AU2006289666B2 - Composition of whey growth factor extract for reducing muscle inflammation - Google Patents

Abstract

The invention relates to the production of compositions containing milk products for use as a nutritional supplement. More specifically, it relates to compositions containing whey growth factor extract, as well as their use in methods for reducing muscle inflammation in individuals undertaking resistance exercise training. According to one aspect of the invention, there is provided the use of a composition comprising whey growth factor extract, isolated from a milk product by cation exchange chromatography, to reduce exercise-induced muscle inflammation.

Description

WO 2007/028211 PCT/AU2006/001323 COMPOSITION OF WHEY GROWTH FACTOR EXTRACT FOR REDUCING MUSCLE INFLAMMATION FIELD OF THE INVENTION The invention relates to the production of compositions containing milk products for use as a nutritional supplement. More specifically, it relates to 5 compositions containing whey growth factor extract, as well as their use in methods for reducing muscle inflammation in individuals undertaking resistance exercise training. BACKGROUND OF THE INVENTION The present invention is to be understood in light of what has previously 10 been done in the field. However, the following discussion is not an acknowledgement or admission that any of the material referred to was published, used or part of the common general knowledge in Australia as at the priority date of the application. The use of nutritional supplements by humans, and even animals, to 15 improve general health or to improve, for example, athletic performance, is known. Nutritional supplements are not intended to provide all the nutrients necessary for a complete diet, but instead are generally intended to complement the dietary intake such that it becomes more nutritionally complete. It is recognised that vitamins, minerals and other substances found in such 20 supplements play important physiological roles and that a deficiency of certain vitamins, minerals and/or other components of supplements has been linked to development of certain diseases, a decrease in general health or lower performance in athletes. Conversely, nutritional supplements are known to enhance a variety of 25 physiological states, under various conditions. There are many targets for nutritional supplements, for example sick patients, convalescing patients, elderly persons and persons undergoing strenuous exercise regimes who wish to improve their performance and/or recovery from such exercise. The nutritional requirements of bodybuilders and persons engaged in 30 strenuous physical exercise are quite particular, whether to decrease body fat and increase lean muscle mass, and or to improve recovery from the strenuous exercise. Recovery includes the ability to overcome exercise-induced inflammation.

WO 2007/028211 PCT/AU2006/001323 2 Inflammation is a localised response to injury or destruction of tissues. It is characterised in the acute form by pain, heat, redness, swelling, and loss of function. Accordingly, reduced muscle inflammation includes, but is not limited to a reduction in any one or more of the aforementioned symptoms in muscle. The 5 inflammatory response antagonises muscle protein synthesis and contributes to much of the physical discomfort experienced by persons 1 to 2 days after engaging in strenuous exercise, including resistance exercise training (Macintyre DL, et al., Sports Med. 1995; 20(1):24-40; MacIntyre DL, et al., Eur J Appl Physiol. 2001; 84(3):180-6). If the inflammation is severe, anti-inflammatory 10 drugs such as corticosteroids may be prescribed. However, the use of such drugs can have deleterious side-effects and, in many cases, is prohibited for athletes in competition. Weight-bearing or resistance training exercise elicits a wide-range of responses in the activated muscle. There are anabolic signalling pathways 15 activated by the stretch and strain of muscles and by growth factors which may be synthesised locally or circulate in the blood stream. Modulation of the expression of various genes is an essential process in regulating the sequence of cellular events needed to activate stem cells that are located within the muscle bed (satellite cells) (Anderson & Wozniak, Can J 20 Physiol Pharmacol. 2004; 82(5):300-10) to rapidly proliferate, before maturing and ultimately fusing with existing muscle fibres or joining together to create new muscle fibres. Regulators of the activation of satellite cells include Syndecan-3 (a transmembrane heparin sulphate proteoglycan essential for satellite cell proliferation), and Pax-7 (a protein that is essential for satellite cell activation and 25 necessary for muscle tissue repair) (Seale et al., Dev Biol. 2004; 275(2):287-300; Cornelison et al., Dev Biol. 2001; 239(1):79-94). Other genes involved in the exercise-induced muscle repair and inflammation process include the small molecular weight chemokines (CCL2 and CCL4) and the early response genes, jun-B and c-fos. 30 If a natural food product, such as a product derived from milk, could be obtained having beneficial effects on exercise recovery it would be a readily available and safe therapeutic supplement.

