, prostate cancer and breast cancer, and a broad range of neurodegenerative conditions, such as Alzheimer"s disease and Parkinson"s disease. The composition can be administered parenterally, orally, or topically.

Description

WATER SOLUBLE BOSWELLIC ACIDS , THEIR PREPARATION AND USE FOR TREATING INFLAMMATORY CONDITIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 60/268,713, filed February 15, 2001.

[0002] The present invention concerns a new composition of boswellic acids, methods of using the composition to treat inflammatory conditions, and methods of producing the new composition. BACKGROUND OF THE INVENTION:

[0003] Boswellia serrata is a large, branching, deciduous tree, which grows abundantly in the dry, hilly parts of India. The gum resin exudate of this tree, known in the vernacular as "Salai guggal", has been used in the Ayurvedic system of medicine for the management of arthritis, respiratory diseases, and liver disorders. The major use of Boswellia serrata in modern medicine is as an anti-arthritic and anti-inflammatory pharmacological agent.

[0004] The active principles found in the gum resin, specifically a combination of boswellic acids, have emerged as effective non-steroidal anti-inflammatory compounds (NSAID's) with broad biological activities and also a low ulcerogenic index. Compared experimentally with the anti-inflammatory drug phenylbutazone, boswellic acids did not produce injury to the gastrointestinal mucosa. The most popular NSAID, aspirin, although much better tolerated than its parent compound salicylates, still has serious side effects, e.g. gastrointestinal irritation and bleeding which limit its long-term use. In addition, aspirin is contraindicated in patients who have experienced asthma, urticaria (in general allergic reactions), and should be administered with caution in children and teenagers due to the risk of Reye syndrome. [00005] One way in which to explain how boswellic acids function as NSAIDs in the treatment of inflammatory conditions is to compare these natural compounds to aspirin without the typical gastrointestinal irritation. Similar to aspirin, boswellic acids inhibit the pathway leading from arachidonic acid (a derivative of our body's phospholipids) to its metabolic derivatives called leukotrienes and prostaglandins. An excess of leukotrienes and prostaglandins may be responsible, directly or indirectly, for the classic signs of inflammation; redness (due to dilated vessels), swelling (due to the blood vessels leaking out), pain (due to activation of the pain receptors) and increased heat over the affected part of the body. The specific biochemical mechanism of boswellic acids differs from that of aspirin, however both compounds result in the diminishment of the mediators of inflammation, leukotrienes or prostaglandins, and the inflammation is subdued.

[0006] Studies designed to determine the anti-inflammatory mechanism of boswellic acids indicate that their primary mode of action involves the inhibition of 5- lipoxygenase, the key enzyme responsible in the formation of leukotrienes. Additionally, boswellic acids do not appear to impair the peroxidation of arachidonic acid by iron and ascorbate. These results suggest that boswellic acids are safe, specific, non-redox inhibitors of leukotriene synthesis that operate through a well defined mechanism.

[0007] One of the most interesting properties of boswellic acids is their anti- complementary activity. In in vitro experiments boswellic acids prevented a well- known inflammatory "chain-reaction" involving several protein compounds collectively known as "complement". This is due to the inhibition of an enzyme that activates one of the components of complement, C3 convertase. The domino effect of the complement in the course of rheumatoid arthritis (or a similar chronic inflammatory process) leads to a subsequent elevation of the enzymes (e.g. cathepsins, glucuronidase and human leukocyte elastase (HLE)) causing excessive catabolism (wasting) of the joint-forming glycoproteins and glycosaminoglycans. This tissue destructive process leads to continuously worsening joint disfigurement, pain, and limited mobility. As a consequence of complement-mediated tissue destruction, there is an increased release of markers (metabolites) of the connective tissue, e.g. hydroxyproline, hexosamine and uronic acid. Boswellic acids have been found to decrease the levels of tissue destructive enzymes and also of the levels of urinary hydroxyproline, hexosamine and uronic acid in the acute and chronic phases of experimental arthritis.

