TECHNICAL FIELD

The invention relates to an active composition and methods of use thereof. More specifically, the invention relates to a harvested hard deer antler which increases osteoblast cell activity and also decreases / inhibits osteoclast cell activity. The composition may be useful in treatment of bone diseases such as osteoporosis and in treament of bone fractures.

BACKGROUND ART

Osteoporosis is defined as a depletion of bone mass compounded with micro-architectural deterioration of bone tissue leading to increased fragility and therefore higher susceptibility to fracture. Increased bone resorption coupled with a reduced rate of new bone formation to replace the lost bone leads to decreased bone mineral density and mass and is characteristic of secondary osteoporosis. Osteoporosis affects a significant number of people worldwide with one in three women and one in five men being at risk of osteoporosis.

Suppressing the activity of the osteoclast, the cell responsible for bone resorption, is one way of treating osteoporosis, which if coupled with bone formation, leads to increased bone mineral density (BMD) and should mean a reduction in the frequency of fracture. Long-term suppression of bone resorption has been ineffective in bone remodelling. The use of growth factors, hormone therapy and fluoride compounds, to stimulate bone formation are limited in their application. Safe and effective treatment for osteoporosis should aim at decreasing osteoclast activity and hence bone resorption and facilitate bone repair.

Other bone diseases are also associated with changes in normal osteogenic activity. Similarly, bone healing post an injury also relies (at least in part) on osteogenic effects such as osteoblast cell function hence inhibiting osteoclast cell development and/or stimulating osteoblast cell proliferation may have many applications around bone health.

The deer antler is highly vascular tissue and part of this means that antler grows rapidly and tissue damage regenerates rapidly. The rapid growth of this bony structure is due to the action of large numbers of osteoblasts as well as the hematopoetically derived osteoclasts progenitors circulating in the complex blood supply of the antlers. This makes the deer antler a rare example of mammalian epimorphic regeneration. The antler tips grow at a rapid rate, associated with extensive osteoclastic resorption of mineralised cartilage from matrix. Various factors are associated with bone regeneration. Insulin like growth factor (IGF-1) is one such factor and influences mitogenesis. The role of parathyroid hormone in bone formation is evident by increased bone sensitivity to PTH in ovariectomized rats. Deer antlers undergo regeneration annually. This phenomenon has been attributed to the presence of nucleus derived, parathyroid hormone - related protein receptor (PTHrP) that influences osteoclasts differentiation and the regulation of skeletal tissue regeneration.

Deer antler has been a traditional oriental medicine. In the past, the emphasis of almost all research has been on use of soft antler or velvet antler and hence the cost of velvet is significantly higher than that of hard antler. There is extensive art about various uses of velvet including for treatment of bone disease in humans. Less well studied is the use of hard antler for treating bone ailments. The studies that have been completed are of cast antler. Harvesting hard antler from the deer before casting but after stripping and transformation to a mineralised form is relatively unusual as most deer are not farmed and instead are wild. The New Zealand deer industry is largely made up of farmed deer where harvesting of antler at different times in the antler lifecycle is both practical and economically viable.

It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice. - '

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non- specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

Further aspects and advantages of the present invention will become apparent from the ensuing description that is given by way of example only. SUMMARY OF THE INVENTION

The invention broadly relates to harvested hard deer antler powders and extracts and various methods and uses of the harvested hard deer antler around inhibiting osteoclast cell development and also increasing osteoblast cell function. The harvested hard deer antler has an unexpectedly useful osteogenic activity compared to cast hard antler and traditional remedies using velvet.

In a first embodiment there is provided a formulation including hard deer antler that, on administration to an animal, (a) increases osteoblast cell function; or

(b) decreases osteoclast cell function; or

(c) increases osteoblast cell function and decreases osteoclast cell function;

wherein the hard deer antler is harvested from deer in the time period between stripping and one month before casting.

