US 20120053240A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0053240 A1 Rathmacher et al. (43) Pub. Date: Mar. 1, 2012

(54) METHOD OF ADMINISTERING Publication Classification BETA-HYDROXY-BETA-METHYLBUTYRATE (51) Int. Cl. (HMB) A6II 3/19 (2006.01) A6IP 25/00 (2006.01) (75) Inventors: John Rathmacher, Story City, IA A6IP3/00 (2006.01) (US); John Fuller, JR. Zearing, IA A6IP 2L/00 (2006.01) (US); Shawn Baier, Polk City, IA A6IP37/00 (2006.01) (US); Steven Nissen, Ames, IA (52) U.S. Cl...... 514/557 (US); Naji Abumrad, Nashville, IA (US) (57) ABSTRACT A method of administering beta-hydroxy-beta-methylbutyric (73) Assignee: METABOLC acid (HMB) is described, and specifically administering TECHNOLOGIES, INC., Ames, HMB-acid to a person such that the administration of free IA (US) acid HMB results in an increase ineffectiveness of HMB over administration of other forms of HMB, including a calcium (21) Appl. No.: 12/973,803 salt HMB composition is described. Administration of HMB acid results in an increase in the peak level of HMB in plasma (22) Filed: Dec. 20, 2010 compared with administration of a similar dose of a calcium salt HMB composition. Administration of HMB-acid results Related U.S. Application Data in a faster time to reach peak plasma levels of HMB relative to (60) Provisional application No. 61/287,857, filed on Dec. administration of a similar dosage of a calcium salt HMB 18, 2009. composition. Patent Application Publication Mar. 1, 2012 Sheet 1 of 8 US 2012/0053240 A1

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METHOD OF ADMINISTERING 0007. Once produced or ingested, HMB appears to have BETA-HYDROXY-BETA-METHYLBUTYRATE two fates. The first fate is simple excretion in urine. After (HMB) HMB is fed, urine concentrations increase, resulting in an approximate 20-50% loss of HMB to urine (26:52). Another fate relates to the activation of HMB to HMB-CoA (4; 6:16: 0001. This application claims priority to U.S. Patent 17:20; 35:36: 41; 43:54). Once converted to HMB-CoA, Application Ser. No. 61/287,857 filed Dec. 18, 2009, which is further metabolism may occur, either dehydration of HMB herein incorporated by reference in its entirety. CoA to MC-CoA, or a direct conversion of HMB-CoA to HMG-CoA (42), which provides substrates for intracellular FIELD OF THE INVENTION cholesterol synthesis. Several studies have shown that HMB is incorporated into the cholesterol synthetic pathway (2-4, 0002 The invention relates generally to a more efficient 16) and could be a source of cholesterol for new cell mem and more effective delivery system for B-Hydroxy-3-meth branes that are used for the regeneration of damaged cell ylbutyrate (HMB) and more specifically to administration of membranes (29). Human studies have shown that muscle HMB free acid resulting in a more efficient and more effective damage following intense exercise, measured by elevated way of administrating HMB over administration of a similar plasma CPK (creatine phosphokinase), is reduced with HMB dosage of the calcium salt form of HMB (CaFHMB). supplementation. The protective effect of HMB has been shown to manifest itself for at least three weeks with contin BACKGROUND OF THE INVENTION ued daily use (14; 22:23). 0003 HMB has been found to be useful within the context 0008. In vitro studies in isolated rat muscle show that of a variety of applications. Specifically, in U.S. Pat. No. HMB is a potent inhibitor of muscle proteolysis (32) espe 5,360,613 (Nissen), HMB is described as useful for reducing cially during periods of stress. These findings have been blood levels of total cholesterol and low-density lipoprotein confirmed in humans; for example, HMB inhibits muscle cholesterol. In U.S. Pat. No. 5,348,979 (Nissen et al.), HMB proteolysis in Subjects engaging in resistance training (26). is described as useful for promoting nitrogen retention in The results have been duplicated in many studies (14; 22:33; humans. U.S. Pat. No. 5,028,440 (Nissen) discusses the use 46; 53) (9-11; 47; 48: 48). In C2C12 muscle cells, HMB fulness of HMB to increase lean tissue development in ani attenuates experimentally-induced catabolism (e.g. mals. Also, in U.S. Pat. No. 4,992.470 (Nissen), HMB is 0009. The molecular mechanisms by which HMB described as effective in enhancing the immune response of decreases protein breakdown and increases protein synthesis mammals. U.S. Pat. No. 6,031,000 (Nissen et al.) describes have been reported (10; 44). In mice bearing the MAC16 use of HMB and at least one amino acid to treat disease cachexia-inducing tumor, HMB attenuated protein degrada associated wasting. In U.S. Pat. No. 6,103,764, HMB is tion through the down-regulation of key activators of the described as increasing the aerobic capacity of muscle of an ubiquitin-proteasome pathway (47). Furthermore, HMB animal without a Substantial increase in the mass of the attenuated proteolysis-inducing factor (PIF) activation and muscle. In addition, HMB has been described as useful for increased gene expression of the ubiquitin-proteasome path improving a human's perception of his emotional State in U.S. way in murine myotubes, thereby reducing protein degrada Pat. No. 6,291525. tion (48). PIF inhibits protein synthesis in murine myotubes 0004 HMB has been shown to have positive effects on by 50% and HMB attenuates this depression in protein syn maintaining and increasing lean muscle mass in cancer thesis (9). Eley etal demonstrated that HMB increases protein cachexia and AIDS wasting. In addition, a positive effect on synthesis by a number of mechanisms, including the down muscle damage and the resulting inflammatory response regulation of eukaryotic initiation factor 2 (eIF2) phosphory caused by exercising which leads to muscle Soreness, strength lation through an effect on dsRNA-dependant protein kinase loss, and an increase in pro-inflammatory cytokines is seen (PKR) and upregulation of the mammalian target of rapamy with use of HMB. cin/70-kDa ribosomal S6 kinase (mTOR/p70°) pathway. 0005. It has previously been observed that HMB alone or The net result is increased phosphorylation of 4E-binding in combination with other amino acids is an effective Supple protein (4E-BP1) and an increase in the active eIF4GeIF4E ment for restoring muscle strength and function in young complex. Leucine shares many of these mechanisms with athletes. Further, it has been observed that HMB in combina HMB, but HMB appears to be more potent in stimulating tion with two amino acids, arginine and lysine, is effective in protein synthesis (9). increasing muscle mass in elderly persons. 0010 HMB can also increase protein synthesis by attenu 0006 HMB is an active metabolite of the amino acid leu ating the common pathway that mediates the effects of other cine. The use of HMB to suppress proteolysis originates from catabolic factors such as lipopolysaccharide (LPS), tumor the observations that leucine has protein-sparing characteris necrosis factor-C/interferon-Y (TNF-C/IFN-Y), and angio tics (18; 24). The essential amino acid leucine can either be tensin II (Ang II) (10; 11). HMB acts by attenuating the used for protein synthesis or transaminated to the O-ketoacid activation of caspases-3 and -8, and the Subsequent attenua (C.-ketoisocaproate, KIC) (24). In one pathway, HMB is tion of the activation of PKR and reactive oxygen species formed in the liver via oxidation of the leucine transamination (ROS) formation via down-regulation of p38 mitogen acti product C.-ketoisocaproate. Approximately 5% of leucine vated protein kinase (p38 MAPK). Increased ROS formation oxidation proceeds via this pathway (28). HMB is superior to is known to induce protein degradation through the ubiquitin leucine in enhancing muscle mass and strength. The optimal proteasome pathway. HMB accomplishes this attenuation effects of HMB can beachieved at 3.0 grams per day, or 0.038 through the autophosphorylation PKR and the subsequent g/kg of body weight per day, while those of leucine require phosphorylation of eIF2C, and in part, through the activation over 30.0 grams per day (29). of the mTOR pathway. US 2012/0053240 A1 Mar. 1, 2012