[0008] There are four major b-boswellic acids involved in the inhibition of 5- lipoxygenase and related anti-inflammatory events. These are: b-Boswellic Acid (BBA), Acetyl-b-Boswellic Acid (ABBA), 11-keto-b-Boswellic Acid (KBBA), Acetyl-11- keto-b-Boswellic (AKBBA), listed here in the order of increasing anti-inflammatory properties.

[0009] Boswellic acids have been found to be effective in alleviation of numerous inflammatory conditions, including, rheumatoid arthritis and osteoarthritis. [0010] A standardized extract of boswellic acids (200 mg tid) was evaluated in a four week double blind, cross-over trial in 30 patients suffering from rheumatoid arthritis. The mean arthritic score (sum of symptoms) and the biochemical index of inflammation in the group receiving boswellic acids came down significantly after the treatment. However, when the placebo was substituted (crossover), the subjective and objective indices of arthritis rose again. (See Majeed, M, Badmaev, V, Gopinathan, S, Rajendran, R, Norton, T. Boswellin The Anti-inflammatory Phytonutrient. Nutriscience Publishers, inc. Piscataway, NJ. 1996. pp. 78.) [0011] In another 20 patient, double blind, crossover study a boswellia gum resin extract (200 mg tid) combined in an herbomineral formula was evaluated in the treatment of rheumatoid arthritis and separately in osteoarthritis. Active and placebo treatments were given for a period of three months. After a washout period of two weeks, the regimens were crossed-over. The three month active therapy resulted in a significant decrease in severity of pain, morning stiffness, improved joint mobility score, grip strength score and the overall disability score compared to the placebo group. The biochemical index of inflammation was also significantly improved due to the treatment. (See Kulkarni, RR, Patki, PS, Jog, VP, Patwardhan, G&B. Efficacy Of An Ayurvedic Formulation In Rheumatoid Arthritis: A Double-Blind, Placebo- Controlled, Cross-Over Study. Ind J Pharmacol. 1992;24:98-101.) [0012] Ulcerative colitis is an example of a chronic inflammatory process in the bowel, which may be caused and/or aggravated by excessive leukotriene production. Effects of Boswellia serrata gum resin (350 mg thrice daily for 6 weeks) vs. the NSAID sulfasalazine was studied in patients with ulcerative colitis. The tested parameters, including stool properties, istolopathology of rectal biopsies, and blood biochemistry improved after treatment with the gum resin. As a result of the treatment, 82% of patients went into remission, as compared to a 75% remission rate obtained with sulfasalazine. (See Gupta, I, Parihar, A, Malhotra,P, Singh, GB, Ludtke, R, Safayhi, H, Ammon, HP. Effects of Boswellia serrata gum resin in patients with ulcerative colitis. Eur J ed Res. 1997 Jan; 2(1):37-43.) [0013] Boswellic acids were also tested in the management of asthma, since a new generation of anti-asthmatic drugs is based on the premise of being leukotriene inhibitors. In a double blind, placebo-controlled study 40 patients with a several years' history of bronchial asthma were treated with 300 mg tid of boswellia gum resin for a period of six weeks. Seventy percent of the patients responded to the treatment as evidenced by a reduction in dyspnea, ronchi, and number of attacks, improvement in lung tests and blood biochemistry. Only 27% of the patients receiving placebo showed clinical improvement. (See Gupta, I, Gupta, V, Parihar, A, Gupta, S, Ludtke, R, Safayhi, H, Ammon, HP. Effects Of Boswellia Serrata Gum Resin In Patients With Bronchial Asthma: Results Of A Double-Blind, Placebo- Controlled, 6-Week Clinical Study. Eur J Med Res. 1998; 3:511-514.) [0014] Finally, boswellic acids also have use in the veterinary field. Several veterinarians found success using boswellic acids in the treatment of chronic inflammatory conditions in horses such as stifle problems, sore backs, bowed tendons and bone spurs. In addition, a preliminary study of boswellic acids in aging pet dogs and cats showed beneficial effects in alleviating arthritic conditions. (See Majeed, M, Badmaev, V, Gopinathan, S, Rajendran, R, Norton, T. Boswellin The Anti-inflammatory Phytonutrient. Nutriscience Publishers, Inc. Piscataway, N.J. 1996. pp. 78.)