In a second embodiment there is provided a method of treating an animal in need thereof for diseases and conditions associated with:

(a) increasing osteoblast cell function; or

(b) decreasing osteoclast cell function; or

(c) increasing osteoblast cell function and decreasing osteoclast cell function;

by the step of administering a formulation containing hard deer antler harvested from the deer during the time period between stripping and casting

In a third embodiment there is provided the use of hard deer antler harvested from deer in the time period between stripping and before casting in the manufacture of a formulation for the treatment of diseases or conditions associated with:

(a) increasing osteoblast cell function; or

(b) decreasing osteoclast cell function; or

(c) increasing osteoblast cell function and decreasing osteoclast cell function. BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings in which:

Figure 1 illustrates the peak activity for velvet, harvested hard antler and cast antler.

Figure 2 illustrates the effect of the cast antler extract on RAW 264.7 osteoclastogenesis.

Significance (p < 0.05) from the 0 control is indicated by an asterisk. Means ± 95% confidence interval (CI);

Figure 3 illustrates the effect of the harvested hard antler extract on RAW 264.7

osteoclastogenesis. Significance (p < 0.05) from the 0 control is indicated by an asterisk. Means ± 95% CI;

Figure 4 illustrates the effect of the harvested hard deer antler pre-treated with an ethanol wash on RAW 264.7 osteoclastogenesis. Significance (p < 0.05) from the 0 control is indicated by an asterisk. Means ± 95% CI; Figure 5 illustrates the effect of the velvet extract on RAW 264.7 osteoclastogenesis.

Significance (p < 0.05) from the 0 control is indicated by an.asterisk. Means ± 95% CI;

Figure 6 illustrates the composite graphs showing the effect of harvested hard antler and velvet powdered fractions on RAW 264.7 osteoclastogenesis. Significance (p < 0.05) from the 0 control is indicated by an asterisk. Means ± 95% CI; and

Figure 7 illustrates the effect of harvested hard antler and velvet powdered fractions on osteoblast proliferation. Significance (p < 0.05) from the 0 control is indicated by an asterisk. Means ± 95% CI.

DETAILED DESCRIPTION

As noted above, the invention broadly relates to harvested hard deer antler powders and extracts and various methods and uses of the harvested hard deer antler around inhibiting osteoclast cell development and also increasing osteoblast cell function. The harvested hard deer antler has an unexpectedly useful osteogenic activity compared to cast hard antler and traditional remedies using velvet.

For the purposes of this specification, the term 'hard deer antler' refers to calcified deer antler post stripping.

The term 'harvested hard deer antler ¹ - refers to deer antler being removed e.g. by cutting, from the deer in the time period between stripping and approximately one month prior to natural casting.

The term 'stripping' refers to the time period characterised by a rapid increase in antler mineral content and stripping of the soft outer covering of the antler. As may be appreciated by those skilled in the art, at this stripping stage (typically in the autumn season) the stag rubs the antler in shrubs and trees and as a result, the antler(s) become hard and polished.

The term 'casting' refers to the natural process where the hard calcified antler naturally drops from the deer, typically in the spring season.

In a first embodiment there is provided a formulation including hard deer antler that, on administration to an animal,

(a) increases osteoblast cell function; or

(b) decreases osteoclast cell function; or

(c) increases osteoblast cell function and decreases osteoclast cell function;

wherein the hard deer antler is harvested from deer in the time period between stripping and one month before casting. Preferably, the harvested hard deer antler formulation of the embodiment above may be characterised by a double action on administration of increasing osteoblast cell function and decreasing osteoclast cell function.

Preferably, the hard deer antler may have an ash content of greater than or equal to 26% by weight. More preferably, the ash content may be greater than or equal to 35% by weight. Still more preferably, the ash content may be greater than 50% by weight.

Preferably, the hard deer antler may have a protein content of greater than or equal to 30-50% by weight. More preferably, the protein content is greater than 50% by weight.

In one embodiment the hard deer antler is in the form of a powder. This should not be seen as limiting as other forms may be used for example a tonic or liquid elixir.

Preferably, the powder hard antler may be micronised. In one embodiment, the powder size may be less than approximately 150 micron. More preferably, the powder may be less than 75 micron.

The inventors envisage that the powder may be administered to a subject by known methods such as oral or parenteral methods. A preferred method is likely to be via oral administration due to ease of administration although this should not be seen as limiting.