WO 2007/028211 PCT/AU2006/001323 3 It is known in the prior art to produce a milk product from cows maintained in a specific hyperimmune state, created by administering periodic booster immunisations with sufficiently high doses of antigens, which can be used to treat various diseases including arthritis, dermatitis and neoplastic diseases (EP 0 5 064103 Al, Beck LR). Normal cow's milk however does not contain the specific 'anti-inflammatory factor' present in milk produced from the aforementioned hyper-immunised animals, although it is believed to contain low levels of other anti-inflammatory factors. A person of skill in the art would appreciate that production of the anti-inflammatory milk products according to EP 0 064103 Al is 10 very expensive and time consuming. It is also known in the prior art to produce anti-inflammatory milk products comprising colostrum which is rich in immunoglobulins. Colostrum is the pre-milk produced immediately after birth before the breast secretions stabilize into milk. Prime colostrum from cows is obtained within the first six hours after calving and 15 contains four times the protein found in milk from the same cow obtained 48 hours later. The immunoglobulin fraction of colostrum is loaded with antibodies, lactoferrin and immune enhancers. Lactoferrin is thought to enhance the anti inflammatory effect of colostrum, and is the subject of US Patent 6,475,511 (Gohlke MB). Immunoglobulins are thought to act locally on immune function in 20 the gut. Protein supplementation has been used widely by persons undertaking resistance exercise training to promote muscle protein synthesis in order to repair muscle tissue and facilitate muscle growth. The nutritional supplementation may be provided in the form of a drink or food and includes protein powders to be 25 mixed with liquid for use, nutritional bars and snack foods, tablets, capsules and other preparations. Suitable protein sources commercially available include hydrolysed milk proteins, caseinates, soy protein isolates and milk protein concentrates prepared from ultra-filtrated skim milk. Nutritional supplements which are based on other protein sources, such as whey protein, are also 30 available and can be provided in the form of fruit juices, but are thought to be inadequate because they do not also provide a lipid source (WO 02/15720). In addition, it has been considered that some milk-derived proteins are not readily WO 2007/028211 PCT/AU2006/001323 4 absorbed by the gut, or do not survive the harsh environment of the digestive system to have a therapeutic effect. Whey growth factor extract (WGFE) is one such milk product which was thought to be susceptible to at least partial loss of biological activity once ingested 5 due to fragmentation of molecules such as growth factors. Moreover, by virtue of the process for its isolation, WGFE contains only low levels of immunoglobulins which, as described above, are thought to be important anti-inflammatory factors. Nevertheless, the capacity of whey protein supplementation to provide a benefit to persons engaged in resistance exercise training has been reported to 10 provide a benefit. Whey protein isolate (WPI) and milk protein isolate (MPI), each having a different composition to whey growth factor extract, have been reported to be effective with bodybuilders in rapidly gaining lean muscle mass while reducing body fat. WPI is high in branched-chain amino acids and considered to be fast-acting, whereas MPI is mainly casein which is more slowly metabolised 15 and is effective in promoting muscle growth. The use of WPI, or whey protein concentrate, in combination with the amino acids glutamine, leucine, isoleucine and valine to improve muscular fatigue in a rat model is the subject of WO 2004/049830 Al (Tsuchita H et al.). The authors measured Tyrosine release from soleus muscle as the indicator of fatigue. 20 The present inventors have found that a composition comprising whey growth factor extract altered the expression of genes involved in exercise-induced muscle inflammation. In a separate study conducted by the inventors, trained athletes administered whey growth factor extract reported a reduction in post exercise soreness. Importantly, in terms of the invention, whey growth factor 25 extract may be isolated from normal bulk milk products without a requirement to maintain cows in a hyperimmune state or to harvest colostrum which is in limited supply relative to other milk products. SUMMARY OF THE INVENTION The invention relates to a WGFE composition and method of using the 30 composition which enables persons undergoing resistance exercise training to reduce their level of muscle inflammation over that obtained with compositions of the prior art.