[0015] Sabinsa Corporation manufactures different grades of Boswellic acids known under the trademark Boswellin®, two of these are Boswellin (standardized for 25% boswellic acids) and Boswellin Forte (standardized for 40% boswellic acids). More specifically, the minimum amount of each boswellic acid that must be present in these grades is:

Boswellin Forte Boswellin

BBA min. 11.0% 6.0%

ABBA min. 8.0% 4.0%

KBBA min. 7.0% 3.0%

AKBBA min. 4.0% 1.5% [0016] Please note that, of course, every acid may not be present in its minimum amount as the required total percentages of boswellic acids (40% and 25%) would not be met if this occurred.

[0017] Such non-water soluble mixtures of BBA, ABBA, KBBA, and AKBBA boswellic acids can be used as a pharmaceutical. Since the ancient times, frankincense has been used in the preparation of cosmetics and perfumes, and also as a fixative in perfumes, soaps, creams, lotions and detergents. Frankincense is a common name for Boswellia gum resin, and Boswellia gum resin is a raw source from which boswellic acids are extracted. The stabilizing effect of frankincense in cosmetic preparations is directly related to the biological properties of boswellic acids. The anti-inflammatory properties of boswellic acids can also yield an interesting applications for topical and cosmetic use of the extract of Boswellia serrata. Boswellia cream for the management of inflammatory conditions has been available for several years in the US market. Its therapeutic composition includes, roughly 5 wt. % boswellic acids, 0.025 wt. % capsaicin, an extract of Capsicum annum fruits, and 10 wt. % methyl salicylate.

[0018] However, a problem associated with these formulations is that they are not soluble in water. Therefore, there is a great need in the field for water soluble boswellic acid mixtures and salts. SUMMARY OF THE INVENTION:

[0019] The new water-soluble composition can be formed through a method comprising the steps of:

(a) dissolving mixtures of boswellic acids in a water and alcohol solution to form a mixture; (b) adding one or more alkali salts to the mixture to form a salt solution;

(c) filtering the solution to separate un-reacted alkali salt from a filtrate; and

(d) recovering the soluble boswellic acid mixture from the filtrate. [0020] Additionally, the new composition can be formed by using super critical carbon dioxide. The new composition can be used to alleviate numerous inflammatory conditions, including, but not limited to, rheumatoid arthritis and osteoarthritis, colon cancer, prostate cancer and breast cancer, and a broad range of neurodegenerative conditions, such as Alzheimer's disease and Parkinson's disease. It can also be used in the prevention and treatment of cardiovascular conditions such as stroke, coronary artery disease or thrombophlebitis. The composition can be administered parenterally, orally, or topically.

DETAILED DISCLOSURE:

[0021] As stated above, the composition can be formed by (a) dissolving mixtures of boswellic acids in a water and alcohol solution to form a mixture, (b) adding one or more alkali salts to the mixture to form a salt solution, (c) filtering the salt solution to separate un-reacted alkali salt from a filtrate, and (d) recovering the soluble boswellic acid mixture from the filtrate.

[0022] Preferably, though, the method involves (a) dissolving boswellin forte in a water and 5% methanol solution to form a mixture, (b) adding one or more potassium salts to the mixture to form a salt solution and then stirring the salt solution at room temperature, (c) filtering the solution with a nutsche filter to separate un-reacted potassium salts from a filtrate, (d) recovering the soluble boswellic acid mixture from the filtrate, (e) drying the filtrate with a vacuum drier at a temperature of no more than 50° C, and (f) powdering the filtrate.