It is envisaged that the amount administered may vary from 5 to 500 mg hard deer antler per day per kg bodyweight. Preferably, the amount administered varies from 20 to 100 mg hard deer antler per day per kg bodyweight. In one embodiment, the dosage may be approximately 50 mg hard deer antler per day per kg bodyweight. The above figures should not be seen as limiting as the amount may vary considerably dependent on the method of preparation, method of extraction (if extraction is completed), form of delivery e.g. power or liquid, mode of

administration, animal species, animal size and individual animal metabolism.

It is also understood by the inventors that the powder may also increase the mineral content and strengt of bones. The inventors understand that this effect may be a function at least in part of the increased osteoblast cell activity and decreased osteoclast cell activity. The effect may also in part be due to the fact that the hard antler powder also includes minerals present in the antler such as calcium. Of significant benefit is the fact that the minerals, and calcium in particular, are provided in the form of microcrystalline hydroyapatite - a form known in bone supplements to be ideal for absorption as the calcium and other nutrients are easily accessed and absorbed by the body. By comparison, alternative calcium sources such as calcium carbonate are poorly absorbed by the body as the calcium needs to be. processed by the body in order to be absorbed. Side effects associated with alternative calcium sources are also avoided. For example, some users of calcium carbonate supplements report bloating which may be a consequence of the carbonate forming carbon dioxide gas in the body.

In one embodiment, the formulation may be produced by drying the harvested hard antler and then extracting / concentrating the dried protein fraction of the antler into a powder. The extraction may reduce the mineral and liquid fractions to less than 10% weight of the formulations. The moisture content of the hard antler before drying is typically 10 to 50% by weight. After drying, the residual water content may less than 10% by weight. The protein . fraction may be extracted via an aqueous extraction method. The resulting extract may then be dried and optionally ground into a powder. This extract may have a greater activity due to the bioactive(s) responsible for the osteogenic activity being in greater concentration or in a profile , that provides a synergy.

In an alternative embodiment, the formulation may be produced by drying the harvested hard antler and grinding the dried antler into a powder. As should be appreciated, this formulation includes the mineral and lipid fractions as well as protein fraction.

In a further embodiment, the harvested hard deer antler may be produced by manufacturing a powder from the raw harvested hard deer antler which may then be washed using alcohol and the resulting washed product passed through a separation process to remove the alcohol. The resulting alcohol washed product may then either be used as is or further extracted, for example, via an aqueous extraction process. The inventors have found that an alcohol wash may not be desirable as this unexpectedly removes osteoblast proliferation observed for harvested hard deer antler that is not washed with alcohol. Nevertheless, osteoclast inhibition remains irrespective of an alcohol wash and therefore, for certain applications, an alcohol wash may have a purpose. For the readers reference, an alcohol wash, such as with ethanol, is common in the art in order to reduce microbial loading.

In a second embodiment there is provided a method of treating an animal in need thereof for diseases and conditions associated with:

(a) increasing osteoblast cell function; or

(b) decreasing osteoclast cell function; or

(c) increasing osteoblast cell function and decreasing osteoclast cell function;

by the step of administering a formulation containing hard deer antler harvested from the deer during the time period between stripping and casting

Preferably, the harvested hard deer antler formulation of the embodiment above may be characterised by a double action on administration of increasing osteoblast cell function and decreasing osteoclast cell function.

In. one embodiment, the harvested hard deer antler may undergo an extraction process to reduce the mineral and lipid fractions to less than 10% weight of the formulation. The extraction process may be an aqueous based extraction process.

In an alternative embodiment, the harvested hard deer antler used in the second embodiment is dried and ground to a powder. As should be appreciated, this method retains the mineral and lipid fractions as well as the protein fraction of the harvested hard deer antler. The inventors envisage that the powder produced by the method of the second embodiment may be administered by known methods such as oral or parenteral methods. A preferred method is likely to be via oral administration due to ease of administration although this should not be seen as limiting.