0011. A recent report tested the hypothesis that HMB, like typical 1 gram dosage was 2 hours (52), thus requiring leucine, would stimulate mTORc1 independent of PI3K sig CaFHMB to be taken before exercise for maximal benefit. naling in C2C12 myotubes (19). HMB stimulated the phos 0017 Thus, the timing of HMB administration and the phorylation of AKTSer473 (+12.9%), S6K1Thr389 (+50%) HMB level in the blood are important to the efficacy of HMB and 4EBP1Thr65/70 (+5.1%). HMB stimulated anabolic sig on muscle. The need exists for a faster and more efficient naling with greater potency than leucine, e.g. S6K1Thr389+ delivery system for HMB. 50% vs. +17%; respectively. As expected, incubation of HMB with rapamycin (mTORc1 inhibitor) ablated increases in SUMMARY OF THE INVENTION mTORC1 signaling, but not AKT phosphorylation (+188%). 0018. The invention is administration of HMB in a free In contrast, incubation with LY290042 (PI3K inhibitor) abol acid form (“HMB-acid). Administration using HMB free ished HMB-induced increases in both AKT and mTORc1 acid improves HMB availability to tissues and thus provides signaling, Suggesting HMB signals to mTORC1 in a PI3K a more rapid and efficient method to get HMB to the tissues dependent manner. These data Suggest that HMB, despite than administration of CaFIMB. The oral intake or sublingual being a leucine metabolite, signals to mTORC1 through administration of a free acid HMB associated with a matrix mechanisms distinct from those of leucine. results in direct and rapid absorption of HMB, offering an 0012. Numerous studies have shown an effective dose of improved method of delivery resulting in an increased avail HMB to be 3.0 grams per day as CaFIMB (-38 mg/kg body ability of HMB to the tissues. weight-day'). As a dietary supplement, HMB has been used (0019. In one example, the HMB free acid is delivered as the mono-hydrated calcium salt, whose empirical formula directly by neutralizing HMB free acid in a soluble matrix is Ca(HMB), H2O. This dosage increases muscle mass and such as a gel. The HMB free acid is administered orally or strength gains associated with resistance training, while mini Sublingually to a person in an effective amount. mizing muscle damage associated with strenuous exercise (14; 26:30; 33). HMB has been tested for safety, showing no BRIEF DESCRIPTION OF THE FIGURES side effects in healthy young or old adults (15:25). HMB in (0020 FIG. 1 shows plasma levels of CPK and LDH after a combination with L-arginine and L-glutamine has also been strenuous exercise bout. shown to be safe when supplemented to AIDS and cancer 0021 FIG. 2 shows muscle strength and subjective sore patients (38). ness after a strenuous bout of exercise. 0013 Studies in humans have also shown that dietary (0022 FIG. 3 shows plasma HMB levels as found in supplementation with 3.0 grams of CaFIMB per day plus Experimental Example 1. amino acids attenuates the loss of muscle mass in various 0023 FIG. 4 shows peak plasma HMB concentration and conditions such as cancer and AIDS (5; 12). A meta-analysis time to peak concentration. of Supplements to increase lean mass and strength with (0024 FIG. 5 shows plasma HMB levels. weight training showed HMB to be one of only 2 dietary 0025 FIG. 6 shows peak plasma HMB concentration and Supplements that increase lean mass and strength with exer time to peak concentration. cise (30). More recently it was shown that HMB and the (0026 FIG. 7 shows percent of HMB dosage excreted in amino acids arginine and lysine increased lean mass in a the urine. non-exercising, elderly population over a year-long study. 0027 FIG. 8 shows a treatment regime of the present 0014 Leucine oxidation increases after exercise, and opti invention. mal levels of HMB during and just after exercise would be (0028 FIG.9 shows changes in CPK after an acute bout of desired for optimal prevention of muscle damage and Subse eccentric exercise. quent recovery. Further, the inflammatory process is stimu lated during an injury, which if left unchecked is deleterious DETAILED DESCRIPTION OF PREFERRED and delays healing. Chronic inflammation and pro-inflamma EMBODIMENTS tory cytokines have been shown to be a major underlying and (0029. The invention is a method of delivery of HMB to a causative factor in cardiovascular disease and type II diabe person, and specifically a method of administering HMB tes, as well as in asthma, autoimmune diseases, inflammatory acid to a person such that the administration of free acid HMB bowel disease, chronic obstructive pulmonary disease and results in an increase in effectiveness of HMB over adminis rheumatoid arthritis. tration of other forms of HMB, including CaFIMB. Use of 0015 Human studies have shown a positive effect of resis HMB-acid results in the improvement of HMB availability to tance exercise on muscle protein synthesis as early as 1-2 a person's tissues. The administration of HMB-acid increases hours post exercise and lasting up to 48 hours (8:34). Studies effectiveness for protecting against muscle damage and have also shown that the timing of nutrient availability to be accompanying inflammatory response over administration of critical for maximal post-exercise stimulation of protein Syn HMB in its other forms. Further, administration of free acid thesis as well as blunting of proteinbreakdown (40). The most HMB may also preserve muscle in cachectic and wasting optimal time for delivery of nutrient appears to be within 2 conditions and act to blunt inflammation, including chronic hours post-exercise. Dreyer at al. (7) demonstrated that the inflammation that may cause a number of diseases, such as ingestion of a leucine-rich nutrient solution within 1 h of cardiovascular disease. The unexpected and Surprising dis post-exercise recovery resulted in significant enhancement of covery that free acid HMB decreases muscle damage better the mTOR signaling pathway and muscle protein synthesis. than CaFIMB indicates that it may also decrease inflamma 0016. The dissociation curve of CaHMB is identical to tory response resulting from the damage. The administration that of calcium acetate (49) resulting in peak plasma HMB of HMB free acid has widespread applications as a nutritional levels ranging from 60 to 120 minutes after ingestion depend or medical Supplement and may affect a large portion of the ing upon the dosage given. Time to peak plasma levels after a population. US 2012/0053240 A1 Mar. 1, 2012