[0023] More preferably, the filtrate can be dried through concentrating the filtrate free of the solvent to obtain a solid, wherein this step also further comprises dissolving the obtained solid in water to obtain a secondary mixture, charcoalizing the secondary mixture, filtering the charcoalized secondary mixture and spray-drying the resulting product.

[0024] Additionally, it is also possible to use the super critical carbon dioxide method of obtaining the boswellic acid mixture. Such a process would comprise the steps of (a) dissolving a mixture of boswellic acids, preferably boswellin forte, in a water and alcohol solution to form a mixture, (b) adding one or more alkali salts to the mixture to form a salt solution, (c) treating the salt solution with supercritical carbon dioxide, (d) allowing the supercritical carbon dioxide to evaporate to leave an oleoresin, (e) passing an alcohol solution of the oleoresin through a column packed with an anion exchange resin, and (f) collecting the soluble boswellic acid mixture from the eluent. Furthermore, it is preferred that the treatment of the salt solution take place for at least 10 hours and that the alcohol solution be 5% methanol and 95% water. Suitable resins include Amberjet 4200(d), Amberlite IRA 410, Amberlite IRA 900, Dowex 1x2-100, Dowex 22cl, Dowex Marathon A2, Dowex MSA 1 , Dowex 550 A, all of which are Rohm-Haas or Dow products. The oleoresin is preferably passed through the resin at a rate of 20-50 L per hour.

[0025] The processes described above produce a water soluble composition, preferably comprising at least 12.5% by weight of the alkali salt of BBA, at least 9.57% by weight of the alkali salt of ABBA, at least 8.15% by weight of the alkali salt of KBBA, and at least 3.72% by weight of the alkali salt of AKBBA, the remainder of the water soluble composition being organic acids or matter, and the alkali salts thereof. This composition of boswellic acids is suitable for the treatment of many inflammatory conditions including rheumatoid arthritis and osteoarthritis, colon cancer, prostate cancer and breast cancer, and a broad range of neurodegenerative conditions, such as Alzheimer's disease and Parkinson's disease. It can also be used in the prevention and treatment of cardiovascular conditions such as stroke, coronary artery disease or thrombophlebitis.

[0026] The composition may be administered to the subject orally, parenterally, or topically. For preparing pharmaceutical compositions containing compounds of the invention, inert, pharmaceutically acceptable carriers are used. The pharmaceutical carrier can be either solid or liquid. Solid form preparations include, for example, powders, tablets, dispersible granules, capsules, creams, and cachets. [0027] A solid carrier can be one or more substances which can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material. In powders, the carrier is generally a finely divided solid which is in a mixture with the finely divided active component. In tablets, the active compound is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

[0028] Powders and tablets preferably contain between about 5% to about 70% by weight of the active ingredient. Suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low-melting wax, cocoa butter and the like. The pharmaceutical compositions can include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component (with or without other carriers) is surrounded by a carrier, which is thus in association with it. In a similar manner, cachets are also included. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration.

[0029] Liquid pharmaceutical compositions include, for example, solutions suitable for oral or parenteral administration, or suspensions, and emulsions suitable for oral administration. Sterile water solutions of the active component or sterile solutions of the active component in solvents comprising water, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration. [0030] Sterile solutions can be prepared by dissolving the active component in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the sterile compound in a previously sterilized solvent under sterile conditions.

[0031] Aqueous solutions for oral administration can be prepared by dissolving the active compound in water or other appropriate solvents and adding suitable flavorants, coloring agents, stabilizers, and thickening agents as desired. Aqueous suspensions for oral use can be made by dispersing the finely divided active component in water together with a viscous material such as natural or synthetic gums, resins, methyl cellulose, sodium carboxymethyl cellulose, and other suspending agents known to the pharmaceutical formulation art. Preferably, the pharmaceutical composition is in unit dosage form. In such form, the composition is divided into unit doses containing appropriate quantities of the boswellic acid mixtures. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, for example, packeted tablets, capsules, and powders in vials or ampules. The unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms.