5 It is thus an object of the present invention to provide a composition and method of using same which is improved/more efficacious than compositions of the prior art for reducing muscle inflammation. According to a preferred embodiment of the invention, there is provided a 5 skeletal muscle inflammation-reducing composition comprising whey growth factor extract, isolated from a milk product by cation exchange chromatography. In a further a preferred embodiment of the invention, there is provided a muscle anti- inflammatory composition comprising whey growth factor extract, isolated from a milk product by a process comprising the steps; 10 a) applying the milk product to a SP Sepharose cation exchange column, b) washing the column with a buffer of low ionic strength, c) eluting the WGFE fraction with a buffer containing in the range 0.4 0.5M NaCl, or equivalent ionic strength, at pH 6.5. 15 According to another a preferred embodiment of the invention, there is provided a muscle anti-inflammatory composition comprising whey growth factor extract, isolated from a milk product by a process comprising the steps; a) applying the milk product to a SP Sepharose cation exchange column, 20 b) washing the column with a buffer of 0.008M NaCl or less, c) eluting the WGFE fraction with a buffer containing 0.4M NaCl, or equivalent ionic strength, at pH 6.5. In a further a preferred embodiment of the invention, there is provided a composition according to the above wherein the whey growth factor extract is 25 isolated from a milk product selected from whole milk, cheese whey, rennet casein whey, acid casein whey, or concentrates thereof, or skim milk. In a further a preferred embodiment of the invention, there is provided a composition according to the above when used as a muscle anti-inflammatory agent. 30 In a further a preferred embodiment of the invention there is provided a method of reducing skeletal muscle inflammation in subjects undertaking resistance exercise training comprising administering to the subjects a daily dose of at least 5 mg/kg body weight whey growth factor extract.

6 In a further preferred embodiment of the invention there is provided a method of reducing resistance exercise induced muscle inflammation in a subject comprising administering to the subject a composition comprising an effective amount of whey growth factor extract, in a daily dose of at least 5 mg/kg 5 body weight whey growth factor extract, once per two or three days up to at least once per day, wherein the whey growth factor extract is isolated from a milk product, selected from the group consisting of whole milk, cheese whey, rennet casein whey, acid casein whey and skim milk, by a process comprising the steps; a) applying the milk product to a cation exchange- column, 10 b) washing the column with a buffer of low ionic strength, c) eluting the WGFE fraction with a buffer containing in the range 0.4 0.5M NaCl, or equivalent ionic strength, at pH 6.5. In a further a preferred embodiment of the invention there is provided a method of reducing skeletal muscle inflammation in subjects undertaking 15 resistance exercise training comprising administering to the subjects a daily dose of at least 5 mg/kg body weight whey growth factor extract, isolated from a milk product by a process comprising the steps; a) applying the milk product to a cation exchange column, b) washing the column with a buffer of 0.008M NaCl or less, 20 c) eluting the WGFE fraction with a buffer containing 0.4M NaCl, or equivalent ionic strength, at pH 6.5. Preferably the cation exchange column is SP Sepharose. In another a preferred embodiment of the invention there is provided a method of reducing skeletal muscle inflammation in subjects undertaking 25 resistance exercise training wherein the amount of whey growth factor extract administered, per daily dose, is at least 12.5mg/kg body weight. Preferably the daily dose of whey growth factor extract is at least 25mg/kg body weight. In a further a preferred embodiment of the invention there is provided a use of whey growth factor extract, isolated from a milk product by cation 30 exchange chromatography, for the production of a medicament for the treatment of a subject in need of reduced skeletal muscle inflammation. In a further a preferred embodiment of the invention there is provided a use of whey growth factor extract, isolated from a milk product by a process 6a described above, for the production of a medicament for the treatment of a subject in need of reduced skeletal muscle inflammation.