0030. In accordance with the present invention, HMB is muscle performance, decreasing muscle damage in muscle administered to humans in its free acid form. The free acid Subjected to stress or damage, decreasing inflammatory HMB may be associated with a carrier, Such as a matrix or gel. response after muscle is subjected to stress or damage, In the preferred embodiment, the free acid HMB is adminis improving the body's immune response after stress or dam tered orally or Sublingually, although any means of adminis age, treating disease associated wasting (such as wasting tering HMB is appropriate. HMB-acid is commercially avail associated with cancer, chronic pulmonary disease, age, able. chronic kidney disease, long-term hospitalization or AIDS). 0031 HMB in its acid form is called 3-hydroxy-3-meth improving a lipid profile Such a low density lipoprotein ylbutyric acid, B-hydroxy-3-methylbutyricacid, or B-hy (LDL) to high density lipoprotein (HDL), and improving a droxy-isovalaryic acid and can be designated “HMB-acid.” person's emotional state. A more effective and more efficient The structural formula is (CH),C(OH)CHCOOH and the way to administer HMB has widespread applications in all of molecule is: these known uses of HMB. 0036 While use of HMB-acid has previously been stated, HMB in free acid form was thought to be equivalent to HMB CH, O in the calcium salt and other salts as proposed administration forms in the prior art. Differences in the effectiveness of --- HMB-acid and HMB salts were not previously tested. OH 0037 Previously, numerous obstacles existed to both extensive testing and commercial utilization of the free acid 0032. In the present invention, HMB-acid is administered form of HMB, and since it was thought there was no differ to a human in an effective amount. An effective amount ence, the calcium salt was adopted as a commercial source of includes a range from about 0.01 grams to about 0.2 grams of HMB. Until recently packaging and, in particular, distribu HMB-acid per kilogram body weight in twenty-four (24) tion of dietary supplements has been better suited to handle hours. HMB-acid may also be administered to a human in an nutrients in a powdered form and therefore the calcium salt of effective amount from about 0.5 grams to about 30 grams of HMB was widely accepted. HMB-acid is a liquid and much HMB-acid per day. An effective amount of HMB-acid will more difficult to deliver or incorporate into products. result in a greater increase in plasma levels of HMB and/or 0038. Unlike other calcium salts, it had been shown that will result in a faster time to reach peak plasma levels of HMB the calcium and HMB components of the molecule dissociate relative to administration of a similar dosage of CaHMBThe very easily, therefore, adding to the assumption that there increase in the effectiveness with administration of HMB would be no physiological difference between HMB free acid acid may be 10%, 20%, 30%, 50%, 75%, 100%,200%, 400%, and the calcium salt of HMB (19) ... (49). 500% or greater than administration of a similar dose of 0039. Additionally, formation and crystallization of the CaHMB. Comparison of HMB-acid with other forms of calcium salt of HMB had been utilized as a final purification HMB may be based on effectiveness or efficiency of HMB stage in the manufacturing process. One compound in par using standard indices known to those of skill in the art. ticular that the crystallization served to limit was 3.3-Dim 0033. In the Examples, the HMB-acid is administered as a ethyl acrylic acid. This compound adds a very off flavor soluble gel, although the invention is not limited to use of a which is hard to mask. Currently, the manufacturing process soluble gel or matrix with HMB-acid. HMB-acid in any phar for HMB has allowed for HMB free acid to be produced in a maceutically acceptable form, including but not limited to purity that allows for oral ingestion of the HMB free acid. Solids, tablets, capsules, and liquids such as oral intravenous Besides currently having a commercial Source that is pure solutions, is within the scope of this invention. The HMB-acid enough for oral ingestion, the HMB-acid needs to be buffered can be administered utilizing any pharmaceutically accept for oral ingestion, a process which only recently was deter able carrier, including but not limited to various starches and mined due to the factors listed above which precluded previ saline solutions. In the preferred embodiment, an effective ous use of HMB-acid. amount of HMB-acid is administered as two or three daily 0040. Because the calcium and HMB in the calcium salt doses, although a single dose of an effective amount of HMB was loosely associated (49), it was previously thought that acid per day will be understood to be within the scope of the there would be no difference in oral administration of HMB invention, as would be any other number of doses of HMB either as a free acid or as a calcium salt (19). As shown in during the day. Example 1, not only is there a surprising difference in plasma 0034. The delivery of HMB-acid, most typically as a levels attained with oral administration of HMB-acid, but HMB-acid soluble matrix Such as agel, results in a significant there is a 25% increase in plasma clearance which indicates improvement in HMB's anabolic effect with marked reduc increased utilization of HMB by tissues with resultant tions in CPK over administration of HMB as a salt, including improved effects on muscle mass and function. When given in CaFHMB or administration of HMB in other forms such as an molar equivalents, HMB in free acid form results in double ester or lactone. In one embodiment, the administration of the plasma level of HMB in about one fourth (4) the time HMB-acid gel results in a doubling of the plasma peak of when compared to the calcium salt of HMB. The improved HMB in about 4 of the time as administration of a similar effect on muscle is clearly shown in Example 2 in that HMB dosage of CaHMB, and has a 25% improved efficiency of administered in free acid form is more protective than delivery as measured by plasma clearance over a similar CaHMB when muscle is subjected to acute exercise. dosage of CaFIMB. 0041 While it is known that CaHMB is soluble in and 0035. This method of delivery has widespread applica easily dissociates in aqueous solutions of neutral to acidic pH, tions. Known uses or benefits of HMB include, but are not one of skill in the art would predicta short lag time in appear limited to, improved nitrogen retention and protein sparing, ance due to this dissociation step. CaHMB administered in a improving lean body mass, improving muscle function and/or gelatin capsule has a dissolution time of between 10 and 15 US 2012/0053240 A1 Mar. 1, 2012