[0032] The specific dosages employed, however, can be varied depending upon the requirements of the patient, and the severity of the condition being treated. The preferred dosage of the alkali boswellic acid salts given is 50-800 mg per day. More preferably, the dosage is 100-600 mg per day. Even more preferred is a dosage of

150-300 mg per day. Most preferred is a dosage of about 200 mg per day, which is preferably administered by doses of 200 mg of the boswellic acid salt composition 3 times a day. The determination of optimum dosages for a particular situation is within the skill of the art.

BRIEF DESCRIPTION OF DRAWINGS:

[0033] Figure 1: This diagram reflects the boswellic acid composition of the

Potassium Boswellin used in Example 1 and the composition of the Boswellin forte used to create the Potassium Boswellin used in Example 1.

[0034] Figure 2: This diagram reflects the presence of the four boswellic acids in the serum of Individual A at 5, 10, 20, 40, 80, and 160 minutes.

[0035] Figure 3: This diagram reflects the presence of the four boswellic acids in the serum of Individual B at 5, 10, 20, 40, 80, and 160 minutes

[0036] Figure 4: This diagram is a graph charting the serum levels of the various four boswellic acids in the serum of Individual A at the 5, 10, 20, 40, 80, and 160 minute intervals.

[0037] Figure 5: This diagram is a graph charting the serum levels of the various four boswellic acids in the serum of Individual B at the 5, 10, 20, 40, 80, and 160 minute intervals EXAMPLES

Example 1:

An initial batch of 1 kg Potassium Boswellin was prepared using 40% Boswellin Forte material (see Figure 1).

Potassium Boswellin and Boswellin 40% were then orally administered to two human volunteers who had spent the previous 16 hours fasting. 1 gram of Potassium Boswellin was given to Individual A and 1 gram of Boswellin 40% was given to Individual B. Both the Potassium Boswellin and the Boswellin 40% were suspended in 35 ml of milk and given to the subjects to consume. 5 ml of blood was withdrawn from the volunteers at intervals of 5, 10, 20, 40, 80, and 160 minutes. The blood samples were collected into sterile centrifuge tubes and left for 120 minutes to retract the clot. The samples were then centrifuged to separate the serum. The serum was transferred to sterile 2 ml vials and stored at 0° C - 4° C overnight.

The serum samples were then brought to room temperature. 1 ml of the serum was placed into a 10 ml stoppered volumetric flask and 1 ml of 3 N HCI was added to it. This mixture was then sonicated for 20 minutes to free the boswellic acid. The volume of the mixture was then brought to 10 ml with methanol and the resulting solution was sonicated for 10 minutes to extract the boswellic acids and to precipitate the serum proteins. This prepared sample was then transferred into capped centrifuge tubes and centrifuged at 4,000 rpm for 10 minutes. The serum proteins were precipitated as sediment and the clear supernatant was filtered through No. 1 filter paper.

The clear supernatants were then subjected to a HPLC assay, the results of which are shown in Figures 2-5. As can be plainly seen, the serum with Potassium Boswellin shows that a greater amount of boswellic acids have been absorbed by the subject's bloodstream, thereby demonstrating the efficacy of the present invention.

Claims

What is claimed is:

1. A method of producing a water soluble boswellic acid mixture comprising

(a) dissolving mixtures of boswellic acids in a water and alcohol solution to form a mixture;

(b) adding one or more alkali salts to the mixture to form a salt solution;

(c) filtering the solution to separate un-reacted alkali salt from a filtrate; and

(d) recovering the soluble boswellic acid mixture from the filtrate.

2. The method of claim 1, wherein the alcohol used in the water and alcohol solution is methanol.

3. The method of claim 2, wherein the water and alcohol solution contains about 5% alcohol.

4. The method of claim 1 , wherein step (b) further comprises stirring the salt solution at room temperature.

5. The method of claim 1, wherein the filtering step of step (c) is performed with a nutsche filter.

6. The method of claim 1, wherein the un-reacted alkali salt separated from the filtrate in step (c) is washed with an alcohol.