WO 2007/028211 PCT/AU2006/001323 7 According to another aspect of the invention, there is provided a skeletal muscle inflammation-reducing composition comprising whey growth factor extract, isolated from a milk product by cation exchange chromatography, and an additional protein source. 5 According to a further aspect of the invention the additional protein source is whey protein, preferably whey protein isolate (WPI), more preferably whey protein isolate wherein the whey protein isolate comprises: Moisture 5.0% Fat 0.5% 10 pH (5% solution) 6.3 Ash 3.7% Lactose 0.5% Protein (TN x 6.38) 90.0% Sodium 0.7% 15 Phosphorous 0.3% Calcium 0.15% In a further aspect of the invention there is provided a method of reducing skeletal muscle inflammation in a subject undertaking resistance exercise training comprising administering to the subject an efficacious amount of a composition 20 comprising whey growth factor extract and an additional protein source, such as whey, preferably WPI. In a further aspect of the invention there is provided a method of reducing muscle inflammation in a subject undertaking resistance exercise training comprising administering to the subject a composition comprising an effective 25 amount of whey growth factor extract, isolated from a milk product by cation exchange chromatography, and an additional protein source taken separately. Preferably the amount of additional protein source administered, per daily dose, is at least 225mg/kg body weight (dry weight) and preferably at least 435mg/kg body weight (dry weight). 30 In a further aspect of the invention the administration of the composition of the invention is once per two or three days up to at least once per day, preferably before and/or immediately after resistance exercise training, more preferably WO 2007/028211 PCT/AU2006/001323 8 immediately after resistance exercise training, most preferably between 20 minutes and two hours after exercise. In a further aspect of the invention there is provided a use of whey growth factor extract, isolated from a milk product by cation exchange chromatography, 5 and an additional protein source for the production of a medicament for the treatment of a subject in need of reduced skeletal muscle inflammation. In a further aspect of the invention, there is provided a food or drink comprising the composition of the invention for use in a method to reduce skeletal muscle inflammation in a subject undertaking resistance exercise training. 10 In yet a further aspect of the invention there is provided a use of the composition of the invention for the manufacture of a food or drink to reduce skeletal muscle inflammation in a subject undertaking resistance exercise training. DESCRIPTION OF THE DRAWINGS 15 Figure 1: Fold change in Myo D, myogenin, and myostatin gene (mRNA) expression in the vastus lateralis muscle of adult subjects, at rest and following 3 hours of resistance exercise training performed at the start of the trial [pre training] and following 12 weeks of treatment [post-training] with 20g WPI (Group A), 1g WGFE + 20g WPI (Group B1), or 2g WGFE + 20g WPI (Group B2). 20 Results are presented as mean ± SEM. Figure 2: Fold change in Pax 7 and Syndecan 3 gene (mRNA) expression in the vastus lateralis muscle of adult subjects, at rest and following 3 hours of resistance exercise training performed at the start of the trial [pre-training] and following 12 weeks of treatment [post-training] with 20g WPI (Group A), 1g WGFE 25 + 20g WPI (Group B1), or 2g WGFE + 20g WPI (Group B2). Results are presented as mean ± SEM. Figure 3: Percentage of Pax 7 positive nuclei in tissue sections from the vastus lateralis muscle of adult subjects, following 3 hours of resistance exercise training performed at the start of the trial [pre-training] and following 12 weeks of 30 treatment [post-training] with 20g WPI (Group A), 1g WGFE + 20g WPI (Group B1), or 2g WGFE + 20g WPI (Group B2) as analysed by immunohistochemical staining.

WO 2007/028211 PCT/AU2006/001323 9 Figure 4: Fold change in IGF-1 Binding Protein 4 and 5 gene (mRNA) expression in the vastus lateralis muscle of adult subjects, at rest and following 3 hours of resistance exercise training performed at the start of the trial [pre training] and following 12 weeks of treatment [post-training] with 20g WPI (Group 5 A), 1g WGFE + 20g WPI (Group B1), or 2g WGFE + 20g WPI (Group B2). Results are presented as mean ± SEM. Figure 5: Fold change in Jun B and c-fos gene (mRNA) expression in the vastus lateralis muscle of adult subjects, at rest and following 3 hours of resistance exercise training performed at the start of the trial [pre-training] and 10 following 12 weeks of treatment [post-training] with 20g WPI (Group A), 1 g WGFE + 20g WPI (Group B1), or 2g WGFE + 20g WPI (Group B2). Results are presented as mean ± SEM. Figure 6: Fold change in CCL2, 4 and 7 gene (mRNA) expression in the vastus lateralis muscle of adult subjects, at rest and following 3 hours of 15 resistance exercise training performed at the start of the trial [pre-training] and following 12 weeks of treatment [post-training] with 20g WPI (Group A), 1 g WGFE + 20g WPI (Group B1), or 2g WGFE + 20g WPI (Group B2). Results are presented as mean ± SEM. Figure 7: TNF-alpha production (ng/ml) by RAW cells exposed to 20 lipopolysaccharide (LPS) and cultured in the presence of colostrum (sample 2BC), WPI (sample 4BC) at 100 mg/ml, WGFE (sample 6BC) at 100 mg/ml, various milk fractions, or LPS alone (control). WGFE and LPS were also added to the culture simultaneously (sample WGFE + LPS). The values plotted represent the arithmetic mean ±SEM of the average response obtained in 3 25 independent experiments. Figure 8: Comparison of TNF-alpha production (ng/ml) by RAW cells exposed to lipopolysaccharide (LPS) and cultured in the presence of 100 mg/ml WPI or 100 mg/ml WGFE. DETAILED DESCRIPTION OF THE INVENTION 30 The invention relates to a composition containing WGFE and uses thereof to enable a subject undergoing resistance exercise training to reduce their level of muscle inflammation over that obtained with compositions of the prior art.