It is envisaged that the amount administered according to the method of the second embodiment may vary from 5 to 500 mg powdered hard deer antler per day per kg bodyweight. Preferably, the amount administered varies from 20 to 100 mg powdered hard deer antler per day per kg bodyweight. In one embodiment, the dosage may be approximately 50 mg powdered hard deer antler per day per kg bodyweight. The above figures should not be seen as limiting as the amount may vary considerably dependent on the method of preparation, method of extraction (if extraction is completed), form of delivery e.g. powder or liquid, mode of administration, animal species, animal size and individual animal metabolism.

Preferably, the animal may be a human although non-humans such as horses, dogs, cats, livestock and so on may also be treated.

In one embodiment the disease or condition maybe osteoporosis. In an alternative embodiment, the condition may be one or more bone fractures.

In a third embodiment there is provided the use of hard deer antler harvested from deer in the time period between stripping and before casting in the manufacture of a formulation for the treatment of diseases or conditions associated with:

(a) increasing osteoblast cell function; or

(b) decreasing osteoclast cell function; or

(c) increasing osteoblast cell function and decreasing osteoclast cell function.

Preferably, the hard deer antler formulation of the embodiment above may be characterised by a double action on administration of increasing osteoblast cell function and decreasing osteoclast cell function.

In one embodiment, the powered hard deer antler may undergo an extraction process to reduce the mineral and lipid fractions to less than 10% weight of the formulation. The extraction process may be an aqueous based extraction process.

In an alternative embodiment, the harvested hard deer antler used in the second embodiment is dried and ground to a powder. As should be appreciated, this method retains the mineral and lipid fractions as well as the protein fraction of the harvested hard deer antler.

The inventors envisage that the hard deer antler of the third embodiment may be administered by known methods such as oral or parenteral methods. A preferred method is likely to be via oral administration due to ease of administration although this should not be seen as limiting. It is envisaged that the amount administered according to the third embodiment may vary from 5 to 500 mg powdered hard deer antler per day per kg bodyweight. Preferably, the amount administered varies from 20 to 100 mg powdered hard deer antler per day per kg bodyweight. Note this may vary as discussed above.

Preferably, the animal may be a human although non-humans such as horses, dogs, cats, livestock and so on may also be treated.

In one embodiment the disease or condition maybe osteoporosis. In an alternative embodiment, the condition may be one or more bone fractures.

As should be appreciated, the above formulations and associated methods and uses may have a variety of potential applications, particularly in treatment of osteoporosis and other bone diseases, as well as in recovery from bone fractures. It is acknowledged that, although deer velvet is known to assist in treatment of bone disease, hard deer antler is less well studied and certainly not hard antler that has been harvested from the deer prior to casting. The natural lifecycle of the antler means that the exact composition of the antler changes significantly during the various parts of the cycle and hence the bioactives and their concentrations vary considerably as well.

Unexpectedly, the inventors have found that hard deer antler has significant and reproducible effects on osteoblast and osteoclast cell function. This is somewhat unexpected as the prevailing art instead centres on use of velvet i.e. antler prior to stripping. The inventors also unexpectedly found that velvet did not inhibit osteoclast development unlike hard deer antler. This unexpected result is further reiterated by the fact that the value of velvet is an order of magnitude higher than hard antler in part due to the perceived improved benefits of velvet over hard antler. The findings by the inventors therefore differ considerably to the art.

A main advantage for the powder may be a potential double effect by encouraging calcium deposition in the bone as well as inhibiting calcium removal. The inventors found that only harvested hard deer antler had this effect which again was unexpected in view of the art.

Current osteoporosis treatments including prescription drugs only have one effect.

A further advantage of the invention formulation over the art is that, as the powder/extract is manufactured from hard antler, the cost of sourcing the raw material is significantly lower than using velvet. In addition, as the extraction process is simple, unwanted chemical treatments may be avoided such as use of alcohols in extraction and/or pre-treatment washing and the steps required to remove such alcohols from the extracts may also be avoided. This change also reduces processing costs and potentially other aspects of the extract such as avoiding the bitter taste can be present in alcohol extracted materials. Despite the above and as

demonstrated by the inventors below, alcohol washing techniques may be used in selected circumstances and hence are not excluded from the invention.

