WO2006009477A1

WO2006009477A1 - Anti-angiogenic compositions containing beeswax

Info

Publication number: WO2006009477A1
Application number: PCT/NZ2005/000179
Authority: WO
Inventors: Paul Frank Davis
Original assignee: University Of Otago; Immuno Research Limited
Priority date: 2004-07-22
Filing date: 2005-07-21
Publication date: 2006-01-26
Also published as: CN101027068A; TW200607516A; KR20070047775A; TWI369989B; JP2008507511A

Abstract

The invention relates to compositions comprising synergistically effective amounts of an anti-angiogenic extract and beeswax. The invention relates particularly to compositions containing an extract from shark meat or an extract from mussel meat, optionally including a mushroom extract, together with beeswax. The invention also relates to a method of treating or preventing diseases or disorders where it is desirable to inhibit angiogenesis using a composition of the invention.

Description

ANTI-ANGIOGENIC COMPOSITIONS CONTAINING BEESWAX

TECHNICAL FIELD

This invention relates to compositions comprising synergistically effective amounts of an anti-angiogenic extract and beeswax. In particular, the invention relates to compositions containing an extract from shark meat, or an extract from mussel meat, together with beeswax. The invention also relates to a method of treating or preventing diseases or disorders where it is desirable to inhibit angiogenesis using a composition of the invention.

BACKGROUND

Angiogenesis is the growth and proliferation of new blood vessels. Angiogenesis plays an important role in many physiological and pathological conditions. In a healthy body, angiogenesis occurs during the proliferative phase of wound healing, for restoring blood flow to tissues after injury or insult, and in females during the monthly reproductive cycle and during pregnancy.

The healthy body controls angiogenesis through a series of "on" and "off' switches. The "on" switches are known as angiogenesis stimulating growth factors and the "off' switches are called angiogenesis inhibitors. In the normal healthy body, the expression of angiogenesis growth factors and angiogenesis inhibitors is finely balanced in order to facilitate activation and suppression of angiogenesis.

Loss of control of angiogenesis can lead to many serious disease states. There are a number of known conditions which result from so-called "excessive angiogenesis". Such conditions include arthritis (Giatromanolaki et al., Arthritis Research & Therapy, 5: R193- 201; DeBusk et al., Arthritis & Rheumatism, 48: 2461-71), retinopathy (Bainbridge et al., Clinical Science, 104: 561-75; Umeda et al., Ophthalmic Research, 35: 217-23), macula degeneration (Bainbridge et al., Clinical Science, 104: 561-75; Yamaza et al., Journal of Electron Microscopy, 51: 127-31 ; Ohno-Matsui et al., Journal of Cellular Physiology, 189: 323-33) and tumours (Sivridis et al., Journal of Pathology, 201 : 173-80; Longo et al., Angiogenesis, 5: 237-56). Inhibitors of angiogenesis will retard the progression of these conditions. There are a number of known anti-angiogenic compounds and preparations (Scappaticci, Expert Opinion on Investigational Drugs, 12: 923-32; Caceres and Gonzalez, Puerto Rico Health Sciences Journal, 22: 149-51) including lipid-based preparations. One of these is derived from the muscle tissue of several species of shark.

It has been known for some time that shark cartilage has anti-angiogenic activity. More recent work has shown that extracts obtained from shark meat have significant anti- angiogenic activity when evaluated by both in vitro and in vivo methodology (see PCT/NZ01/00285, published as WO 02/49654).

The extract of shark meat obtained by solvent extraction, and then removal of the solvent, is a viscous oil with some particulate matter which tends to settle on standing. Such sedimentation in gelatin capsules can lead to distortion of the capsule shape. In order to address this problem, beeswax was added to the extract.

Surprisingly, however, it was found that not only did the beeswax minimise distortion of the capsule shape, but the combination of beeswax and extract enhanced the anti-angiogenic effect of the extract. This would not have been predicted from the known properties of beeswax.