WO 2007/028211 PCT/AU2006/001323 10 According to one aspect of the invention, there is provided a skeletal muscle inflammation-reducing composition comprising whey growth factor extract, isolated from a milk product by cation exchange chromatography. The whey growth factor extract for use in the invention may be isolated 5 from milk, skim milk, milk derivatives, whey, colostrum, and colostrum derivatives by, for example, the method described in Australian Patent No. 645589 (PCT/AU91/00303) which is incorporated herein by reference. This method essentially relies on strong cation exchange chromatography to selectively extract basic proteins from the starting material to constitute whey growth factor extract. 10 PROCESS FOR PRODUCING WGFE FRACTION A preferred method of producing WGFE for use in the invention is to use a column packed with SP (sulphopropyl) Sepharose To the column a flow of a dairy product, preferably skimmed milk, is applied until the volume of milk applied is up to 1000 times the volume of the resin packed into the column. The milk 15 remaining in the column is removed with a buffer of low ionic strength (<0.008M NaCI or equivalent) for 10 min. The WGFE fraction is eluted from the column with a buffer containing sodium ions equivalent to 0.4-0.5M NaCl (though other cations would be suitable), most preferably 0.4M NaCl. The mobile phase may have a pH within a broad range, such as 4.5-9.0, 20 preferably 5.5-7.5, most preferably about 6.5. At the upper and lower limits both protein stability and the ability of proteins to bind to the cation exchange resin become influenced. A pH in the range 5.5-7.5 provides the highest WGFE yields. The type of cation exchange resin suitable for adsorption of the WGFE components may include resins such as Sepharose cation exchange resin beads. 25 For example, SP Sepharose Big Beads and CM Sepharose beads (products of GE Healthcare) which contain sulfopropyl functional groups and carboxymethyl groups, respectively, are suitable. The size of the cation exchange resin beads is preferably in the range from 45-300pm. Both SP Sepharose beads in the range 45-165pm and in the range 100-300pm are suitable for WGFE purification 30 according to the invention. One of the further treatments to which the WGFE fraction can be subjected is desalting by, for example, dialysis or ultrafiltration.