minutes in the gut. Therefore, one skilled in the art would capsule was taken with 355 mL of water (approx. 12 oz.). The expect similar absorption and thus peak plasma levels of free acid gel dosage was 0.80g and was equivalent to the free HMB whether the HMB was administered as CaFIMB or as acid contained in the CaHMB in the capsule. The free acidgel HMB-acid, however, slightly delayed for the differences treatments were either swallowed (FASW) or held sublingual described. Contrary to these expectations, however, it was for 15 seconds and then swallowed (FASL). FASW consisted found that there was a significant difference in plasma peak of expelling the entire dose into the mouth in a 3 ml syringe, between the two forms. In some instances, the difference in swallowing, and then following this with 355 mL of water. peak plasma times was ninety (90) minutes, which is several FASL subjects were instructed to place the entire dosage fold longer than should be accounted for by capsule dissolu tion and disassociation of the CaFIMB. under the tongue and hold the dosage for 15 sec before swal 0042. One skilled in the art would also predict similar lowing. They then rinsed and followed the dose with 355 mL plasma peaks and areas under the curve since a similar of water. amount of the nutrient HMB was being released into the gut 0048 Study 1 design. For study 1 the subjects reported to with the CaFIMB curve shifted further out in time. An unex the laboratory in the morning following an overnight fast. pected result of administering HMB-acid is the doubling in Before ingestion of one of the Supplemental treatments, a peak plasma HMB levels. flexible sterile polyethylene catheter was inserted into a fore 0043. An additional unexpected result is the greater clear arm vein using sterile procedures and a pre-ingestion blood ance (utilization) of HMB once in the plasma. Many nutrients sample was drawn. Subsequent blood samples were taken at have similar plasma and urinary profiles unless there is a 0, 2, 5, 10, 15, 25, 35, 45, 60, 90, 120 and 180 min after conservation mechanism in the kidney for that nutrient. None ingestion of the treatment. Plasma was separated and samples is presently known for HMB and thus one skilled in the art were stored frozen at -70° C. for analysis of HMB concen might presume a much higher percentage of the dosage would tration. In addition, a portion of the pre-ingestion and 180 be excreted in the urine with the doubling of the plasma peak minute blood samples were used for measurements (Lab in HMB concentrations. Again, this was not observed and is Corp, Kansas City, Mo.) of glucose, uric acid, blood urea an unexpected finding. This coupled with the higher clear nitrogen (BUN), creatinine, Sodium, potassium, chloride, car ance rate show improved utilization of HMB by tissues which bon dioxide, phosphorous, protein, albumin, globulin, albu again was a Surprising finding. min:globulin ratio, total bilirubin, direct bilirubin, alkaline 0044 As shown in the Experimental Examples, and spe phosphatase, lactate dehydrogenase, aspartate aminotrans cifically in Example 2, administering HMB-acid is more ferase (AST), alanine aminotransferase (ALT), gamma quickly effective in minimizing muscle damage after exercise glutamyl transpeptidase (GGT), iron binding capacity than CaHMB. This second example shows a benefit that could (TIBC), unsaturated iron binding capacity (UIBC), iron, iron not have been predicted directly from the findings of Example saturation, total cholesterol, triglycerides, high density lipo 1. protein (HDL), low density lipoprotein (LDL), and choles 0045. The method of this invention is further illustrated by terol ratio. A complete blood count (CBC) was also per the following experimental examples. formed before and after the 180 min treatment period. Subjects also completed a brief questionnaire to report any EXPERIMENTAL physical symptoms (such as nausea, headache, etc) they may Example 1 have experienced during the experiment. 0049 Study 2 design. Study 2 was conducted similar to Absorption of HMB-Acid Gel Compared with study 1 with the following modifications. In study 2, plasma CaFIMB in Capsule Form levels of HMB were measured for 1440 min (24 h) and total Materials and Methods urine collection was also performed for measurement of uri nary HMB excretion during this period. After the 180 min 0046 Human subjects. In study 1, four male and four blood sample, the subjects were allowed to leave the labora female college-aged subjects were studied. In study 2, an tory and were instructed to return to the laboratory for addi additional four male and four female college-aged Subjects tional blood samples at 360, 720, and 1440 min after the were studied. The protocols for both studies were approved ingestion of the Supplement. As in study 1, each Subject took by the Iowa State University IRB and each subject gave each of the treatments with at least a one week washout period informed consent to participate in the study. Due to the nature between treatments. Samples were again stored frozen at of the treatments, neither the subjects nor the researchers -70° C. for analysis of plasma and urinary HMB concentra could be blinded. tions. Pre-ingestion, 180 min and 1440 min blood samples 0047 Treatments. The same treatments were given to the were again assayed for the same measurements already for subjects in both study groups. The three treatments were study 1. Subjects were provided with a standardized lunch given in random order to each subject with at least a one week after the 180 min blood sampling and instructed to eat this at washout period between treatments. The treatments were approximately 240 min postingestion. Following the 720 min supplied by Metabolic Technologies, Inc. (MTI, Ames, Iowa) blood sample, Subjects were instructed to eat a normal and were prepared with food grade ingredients. One gram of evening meal before 10 pm. Subjects reported back to the CaHMB or the equivalent HMB in free acid in a gel form was laboratory the following morning for the fasted 1440 min administered to the subjects. The CaFIMB capsules were blood sample. A urine collection container was provided and obtained from a commercial Supplement manufacturer (Opti Subjects collected all urine produced during the experimental mum Nutrition, Aurora, Ill.) while the HMB-acid gel was 24h experimental period. The urine was stored refrigerated prepared at MTI laboratories. Briefly, the HMB-acid was when not being collected. Urine volumes were measured and adjusted to a pH 4.5 with potassium carbonate (KCOs) and samples of the total urine collection were taken and stored flavors and sweeteners were then added. The 1.0 g CaHMB frozen at -70° C. until analyzed for HMB. US 2012/0053240 A1 Mar. 1, 2012