7. The method of claim 6, wherein the alcohol is methanol.

8. The method of claim 1 , wherein the solid is dried with a vacuum drier.

9. The method of claim 8, wherein the solid is dried at a temperature below 50° C.

10. The method of claim 1 , further comprising powdering the solid.

11. The method of claim 1 , wherein step (d) further comprises concentrating the filtrate free of the solvent to obtain a solid.

12. The method of claim 1, wherein the concentration step of step (d) further comprises dissolving the obtained solid in water to obtain a secondary mixture, charcoalizing the secondary mixture, filtering the charcoalized secondary mixture and spray-drying the resulting product.

13. A water soluble composition comprising at least 12.5% by weight of the alkali salt of BBA, at least 9.57% by weight of the alkali salt of ABBA, at least 8.15% by weight of he alkali salt of KBBA, and at least 3.72% by weight of the alkali salt of AKBBA, the remainder of the water soluble composition being organic acids or matter, and the alkali salts thereof.

14. A pharmaceutical composition comprising a pharmaceutically effective amount of the compound produced by the method of claim 1 and pharmaceutically acceptable excipients.

15. A method of treating an inflammatory condition comprising administering to a subject in need of such treatment an effective amount of a composition produced by the method of claim 1 , wherein said effective amount is 50-800 mg per day.

16. The method of claim 15, wherein the composition of claim 1 is administered orally.

17. The method of claim 15, wherein the composition of claim 1 is administered topically.

18. The method of claim 15, wherein the composition of claim 1 is administered parenterally.

19. The soluble composition produced by the process of claim 1.

20. The method of claim 1 , wherein the alkali salts are potassium salts.

21. A method of producing a water soluble boswellic acid mixture comprising

(a) dissolving mixtures of boswellic acids in a water and alcohol solution to form a mixture;

(b) adding one or more alkali salts to the mixture to form a salt solution;

(c) treating the salt solution with supercritical carbon dioxide; (d) allowing the supercritical carbon dioxide to evaporate to leave an oleoresin;

(e) passing an alcohol solution of the oleoresin through a column packed with an anion exchange resin; and

(f) collecting the soluble boswellic acid mixture from the eluent.

22. The process of claim 21, wherein the salt solution is treated with supercritical carbon dioxide for 10 hours.

23. The process of claim 22, wherein the alcohol solution is a 5% alcohol solution.

24. The process of claim 23, wherein the alcohol in the alcohol solution is methanol.

25. The method of claim 1 wherein the mixture of boswellic acids is BBA, ABBA, KBBA, and AKBBA in a total concentration of 25%.

26. The method of claim 1 wherein the mixture of boswellic acids is BBA, ABBA, KBBA, and AKBBA in a total concentration of 40%.

27. The method of claim 21 wherein the mixture of boswellic acids is BBA, ABBA, KBBA, and AKBBA in a total concentration of 25%.

28. The method of claim 1 wherein the mixture of boswellic acids is BBA, ABBA, KBBA, and AKBBA in a total concentration of 40%.

29. The composition of claim 15, wherein the alkali in the alkali salt is potassium.

30. The method of claim 15, wherein the inflammatory condition is arthritis.

31. The method of claim 15, wherein the effective amount is 100-600 mg per day.

32. The method of claim 31, wherein the effective amount is 150-300 mg per day.

33. The method of claim 32, wherein the effective amount is 200 mg per day.

34. A method of treating an inflammatory condition comprising administering to a subject in need of such treatment an effective amount of a water soluble mixture of boswellic acids, wherein said effective amount is 50-800 mg per day.

35. The method of claim 34, wherein the effective amount of a water soluble mixture of boswellic acids is administered orally.

36. The method of claim 34, wherein the effective amount of a water soluble mixture of boswellic acids is administered topically.

37. The method of claim 34, wherein effective amount of a water soluble mixture of boswellic acids is administered parenterally.