Consideration was then given to whether the presence of beeswax might enhance the activity of other natural product extracts having anti-angiogenic activity. The applicant discovered that beeswax can enhance the anti-angiogenic activity of an extract from the meat of mussel shell fish. The applicant also found that the anti-angiogenic activity of a shark meat extract combined with a mushroom extract is enhanced in the presence of beeswax.

The surprising discovery that beeswax enhances the angiogenic activity of a range of natural product extracts forms the basis of this invention.

It is therefore an object of this invention to provide an anti-angiogenic composition comprising synergistically effective amounts of an anti-angiogenic extract and beeswax, or at least to provide a useful choice. STATEMENTS OF INVENTION

In a first aspect, the invention provides an anti-angiogenic composition containing synergistically effective amounts of an anti-angiogenic extract and beeswax.

In one embodiment of the invention the extract is a shark meat extract, preferably where the meat is muscle tissue from the shark. Alternatively, the extract may be an extract of mussel meat, preferably from the New Zealand green-lipped mussel, Perna canaliculus.

In a further embodiment the anti-angiogenic extract is a combination of a shark meat extract and a mushroom extract. The mushroom extract may be the extract of any mushroom that has anti-angiogenic activity, but is preferably the Korean mushroom (Phellinus linteus) or the Maitake mushroom (Grifola frondosa).

Preferably, the composition further contains an ingestible oil, such as olive oil. It is also preferred that the composition contains vitamin E.

The composition may contain amounts of the anti-angiogenic extract and beeswax in any ratio that gives the synergistic ant-angiogenic effect, but the ratio is preferably in the range of 1 : 1 to 10: 1 , most preferably about 4:1 or 5: 1.

In a second aspect of the invention there is provided a method of treating or preventing a disease or disorder in which it is desirable to inhibit angiogenesis, comprising administering to a subject an anti-angiogenic composition containing synergistically effective amounts of an anti-angiogenic extract and beeswax.

The invention also provides the use of an anti-angiogenic composition containing synergistically effective amounts of an anti-angiogenic extract and beeswax for the preparation of a medicament for preventing or treating a disease or disorder in which it is desirable to inhibit angiogenesis.

In a preferred embodiment of the invention the disease or disorder is arthritis, retinopathy, macular degeneration, or cancer. Preferably the subject is a human. DETAILED DESCRIPTION

The invention provides anti-angiogenic composition comprising synergistically effective amounts of an anti-angiogenic extract and beeswax. In particular, the invention relates to anti-angiogenic compositions comprising synergistically effective amounts of an extract from shark meat or an extract from mussel meat, optionally with a mushroom extract, together with beeswax.

The term "extract" as used herein means a substance obtained by any extraction method, whether or not the crude extract has been fractionated or purified by usual methods such as chromatography and filtration. Extraction methods include, but are not limited to, solvent extraction. Any suitable solvent may be used. The solvent may be an organic solvent or an aqueous solvent, or a mixture of organic and aqueous solvents. Ethanol is typically used as the organic solvent (or an aqueous ethanol mix), but other organic solvents may be used. The extraction solvent may also be a supercritical fluid such as supercritical CO₂.

The term "shark meat" as used herein means any meat or flesh of any shark or shark-like organism including, but not limited to, dogfish, rig (lemonfish), school shark, ghost shark, mako, blue shark, elephant fish, salmon shark, and blacktip reef shark.

As used herein, the term "mussel extract" is intended to mean an extract obtained from the meat of any mussel shell fish variety including, but not limited to, the New Zealand green- lipped mussel (Perna canaliculus) and the blue mussel (Mytilus edulis).

The term "mushroom extract" as used herein means an extract obtained from any mushroom including, but not limited to, the Korean mushroom (Phellinus linteus) and the Maitake mushroom (Grifola frondosa).

As used herein, the term "beeswax" means any composition or preparation that contains any type of beeswax. The beeswax may be cappings wax, melt out wax, or any other suitable form or type of beeswax. The applicant has surprisingly found that compositions containing beeswax together with an anti-angiogenic extract, such as a shark meat extract or a mussel extract, show remarkably enhanced anti-angiogenic activity compared to the extract without beeswax.