0050 HMB analysis. Plasma and urine HMB were ana subjects completed all 3 testing protocols. No adverse treat lyzed by gas chromatography/mass spectrometry (GC/MS) ment effects, such as nausea after taking the treatments, were as previously described. (27) reported in either study. 0051 Calculations and statistics. Areas under the curves 0057 Study 1 results (Tables 2-4). Table 2 illustrates the were calculated for each Subject using the trapezoidal method peak plasma concentrations (C) and time to reach peak that sums the area above baseline. (51) Half-life of plasma plasma concentrations (t). CaHMB taken by capsule HMB was calculated for study 2. The following equations resulted in a peak plasma HMB level of 131+10 nmol/mL, were used: whereas HMB taken either by free acid gel FASW or FASL k(ln(Ceak)-ln(Crogh))/Tineral delivery resulted in significantly greater (259-24 and 231-21 nmol/mL, respectively, p<0.0001) and earlier (33.1+4.6 and t2=0.693/k 36.3+1.3 min, respectively) peaks in plasma HMB levels 0052 Peak plasma concentrations for each subject were compared with CaHMB by capsule at 121.9+15.6 h (p<0. used for C. Trough concentrations, C., were the con 0001). At 180 min, plasma HMB for all delivery methods was centrations measured at 720 min, because the 720 min plasma still elevated above baseline, and there were no treatment concentrations were not significantly different from baseline. differences among the three groups. Consequently, Areas T. was the time at which C was measured and T. under the curve (AUC) for plasma HMB levels following the is the time from T. until the time at C (720 min). The 3 treatments are also shown in Table 2. HMB administered in extracellular fluid compartment was assumed to be 20% of free acid gel form resulted in 97% and 91% greater areas body weight and calculated using equation 3 below (1). The under the curve (AUC) for FASW and FASL, respectively plasma clearance of HMB was then calculated by multiplying (p<0.0001). There were no differences between HMB-acid the extracellular fluid compartment, V, by the elimination gel delivered by FASW or by FASL. constant, K, as shown in equation 4 Thalhammer, 1998 0058 Table 3 shows blood chemistries for study 1. There 9588/id}. were no significant main effect treatment differences for any of the measured time points or differences. Table 4 shows V=Body wit(0.20) bloodhematology measured in study 1. The FASW group had significantly greater decrease in absolute lymphocyte num Clearance=V(K)(50). bers over the measurement period (p<0.04) due primarily to 0053. The data were analyzed using a crossover design the fact that the FASW group tended to have higher lympho with Proc GLM in SAS. (45) For the timed sampling of cyte numbers at the start of the study (p<0.09). There were no plasma HMB a repeated measures polynomial model was significant differences in lymphocyte numbers at the end of used. The model included subject, order, and treatment main the study and all the means were within normal limits for effects and time by treatment interaction where appropriate. lymphocyte number. For other parameters Proc GLM was also used with subject, 0059 Study 2 results (Tables 2, 5-7). Study 2 was con order, and treatment main effects and the p values are reported ducted to look at plasma HMB for a 24 hour period as well as for the treatment main effect. Statistical significance was to measure urinary losses during this same time period. Simi determined for p-0.05 and a trend was determined for lar to study 1, a rapid increase in and significantly greater 0.05