WO 2007/028211 PCT/AU2006/001323 11 Accordingly, in a further aspect of the invention, there is provided a composition according to the above wherein the whey growth factor extract is isolated from whey or skim milk. The whey used as starting material may be cheese whey, rennet casein whey, acid casein whey, or concentrates thereof. 5 The amounts of whey growth factor extract and protein source to use according to the invention are to be sufficient for reduced muscle inflammation or to have a therapeutic effect. In another aspect of the invention there is provided a dosage regime wherein the amount of whey growth factor extract administered, per daily dose, is 10 at least 5mg/kg body weight to 12.5mg/kg body weight. Preferably the daily dose of whey growth factor extract is at least 25mg/kg body weight. In a further aspect of the invention there is provided a method of reducing skeletal muscle inflammation in subjects undertaking resistance exercise training comprising administering to the subjects an efficacious amount of a composition 15 comprising whey growth factor extract. When the composition includes an additional protein source, which may be any protein source suitable for consumption such as WPI, post-exercise inflammation is reduced compared to subjects administered a protein source alone. The protein source may be obtained from whole milk, preferably whey 20 protein and more preferably whey protein isolate (WPI). One such whey protein isolate is available commercially under the trade name NatraProTM by Murray Goulburn Co-Op Company Ltd. A typical composition of NatraProTM WPI includes: Moisture 5.0% 25 Fat 0.5% pH (5% solution) 6.3 Ash 3.7% Lactose 0.5% Protein (TN x 6.38) 90.0% 30 Sodium 0.7% Phosphorous 0.3% Calcium 0.15% WO 2007/028211 PCT/AU2006/001323 12 Preferably the composition according to the invention also contains a protein source and, in a dosage regime, the amount of additional protein source per daily dose administered is at least 225mg/kg body weight (dry weight) and preferably at least 435mg/kg body weight (dry weight). The additional protein 5 source could be formulated with the WGFE composition or formulated to be administered separately to the WGFE composition. In a further aspect of the invention there is provided a use of whey growth factor extract for the production of a medicament for the treatment of a subject in need of reduced skeletal muscle inflammation. For example, athletes may 10 require resistance exercise training to build up their muscle strength to enhance their performance, or to overcome a debilitating injury which has caused muscle wasting. The muscle inflammation may be caused by, for example, a 'corked' thigh, or a torn muscle. A medicament comprising the composition of the invention may further assist in the subject recovering more rapidly to return to 15 their training regime and/or to compete. Muscle inflammation may also occur in those with a propensity for bruising, such as the elderly. In a further aspect of the invention there is provided a method of reducing skeletal muscle inflammation in subjects undertaking resistance exercise training comprising administering to the subjects an efficacious amount of a composition 20 comprising whey growth factor extract and an additional protein source. It will be apparent to those skilled in the art that the administration of the composition of the invention may be on the days of training or on the days of training and/or other days, providing the regimen of administration results in reduced skeletal muscle inflammation. Preferably administration is on the days of 25 exercise and more preferably administration occurs either just before and/or immediately after the exercise. More preferably, administration occurs between 20 minutes and 2 hours after exercise. Accordingly, in a preferred aspect of the invention, there is provided a method as described above wherein the administration is immediately after the exercise. Subjects administered 1g WGFE 30 plus 20g WPI, or 2g WGFE plus 20g WPI immediately after resistance exercise training showed increased modulation of the expression of genes involved in inflammation compared to subjects administered WPI alone (see Examples and Results). In addition, subjects administered WGFE reported reduced post- WO 2007/028211 PCT/AU2006/001323 13 exercise soreness compared to those administered WPI, supporting a role for WGFE in reducing post-exercise muscle inflammation. Moreover, the composition of the invention may be used to reduce muscle inflammation in non-human mammals such as horses, greyhounds and others in 5 which reduced muscle inflammation is desirable. In a further aspect of the invention, there is provided a food or drink comprising the composition of the invention for use in a method to reduce skeletal muscle inflammation in subjects undertaking resistance exercise training. In yet a further aspect of the invention there is provided a use of the 10 composition of the invention for the manufacture of a food or drink to reduce skeletal muscle inflammation in subjects undertaking resistance exercise training. WPI typically contains about 90% w/v protein; therefore 20g of WPI contains about 18g of protein as a nutritional source. Whey growth factor extract typically contains about 85% w/v protein; 15 therefore 2g of whey growth factor extract contains about 1.7g of protein as a nutritional source. It will be appreciated that the present invention described herein is not to be limited to specific examples of features disclosed. EXAMPLES 20 Example 1: Clinical Trial A clinical trial was conducted in which 20 young males participated in a three month randomised double-blinded resistance training program. Whey growth factor extract was prepared according to the method broadly described in Australian Patent No. 645589 (PCT/AU91/00303), more specifically described 25 supra. Each whey protein formulation contained