Also as noted above, not only does the powder potentially provide osteoblast and osteoclast effects, but the powder also provides important minerals to health and in particular calcium. As the calcium is provided as microcrystalline hydroxyapatite, the bioavailability of the calcium from the powder is very high hence the powder provides both cell effects but a potent calcium source as well.

WORKING EXAMPLES

The invention is now described with reference to three examples completed to illustrate ways to manufacture the formulation and the efficacy of the formulaiton.

EXAMPLE 1

The efficacy of harvested hard deer antler as a source of calcium and growth factors, in treating secondary osteoporosis is unexploited. Hence this first example was an initial study to assess the efficacy of a combination powdered harvested hard deer antler and deer bone product (whole, dried and ground to a powder), against calcium carbonate and milk calcium as established sources. The ovariectomized (OVX) rat is the FDA approved model for preclinical testing of nutrients or drugs to prevent bone loss, showing least intra-species variability.

Sixty female Sprague Dawley rats were either sham-operated (n=15) or ovariectomized (OVX) at 9 months of age and these animals were randomized into three groups (n=15 each). The animals were fed a balanced semi-synthetic diet consisting of 15% cellulose, 5% corn oil, 0.5% calcium, 62% starch and added vitamins and minerals as needed until ovariectomy or sham surgery. Animals had ad libitum access to de-ionized water.

Post-surgery each group was fed a low calcium diet (0.1 % calcium) for 4 weeks (28 days) to exacerbate ovariectomy induced bone-loss, to mimic osteoporosis. After this period, animals were fed a casein-based semi-synthetic diet with either the harvested hard deer antler and ^" deer bone test product, milk calcium, or calcium carbonate serving as the only source of calcium for the remaining 28 weeks of the study. The sham control and OVX control group received base diet. At week -2, 4, 10, 17, 22 and 32 the 60 rats underwent in vivo dual energy X-ray absorptiometry (DEXA) scanning of bone density under anaesthesia. Blood samples were taken at week -1 , 4 and 32. At week 32 (following their DEXA scanning) the rats were fasted overnight and then euthanized by exsanguination (heart puncture under anaesthesia). The animals were anaesthetised before heart puncture. After heart puncture, the animals were exposed to 100% CO2. The bodies were dissected and various bones collected and fixed for further analysis.

The results of the study found that the harvested hard deer antler and bone mixture had a similar effect to calcium carbonate or milk. Surprising though was the finding that the mixture had a slightly lower mineral uptake than calcium carbonate and milk suggesting the bone healing effects were present. EXAMPLE 2

The purpose of this study was to provide a basic assessment of the relative active components in velvet against harvested hard deer antler and cast antler. A comparative test was also included to determine if the traditional ethanol wash step used in processing antler had any effects on the powder efficacy.

The samples were dried, ground and analysed by HPLC in a standard protein assay (Invitrogen).

Figure 1 shows the size exclusion chromatography profiles of solutions of the extracts, each dissolved at 8 mg/ml in ammonium bicarbonate. The overlaid plots for each sample measured are standardised to be on the same scale to allow direct comparison.

As can be seen there are some significant differences in the sizes and relative proportions of the peaks which are understood to be due to the markedly different protein compositions of the samples which altered their absorbance at 280 nm (and hence their detection by HPLC).

More specifically, velvet as expected showed a strong peak. The harvested hard deer antler also showed a similar strong peak activity to velvet. Unexpectedly, the cast antler showed significantly less activity with only a small peak compared to that observed for velvet and harvested hard antler. Also unexpectedly, the ethanol washed sample showed minimal activity as well suggesting that this standard ethanol wash step removes potential bioactive.

The above results, while preliminary in nature illustrate the presence or otherwise of potential bioactive(s) in harvested hard deer antler and shows that these are potentially at least comparable to velvet and considerably better than using cast antler. The results also suggest that for best activity the antler should not be ethanol washed as per present practice as this appears to alter the presence of bioactive(s).

EXAMPLE 3

This example describes an experiment completed by the inventors to further define the activity of hard antler powder fractions harvested from live deer on osteoclastogenesis and osteoblast proliferation compared to cast antler and velvet.