approximately 25% increase in HMB clearance with the free CaHMB given twice daily at either 1.5 or 3 grams per day. acid gel form compared with the CaHMB form (P<0.003). They demonstrated the optimal dose of CaHMB to be 3.0 0062 Blood chemistries measured during study 2 are grams per day resulting in decreased release of creatine shown in Table 6 and blood hematology measured in study 2 kinase (CK), an indicator of muscle damage, and 3-methyl is shown in Table 7. Statistical analysis of the change in histine, an indicator of protein breakdown (26). Similar find parameters over the 24 h measurement period showed no significant statistical changes (p<0.05) for any of the chem ings were reported by Gallagher who also showed that daily istry or hematology values measured. There was a strong administration of 6 grams per day would be more beneficial trend for a difference with chloride (p<0.06). FASL showed a than 3 grams per day (14). Taken together, the above studies greater increase over the period compared with FASW; how indicate a benefit to having more HMB available to the ever neither FASW nor FASL was significantly different from muscles during exercise. CaFHMB. A trend was also seen with sodium where FASW 0067. HMB delivery by free acidgel results in a faster and showed a larger increase over the measurement period than greater peak in HMB blood levels as well as equally sustained FASW or CaFIMB (p<0.07). Means for chloride and sodium levels when compared with CaHMB administered in a cap over the study period were well within normal values. sule. This form of delivery is equally safe as those currently 0063 Discussion and previously (15; 25) found with oral administration of 0064. It is clear from the present studies that oral or sub CaMB. lingual administration of HMB in free acid gel form resulted in more rapid and Sustained increases in plasma HMB than when compared HMB administered as the calcium salt TABLE 1 (CaFIMB) in a hard gelatin capsule. Free acid gel administra Subject Descriptors. tion of HMB resulted in significant increases (average +14. 8%) in the AUC for plasma HMB without any major changes in plasma HMB half-lives or urinary losses. Combined this Treatment resulted in significantly increased clearance of HMB and utilization by tissues that was 25% greater than that of the CaMB HMB-Acid Ge HMB-Acid Gel CaHMB form. The data show the improved efficiency of this Capsule Swallow Sublingual form of delivery of HMB. Study 1 0065. The findings from this study related to plasmakinet ics of CaFIMB delivery agree with those previously reported Gender, male/female 44 44 44 by Vukovich etal (52) There is congruence of data related to Age, y 23.8 - 1.3 23.8 - 1.3 23.8 - 1.3 peak plasma levels, time to peak and plasma half lives, Body Weight, kg occurred despite significant differences in mode of delivery of CaHMB (four 250 mg capsules by Vukovich et al vs. 1 All 68.5 - 3.9 68.5 - 3.8 68.64.O gram capsule in the present studies). Interestingly, the deliv Females 63.2 - 4.5 63.5 - 4.5 63.5 - 4.9 ery of HMB as free acid in a gel form resulted in faster peak Males 73.75.6 73.5 5.6 73.85.9 levels (nearly 90 minutes earlier with the use of the free acid Study 2 form) but similar half lives, despite plasma levels reaching almost 2-fold those achieved with oral administration of Gender, male/female 44 44 44 CaHMB. Several studies have supported the use of HMB as a Age, y 22.4 - 1.0 22.4 - 1.O 22.4 - 1.0 nutritional supplement during exercise. HMB has been Body Weight, kg shown to decrease muscle protein and membrane breakdown (22:23; 26) and to enhance protein synthesis. (10) It would All 72.O 4.2 724 - 4.1 723 - 4.1 therefore be advantageous to have high levels of plasma HMB Females 66.83.2 67.4 3.3 67.03.5 during the exercise period, and to have HMB retention to be Males 77.27.2 77.4 7.2 77.7 6.9 as good as or even better than those previously reported (26: 29:52). In this regard, oral administration of CaFIMB would need to be administered at least 2 hours before any serious stressful bout of exercise, whereas HMB free acid gel may be TABLE 2 administered before the exercise bout and have an almost Study 1 Area. Under the Curve, Concentration peak (C) and Time to immediate effect. Reach Concentration Peak (tal). 0066 Delivery of the free acid form of HMB was also associated with significantly higher retention of HMB. Study 1 Treatment Administration of the free acid gel form resulted in a signifi Free Acid Gel Free Acid Gel cant increase in AUC while not significantly increasing uri CaMB Swallow Sublingual p Value? nary excretion which would indicate more HMB retention Creais 1312 - 10.1 259.1 - 23.9 2312. 21.0 O.OOO1 and utilization by the tissues compared with the CaHMB nmol/mL form. The estimated amount of HMB retained was 25% teai min 121.9 156 33.14.6 36.3 1.3 O.OOO1 greater with the HMB-acid gel compared with the CaHMB AUC, 13,997 + 1,534 27,532 + 1,742 26,778 + 1,980 0.0001 form based upon plasma clearance. Previous studies by Vuk nmolf ovich etal showed that the oral delivery of 3 grams of CaHMB 180 min resulted in plasma peak levels that were 3 times higher than 'Meant SEM. those achieved with a 1 gram dose (52). Nissen et al demon *P value for treatment differences. strated a dose-dependent response to oral administration of