an artificial sweetener (NutrasweetTM, Nutrasweet Company, USA). Whey protein formulations were consumed immediately after each exercise session, with each subject completing three supervised exercise sessions per week. Subjects were randomly assigned to one of three supplement groups: 30 Group A: 20g NatraPro (WPI) per dose; n=7 Group B1: 20g NatraPro (WPI) plus 1g WGFE per dose; n=6 Group B2: 20g NatraPro (WPI) plus 2g WGFE per dose; n=7 WO 2007/028211 PCT/AU2006/001323 14 A typical composition of NatraProTM WPI includes: Moisture 5.0% Fat 0.5% pH (5% solution) 6.3 5 Ash 3.7% Lactose 0.5% Protein (TN x 6.38) 90.0% Sodium 0.7% Phosphorous 0.3% 10 Calcium 0.15% Muscle Analysis Muscle samples were collected from the vastus lateralis muscle of the right leg using the percutaneous needle biopsy technique. Excised muscle tissue was visually inspected, dissected free of any fat or connective tissue and blotted to 15 remove excess blood and immediately frozen in liquid nitrogen for subsequent analysis. A portion of the muscle tissue was mounted in an aqueous mounting medium and frozen in isopentane cooled in liquid nitrogen for subsequent immunohistochemical analysis. Immunohistochemistry 20 Serial sections (10pm) of each sample were mounted on microscope slides for analysis of myosin heavy chain fibre type. An immunohistochemical technique based on the fast and slow isoforms of myosin was used to examine fibre type distribution and muscle cross-sectional area based on the protocol of Behan. Cellular localisation of proteins was performed using standard 25 immunohistochemical techniques and antibodies raised against the proteins of interest. RNA extraction & gene expression analysis RNA was extracted from skeletal muscle samples using the ToTALLY RNA kit and reagents (Ambion Inc.) according to the manufacturer's instructions. Total 30 RNA concentrations and quality were determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). Subsequently, RNA was reverse WO 2007/028211 PCT/AU2006/001323 15 transcribed into cDNA using the AMV reverse transcriptase kit protocols and reagents (Promega). Analysis of gene expression was performed on the Applied Biosystems 7500 Real-Time PCR System using gene specific primers designed using Primer Express 2.0 software. 5 Protein expression analysis Standard western blotting techniques and an antibody directed towards a common section of myosin heavy chain protein isoforms (Zymed) was employed to examine total Myosin Heavy Chain protein content. RESULTS 10 Results are presented as mean ± SEM and significance calculated by two way ANOVA using Bonforoni post hoc tests. No significant differences in age, weight, height, or BMI values were observed both pre and post training. Table 1 - Subject Characteristics Subject NatraPro WPI (A) NatraPro WPI (B 1) NatraPro WPI (B 2) Characteristics (n=7) (n= 6) (n= 7 Age 20.4 0.6 19.5 ±0.6 19.0 0.4 Height 182.7 3.3 180 ± 5.2 182.3 i 4.1 Weight Pre Training 79.9 4.0 80.3 ± 4.6 79.3 6.0 Post 80.1 ± 3.2 80.0 i 4.7 80.9 i 5.9 Training BMI Pre Training 24.5 ± 0.8 24.3 1.0 23.9 1.1 Post 24.07 ± 1.1 24.2 1.0 24.4 1.0 Training 15 GENE EXPRESSION ANALYSIS There was no observed, consistent effect of training or treatments on the expression of MyoD and myogenin whilst training significantly increased the expression of muscle LIM protein (MLP) (Figure 1). The expression of Pax 7 and Syndecan 3, two markers of satellite cell recruitment and proliferation, was 16 increased 2-fold and 7-fold, respectively, following 12 weeks of training and is supported by protein expression data (Figures 2 and 3). The expression of IGFBP4 and IGFBP5 (Figure 4) were markedly increased in group B1 at 12 weeks in response to acute exercise. The normal post-exercise up-regulation of 5 the early response genes jun-B and c-fos which are linked to muscle inflammation (Chen YW et al, J. App. Physiol. 2003; 95(6):2485-94) was attenuated in both B1 and B2 supplement groups suggesting a reduced stress response in these groups (Figure 5). At 12 weeks both the resting and post-exercise expression levels of CCL4 and CCL7 were increased following a single bout of resistance 10 exercise in groups B1 and B2 (significantly) compared with control group A (Figure 6). EXAMPLE 2: EFFECT OF WGFE ON LPS-INDUCED TN F-ALPHA EXPRESSION BY RAW CELLS An in vitro study was conducted to test the effect of various milk product 15 fractions on LPS-induced pro-inflammatory TNF-alpha expression by RAW cells. RAW cells were grown in the presence of LPS and then colostrum (sample 2BC), WPI (sample 4BC) at 100 mg/ml, WGFE (sample 6BC) at 100 mg/ml, various milk fractions, was added. In one sample no milk product was added following LPS stimulation ('+ve' control). WGFE and LPS were also added to the culture 20 simultaneously (sample WGFE + LPS). The values plotted represent the arithmetic mean ±SEM of the average response obtained in 3 independent experiments (Figure 7). LPS stimulated the production of 25.3 ng/ml TNF-alpha production; colostrum stimulated 27.7 ng/ml TNF-alpha production - no inhibitory effect; WPI reduced TNF-alpha production by about 25% (20 ng/ml TNF-alpha) 25 compared to WGFE which inhibited TNF-alpha production by about 96% (1 ng/ml TNF-alpha) in the presence of LPS. A comparison was done between the effect of WGFE and WPI on inhibition of TNF-alpha expression (Figure 8). Whey growth factor extract was prepared as for Example 1. The data supports, that WGFE reduces the inflammation response in 30 muscle following resistance exercise training. In vitro data suggests that WGFE reduces inflammation by inhibiting TNF-alpha expression. The data also supports that WGFE in combination with an additional protein source such as WPI decreases the post-exercise inflammatory response to a greater extent than 16a observed when WPI, a protein source known to be used by subjects undertaking resistance exercise training, is administered alone.