Aims and Objectives:

The purpose of the study was to test the effect of four antler extracts on bone osteoblast proliferation (increase in growth and number) or the formation of osteoclasts (bone resorbing cells). The extract samples tested were:

• Cast antler i.e. hard deer antler that had naturally fallen from the deer;

^• Hard antler harvested from the deer before natural casting;

• Hard antler harvested as above but produced using an alcohol wash before aqueous extraction;

^• Velvet or soft antler common in the industry. Methods and materials

Samples were solubilised per unit weight with the velvet, cast and harvested hard antler samples being extracted in aqueous solution. The alcohol wash sample was produced by taking raw harvested hard deer antler, washing this with alcohol, removing the alcohol via evaporation and then completing an aqueous extraction in the same manner as the other samples. Further methodology for producing the extracts are described in Example 1 above.

Testing the effect of the different fractions on osteoclast formation

Osteoclasts can be generated from the murine macrophage RAW 264.7 cell line. RAW 264.7 cells were seeded into 24 well plates and treated with murine RANK-L. RANK-L is an antibody that inhibits osteoclast cell function. The effect of the hard antler powdered fractions on osteoclast formation was tested by the addition of the compound to the RANK-L containing cell culture media at different concentrations (0.1 , 1 , 10, 100 g/ml). The four samples to be test were solubilised in cell culture medium by sample weight.

The solubilised samples were filter sterilised (0.2 μηι) prior to being added to the cells.

Lactoferrin (LF) (1 15 μg/ml) was included as a positive control for its known anti- osteoclastogenic effects. Each experiment (plate) was run in triplicate for replication. The cells were incubated for five days at 37°C in 5% C0 ₂ . The media and treatments were changed with fresh treatments on day 3. The plates were then fixed and stained for tartrate-resistant acid phosphatase (TRAP) and then counterstained with hematoxylin. Osteoclasts appear as large multinucleated cells staining purple red and may form even larger giant cells. TRAP levels in the cell culture media of each well was measured colometrically at 550 nm, and expressed as treatment over the zero control.

Testing the effect of the fractions on osteoblast proliferation

C3T3-E1 preosteoblast cells (MC3T3-E1 subclone 4 murine preosteoblast, CRL-2593; ATCC, Manassas, VA, USA) were maintained under 5% C0 ₂ and 95% air at 37°C in MEMa medium (MEMa; Gibco, Invitrogen NZ) with 10% (v/v) FCS (Gibco, Invitrogen NZ) supplemented with antibiotics (Gentamicin; Gibco, Invitrogen NZ). The effect of the four test samples on osteoblast cell proliferation was determined by treating osteoblasts with different extract concentrations (0.1 , 1 , 10, 100, 1 ,000 pg/ml).

Cells were seeded in 96-well plates at 0.75x10 ⁵ cells / ml (0.1 ml / well) and incubated for 24 hr at 37"C. After 24 hr, cell growth was arrested by incubation in medium with 0.1 % BSA for a further 24 hr. The fractions were dissolved in cell culture medium (w/w) and filter sterilised (0.20 μιη). Lactoferrin is known to stimulate cell proliferation and was included as a positive control (1 ,000 g/ml). Each experiment (plate) was run in triplicate for replication. Cells were incubated with each concentration in medium with 0.1 % BSA. Each plate was incubated for 24 hr at the appropriate temperature and then cell proliferation was quantified via an MTT colorimetric assay.

Statistical analyses All results are presented as means ± 95% CI. TRAP levels and MTT absorbance were analysed by a one way analysis of variance, with a post hoc Dunnett's test comparing the different fraction concentrations with the control containing no sample.

Results

Osteoclastogenesis

A summary of the results is shown in Figures 2-6. The RAW cells are a mouse monocyte cell line that can be transformed into osteoclasts using RANK ligand. On the right hand side of the graphs there is no RANK-L present and therefore no osteoclasts which stains pink with TRAP stain. On the left, the grey bars, the cells have been exposed to RANK-L, lactoferrin (1 15 g/m.l) as positive control, 0 control, and various levels of extracts. The higher the value for TRAP (treatment/control) the more osteoclasts have been formed.