Table 1 (below) shows the anti-angiogenic activity of compositions containing a shark meat extract product and beeswax. The anti-angiogenic activity of the extract more than doubles when beeswax is added. Beeswax alone produces only 47% inhibition at the high concentration of 100 μg/ml. This concentration of beeswax is approximately 40 times the concentration of the beeswax in the shark meat extract-beeswax mixture. Thus, the 52.3% inhibition demonstrated by the extract-beeswax combination indicates that a synergistic anti-angiogenic effect is produced by combining beeswax with shark meat extract.

The green-lipped mussel extract and a composition containing the extract plus beeswax were also assayed for their anti-angiogenic activity. The mussel extract alone has a slightly lower anti-angiogenic effect than shark meat extract. Table 2 (below) shows the activity of the mussel extract and a composition containing the extract plus beeswax. There is a significant (threefold) increase in anti-angiogenic activity when beeswax is added to an extract of green-lipped mussel.

Again, the anti-angiogenic activity of beeswax alone is minimal relative to the anti- angiogenic activity of a combination of beeswax and an extract of green-lipped mussel. A high concentration (100 μg/ml) beeswax solution elicits approximately 50% inhibition while a 40 times more dilute beeswax solution increases the anti-angiogenic activity of the mussel extract by more than threefold.

It was originally thought that beeswax would merely maintain the integrity and shape of gelatin capsules containing a shark meat extract. Until now, there has been no evidence to suggest that beeswax has any anti-angiogenic activity itself. It has been assumed, therefore, that beeswax would be an inert component. However, adding beeswax to the extract was surprisingly found to double the anti-angiogenic activity when added at a ratio of 4:1. This effect of beeswax on anti-angiogenic activity is further supported by the data for the green-lipped mussel extract. The mussel extract has relatively low anti-angiogenic activity (in fact, it is better known for its anti-inflammatory activity). However, surprisingly, the addition of beeswax at a ratio of 4:1 increases the anti-angiogenic activity by more than threefold.

The fact that beeswax modulates the anti-angiogenic activities of both a shark meat extract and an extract from mussels indicates that beeswax can exert a synergistic effect on a range of other natural product extracts having anti-angiogenic activity.

The invention is further described with reference to the following examples. It is to be appreciated the invention is not limited to the examples.

EXAMPLES

Example 1 : In vitro Anti-Angiogenesis Assay

The aorta is removed from a rat and cleaned of adhering fatty and connective tissues before being cut into rings of approximately 3mm size. Fibrinogen is layered in the bottoms of wells of multi-well culture plates and allowed to gel by thrombin action. A ring is then layered on the top of each gel and a further layer of fibrin placed on this. The fibrinogen is prepared in MCDB131 medium supplemented with antibiotics. The double layer of fibrin is then overlaid with MCDB131 containing the test materials. The gels are incubated at 37⁰C in an atmosphere of 3%CO₂/97% air. The rings are examined using an inverted microscope and the growth of microvessels from the perimeter of each ring is observed. Digital pictures are taken of these every 2 days and the extent of microvessel growth relative to the size of the ring is determined using NIH Image software. From this the rate of growth of microvessels can be determined for each well. Each test substance is assayed in triplicate and the mean growth rate is calculated.

Example 2: Shark Meat Extract and Beeswax

An extract from shark meat, obtained by ethanol extraction of powdered or freeze dried shark meat followed by filtration and solvent removal, and the extract plus beeswax were assayed for anti-angiogenic activity in accordance with the procedure of Example 1. The results are shown in Table 1.

Table 1: Percentage Inhibition of Angiogenesis

Shark meat extract (10μg/ml) 24.40%

Shark meat extract (10μg/ml) + Beeswax (2.45μg/ml) 52.27%

Beeswax (1 OOμg/ml) 47.00%

Example 3: Mussel Extract and Beeswax An extract of green-lipped mussel extract, obtained by ethanol extraction of powdered or freeze dried mussel meat followed by filtration and solvent removal, and the mussel extract plus beeswax were assayed for anti-angiogenic activity using the method of Example 1. The results are shown in Table 2.