US 2012/0053240 A1 Mar. 1, 2012

TABLE 4-continued Henatology Values Study 1.1 CaMB HMB-Acid Gel Swallow HMB-Acid Gel Sublingual

% % % p Before After Change Before After Change Before After Change Value? Eosinophils, x10/L O2 (0.04 O.1 0.04 -21.4% O2 (0.04 O.1 O.O2 -41.2% O2 O.O3 O.1 O.O2 -28.6% O.47 Basophils, x 10 L O.OS O.O O.04 O.O -25.0% O.O1 O.O1 O.O4 O.O2 200.0% O.O3 + O.O2 O.O3 + O.O2 O.0% 0.14 'Meant Standard Error of the Mean. *P value for treatment effect of the difference in starting and ending values indicated by 96 change for each treatment,

TABLE 5 TABLE 5-continued Study 2 Plasma HMB Area. Under the Curve, Concentration Peak Study 2 Plasma HMB Area. Under the Curve, Concentration Peak (C), Time to Concentration Peak (t), Half-life, Urinary (C), Time to Concentration Peak (t), Half-life, Urinary HMB Loss, and HMB Retention. HMB Loss, and HMB Retention. Study 2 Treatment Study 2 Treatment HMB-Acid Ge HMB-Acid Gel HMB-Acid Ge HMB-Acid Gel CaMB Swallow Sublingual p Value? CaMB Swallow Sublingual p Value? 24h 14.7 2.0 17.82.9 17.2 2.5 O.18 Cocaie 1312 - 6.O 238.6 16.O 247.6 19.8 O.OOO3 Urine nmol/mL HMB, peakh 13S.O. 17.0 41.95.8 38.82.6 O.OOO1 % initial dose Half-life, 3.17 O.22 2.50 - 0.13 2.51 O.14 O.OO)4 Clearance 53.94.2 67.3 - 3.2 66.9 1.6 O.OO3 h (mL/min) AUC, 46,281 + 2,717 53,395 + 2,862 52,886 + 2,729 0.001 nmolf 'Meant SEM. 1440 min *P value for treatment differences. 24h 1.OO. O.13 1.21 O.19 1.16 O.17 O.18 *At C. total extracellular HMB was estimated using 20% body weight as the extracellular volume. Urine It was assumed for this calculation the plasma and extracellular HMB concentrations were equalized. Amount retained was calculated by subtracting total amount of HMB excreted in HMB, the urine, Relative retention percentage indicated in the table should remain unchanged even mmol if this assumption is not met,

TABLE 6 Blood Chemistry Study 2.' CaMB HMB-Acid Gel Swallow HMB-Acid Gel Sublingual