Claims (15)

1. A method of reducing resistance exercise induced muscle inflammation in a subject comprising administering to the subject a composition comprising an effective amount of whey growth factor extract, in a daily dose of at least 5 mg/kg body weight whey growth factor extract, once per two or three days up to at least once per day, wherein the whey growth factor extract is isolated from a milk product, selected from the group consisting of whole milk, cheese whey, rennet casein whey, acid casein whey and skim milk, by a process comprising the steps; a) applying the milk product to a cation exchange column, b) washing the column with a buffer of low ionic strength, c) eluting the WGFE fraction with a buffer containing in the range 0.4 0.5M NaCl, or equivalent ionic strength, at pH 6.5.

2. Use of a composition comprising whey growth factor extract, isolated from a milk product, selected from the group consisting of whole milk, cheese whey, rennet casein whey, acid casein whey and skim milk, by a process comprising the steps; (a) applying the milk product to a cation exchange column, (b) washing the column with a buffer of low ionic strength, (c) eluting the WGFE fraction with a buffer containing in the range 0.4 0.5M NaCl, or equivalent ionic strength, at pH 6.5, for the manufacture of a medicament for reducing resistance exercise induced muscle inflammation in a subject, wherein the medicament is formulated for administration of at least 5 mg/kg body weight of whey growth factor extract to the subject once per two or three days up to at least once per day.

3. Method according to claim 1, or use according to claim 2, wherein the whey growth factor extract is isolated by a process comprising the steps; a) applying the milk product to a SP Sepharose cation exchange column, b) washing the column with a buffer of 0.008M NaCl or less, 18 c) eluting the WGFE fraction with a buffer containing O.4M NaCl, at pH

6.5. 4. Method or use according to any one of the preceding claims, wherein the subject is administered a composition comprising an effective amount of whey growth factor extract, in a daily dose of at least 12.5 mg/kg body weight whey growth factor extract. 5. Method or use according to any one of the preceding claims, wherein the subject is administered a composition comprising an effective amount of whey growth factor extract, in a daily dose of at least 25 mg/kg body weight whey growth factor extract. 6. Method or use according to any one of the preceding claims, wherein the composition comprises an additional protein source.

7. Method or use according to claim 6, wherein the additional protein source is taken separately.

8. Method or use according to claim 6 or 7, wherein a daily dose of at least 225mg/kg body weight additional protein source is administered.

9. Method or use according to claim 6, 7 or 8, wherein the additional protein source is whey.

10. The method of any one of claims 1 to 9, wherein the administration to the subject is before, and/or immediately after, resistance exercise training.

11. Method or use according to any one of the preceding claims, wherein the administration is immediately after resistance exercise training.

12. Method or use according to any one of the preceding claims, wherein the administration is between 20 minutes and two hours after exercise. 19

13. Method or use according to any one of the preceding claims, wherein the composition is in the form of a food, drink, tablet or capsule.

14. The method or use of claim 13, wherein the food is in the form of a nutritional bar or snack food.

15. A method of treating resistance exercise induced muscle inflammation in a subject according to claim 1, substantially as hereinbefore described with reference to the Examples.

16. The method or use according to any one of the previous claims wherein the whey growth factor extract is isolated from skim milk.

17. A food, drink, tablet or capsule comprising WGFE, when used in a method according to any one of the preceding claims. MURRAY GOULBURN CO-OPERATIVE CO. LIMITED WATERMARK PATENT AND TRADE MARK ATTORNEYS P26113AUPC