The value for the 0 control is approximately 1 in Figure 2 and with lactoferrin (LF) is lower at 0.75. This means LF has suppressed the formation of osteoclasts.

Casf antler

The cast antler fraction inhibited (Figure 2; p < 0.0001 ) osteoclast differentiation at all tested concentrations, with the strongest effect occurring at a sample concentration of 10 and 100 μg/ml. Large multinucleated osteoclasts were notably reduced at 1 μg/ml and largely absent at 10 and 100 pg/ml. At 100 g/mL it was 25% more effective than lactoferrin.

Harvested hard antler

The harvested hard antler fraction inhibited (Figure 3; p < 0.0001) osteoclast differentiation at a slightly wider range of concentration being 1 , 10 and 100 μg/ml. Large multinucleated osteoclasts were absent at 100 pg/ml. A 10 g/mL concentration of hard antler had a similar effect to lactoferrin. At 100 μg/mL hard antler was also more effective than lactoferrin.

Harvested hard antler (ethanol washed)

The ethanol-washed harvested hard antler fraction was less effective and suppressed osteoclast formation only at 10 and 10 pg/mL (Figure 4; p < 0.0001). Large multinucleated osteoclasts were largely absent at 10 pg/ml and completely absent from cells treated with 100 μg/ml.

All three of the above extracts were 25% more effective than lactoferrin at 100 pg/mL.

All three of the hard antler derived powdered fractions had a dose dependent inhibitory effect on osteoclastogenesis (Figure 6). All samples had no cytotoxic effect on cell numbers at the tested concentrations.

Velvet

The velvet fraction had no effect (Figure 5; p = 0.5931 ) on osteoclast differentiation and large multinucleated osteoclasts were present at all test concentrations.

Osteoblast Proliferation In Figure 7, cell growth is compared to a control (0), and positive control (LF or lactoferrin at 1000ug/mL).

Lactoferrin significantly increased the growth of the osteoblasts compared to the 0 control (MTT (treatment/control).

Cast and Ethanol Washed Hard Antler

The cast antler and ethanol-washed harvested hard antler treatment did not have any effect on cell proliferation (Figure 7; p = 0.1524 and p = 0.0878). Cast antler and harvested hard antler (ethanol extracted) did not increase growth compared to the 0 control, and the number of cells remained less than those in the presence of lactoferrin.

Harvested Hard Antler and Velvet

Harvested hard antler and velvet both stimulated cell proliferation (Figure 7; p < 0.0001 and p < 0.0001). The harvested hard deer antler fraction exhibited a dose dependent effect that was stronger than that observed for velvet.

Discussion

From these results it is clear that hard antler contain components that inhibit osteoclast cell development. The cast antler and ethanol-washed harvested hard antler powder fractions do inhibit osteoclast development but, unlike the harvested hard deer antler, do not stimulate osteoblast cell proliferation. In contrast to the harvested hard antler powder fractions, the velvet powder fraction did not have any detectable anti-osteoclastic effect but exhibited an anabolic effect on the osteoblasts resulting in proliferation.

Trial Summary

The purpose of the study was to test the effect of four antler extracts on bone osteoblast proliferation (increase in growth and number) or the formation of osteoclasts (bone resorbing cells). The extracts tested were:

^• Cast antler (aqueous extracted); .

• Harvested hard antler (aqueous extracted);

• Harvested hard antler (ethanol washed and aqueous extracted);

• Velvet (aqueous extracted).

As shown in Table 1 below, the cast antler and ethanol-washed hard antler powder fractions inhibited osteoclast development whilst not influencing osteoblast cell proliferation. The harvested hard antler powder fraction had both an inhibitory effect on osteoclast development and also exhibited an anabolic effect on osteoblast cell proliferation. The velvet powder fraction did not have any detectable effect on osteoclast development but had an anabolic effect on osteoblast cell proliferation. Table 1- Summary of Results

The results observed for hard antler are very positive and differ significantly to the art by inhibiting osteoclast cell development unlike velvet. In addition, only harvested hard deer antler exhibited a dual effect of osteoblast proliferation and osteoclast inhibition. ~

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope of the claims herein.