Table 2: Percentage Inhibition of Angiogenesis

Mussel extract (10μg/ml) 12. 83%

Mussel extract (1 Oμg/ml) + Beeswax (2.45μg/ml) 41. 96%

Beeswax (1 OOμg/ml) 49. 89%

Example 4: Shark Meat Extract/Mushroom Extract and Beeswax

Using the procedure of Example 1 , a mixture of the shark meat extract and an extract from the Korean mushroom (Phellinus linteus) (obtained by ethanol extraction of powdered mushroom followed by filtration and solvent removal), in a 9:1 ratio by weight, was shown to have significant anti-angiogenic activity. When beeswax was added to the mixture to give a beeswax to extract mixture ratio of 1 :4, the activity was further enhanced (Table 3).

A similar preparation was made using an extract from the Maitake mushroom (Grifola frondosa) and the shark meat extract. Again, the addition of beeswax enhanced the anti- angiogenic activity. Table 3: Percentage Inhibition of Angiogenesis

Shark meat extxacUPhellinus linteus extract (9:1)(10μg/ml) 17 .67%

Shark meat extract/ Phellinus linteus extract (9:1)( (10μg/ml)

+ Beeswax (2.45μg/ml) 29 .16%

Shark meat extract/ Grifola frondosa extract (9:1)(10μg/ml) 16 .28%

Shark meat extract/ Grifola frondosa extract (9:1)(10μg/ml)

+ Beeswax (2.45μg/ml) 26 .42%

For both the shark meat extract/ Phellinus linteus extract (9:1) and the shark meat extract/ Grifola frondosa extract (9:1), the addition of the beeswax improved anti-angiogenic activity by between 62 and 65%.

Although the invention has been described by reference to examples, it should be appreciated that variations and modifications may be made without departing from the scope of the invention. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically described in this specification.

INDUSTRIAL APPLICABILITY The anti-angiogenic compositions of the invention are useful for the treatment or prevention of a wide variety of diseases. These include diseases associated with angiogenesis such as arthritis, retinopathy, macular degeneration, and cancer.

Claims

1. An anti-angiogenic composition containing synergistically effective amounts of an anti-angiogenic extract and beeswax.

2. A composition as claimed in claim 1 where the anti-angiogenic extract is a shark meat extract.

3. A composition as claimed in claim 2 where the shark meat extract is an extract of shark muscle tissue.

4. A composition as claimed in claim 1 where the anti-angiogenic extract is a mussel meat extract.

5. A composition as claimed in claim 4 where the mussel meat is obtained from the New Zealand green-lipped mussel.

6. A composition as claimed in claim 3 which additionally contains a mushroom extract.

7. A composition as claimed in claim 6 where the mushroom is the Korean mushroom (Phellinus linteus) or is the Maitake mushroom (Grifola frondosa).

8. A composition as claimed in any one of the preceding claims further containing an ingestible oil.

9. A composition as claimed in claim 8 where the ingestible oil is olive oil.

10. A composition as claimed in any one of the preceding claims further containing vitamin E.

11. A composition as claimed in any one of the preceding claims where the ratio of the anti-angiogenic extract to beeswax is in the range of 1:1 to 10:1.

12. A composition as claimed in claim 11 where the ratio is approximately 4:1 to 5:1.

13. A method of treating or preventing a disease or disorder in which it is desirable to inhibit angiogenesis, comprising administering to a subject an anti-angiogenic composition containing synergistically effective amounts of an anti-angiogenic extract and beeswax.

14. A method as claimed in claim 13 where the disease or disorder is arthritis, retinopathy, macular degeneration, or cancer.

15. The use of an anti-angiogenic composition containing synergistically effective amounts of an anti-angiogenic extract and beeswax for the preparation of a medicament for preventing or treating a disease or disorder in which it is desirable to inhibit angiogenesis.

16. The use as claimed in claim 15 where the disease or disorder is arthritis, retinopathy, macular degeneration, or cancer.