% Before After Change Before After Change Before After Change Value? Glucose, mg/dL 70.8 O.8 8O3 + 2.4 13.4% 74.1 2.0 79.91.9 7.8% 73.01.8 80.8 2.1 10.6% O.62 Uric Acid, mg/dL 48 0.3 4.20.5 - 12.0% 4.7 O.1 48 0.3 1.9% 4.7 O.3 4.7 O.3 O.8% OSO Blood Urea Nitrogen, 14.8 + 1.4 1341.O -9.3% 14.5 1.4 13.91.4 -4.3%. 15.3 1.7 1541.1 O.8% OSS mg/dL Creatinine, mg/dL 1.O. O.1 1.O. O.1 -1.5% 1.O. O.1 1.O. O.1 -0.6% 1.O. O.1 1.O. O.1 7.9% 0.12 Sodium, mEq/L 139.5 - 0.8 1414 - O.S 1.3% 139.90.7 1415 0.9 1.2%. 138.5 - 0.6 142.1 11 2.6% O.O7 Potassium, mEq/L 4.1 + 0.1 4.7 O.1 14.8% 4.1 + 0.1 4.7 O.2 14.0% 4.O. O.1 4.9 O.1 21.7% O.S1 Chloride, mEq/L 102.4 - O.S 103.0 - O.S O.6% 103.10.8 102.8 0.7 -0.4%. 102.1 - O.S 104.3 O.9 2.1% OO6 CO2, mEq/L 24.9 O.6 27.3 O.6 9.5% 24.81.0 27.8 0.9 12.1%. 25.3 O.8 27.9 O.4 10.4% 0.85 Phosphorus, mg/dL 4.O. O.1 4.4 + 0.1 10.6% 4.2 + 0.2 4.50.2 6.2% 4.1 + 0.1 4.6 O2 11.5% O.44 Protein, g/dL 6.40.2 6.5 - O.2 2.8% 6.4 + 0.1 6.7 O.2 S.1% 6.5 - O.2 6.6 O2 2.9% 0.62 Albumin, g/dL 4.1 + 0.1 4.2 + 0.1 O.6% 4.1 + 0.1 43 O.1 4.5% 4.1 + 0.1 4.2 + 0.1 2.7% 0.08 Globulin, g/dL 2.2. O.1 2.4 + 0.1 6.7% 2.3 O2 2.4 + 0.2 6.0% 2.3 0.1 2.4 + 0.1 3.2% 0.93 A:G Ratio? 1.9 O.1 1801 -3.4% 1.90.2 18 0.1 -2.6% 180.1 1801 -1.4% O.83 Total Bilirubin, mg/dL O.6 O.1 OS O2 -17.8% O.S. O.1 O.S. O.1 2.6% O.6 0.1 O.S. O.1 - 13.3%. O.10 Direct Bilirubin, mg/dL O.20.1 O.1 0.04 -37.4% O.1 O.O 3 O.1 0.04 6.0% O.1 O.O3 O.1 0.04 - 12.8% O21 Alkaline Phosphatase, 6S.S. 7.3 66.8 6.9 1.9% 64.8 7.3 74.17.6 14.5% 66.5 - 7.3 62.8 6.7 -5.6%. O.18 IUL Lactate 1618 h 12.6 1424 - 10.5 - 12.0% 155.9 13. 9 136.6 116 - 12.3%. 163.8 - 11.9 1518, 5.2 -7.3% O.86 Dehydrogenase, IUL Aspartate 22.6 t 1.4 20.01.1 -11.6% 23.3 O.9 21.3 - 0.7 -8.6%. 23.93.2 24.8 2.3 3.7% 0.23 Aminotransferase, US 2012/0053240 A1 Mar. 1, 2012

TABLE 6-continued Blood Chemistry Study 2.1 CaMB HMB-Acid Gel Swallow HMB-Acid Gel Sublingual

% % % p Before After Change Before After Change Before After Change Value? Alanine 1541.8 14.92.2 -3.3%. 16.4 16 16.4 1.7 O.0% 15.3 1.5 16.6 1.8 9.0% 0.18 Aminotransferase, IUL Gamma-glutamyl 14.01.9 14.3 + 1.4 1.8%. 13.3 1.2 14.01.0 5.7%. 13.9 1.2 13.91.O O.0% O.SO Iron Binding 371.8 28.6 377S 30.3 1.5%. 378.632.1 395.134.7 4.4%. 371.931.4 385.O. 29.8 3.5% 0.68 Capacity, Lig/dL UIBC, ugldL 272.8 39.5 317.6 38.3 16.5%. 310.6 - 42.4 335.1 42.1 7.9%. 264.333.3 3.12.0 35.9 18.1% 0.44 Iron, Lig/dL 99.02O.O 59.9 12.2 -39.5%. 68.0 - 13.4 53.8 - 10.8 -21.0%. 107.6 13.7 65.0 - 12.1 -39.6% O.37 Iron Saturation, % 28.458 17.5 - 4.2 -38.3%. 20.3 S.O 16.946 -16.7%. 30.15.1 20.1 - 4.2 -33.2% O.38 Total Cholesterol, mg/dL 160.4 + 8.0 162.9 + 7.1 1.6%. 164.6 10.5 176.9 11.8 7.4%. 16169.4 1633 - 9.3 1.0% 0.25 Triglycerides, mg/dL 97.4 12.5 106.9 17.6 9.8%. 112.6 18.4 121.4 - 22.7 7.8% 93.1 10.6 107.6 16.6 15.6% 0.52 HDL, mg/dL 52.3 2.7 54.O 3.5 3.3% 51.42.8 56.429 9.7% S2.33:1 51.3 2.8 -1.9% O24 LDL, mg/dL 886 - 7.2 88.6 5.9 O.O% 90.8 - 7.0 96.18.1 5.9% 91.35.9 90.4 6.8 -1.0% O.18 Cholesterol Ratio 3.10.2 3.10.2 -O.8% 3.20.2 3.20.2 -2.3% 3.10.2 3.20.2 3.6% 0.53 'Meant Standard Error of the Mean. *P value for treatment effect of the difference in starting and ending values indicated by 96 change for each treatment,

TABLE 7 Hematology Values Study 2.' CaMB HMB-Acid Gel Swallow HMB-Acid Gel Sublingual

% % % Before After Change Before After Change Before After Change p Value WBC, x10/L 7.303 6.5 O.3 -10.5% 6.4. O.3 71 - 0.3 9.7% 7.10.4 6.8 O.3 -3.5% O.10 RBC, x 10/L 43 O.2 43 O.2 O.7% 4.2 + 0.1 4.4 + 0.1 3.5% 43 O.1 43 O.1 O.1% 0.27 Hemoglobin, g/dL 13.20.7 13.30.6 O.7%. 13.00.4 1340.4 3.2% 13.20.5 13.20.5 O.4% O.31 Hematocrit, % 38.1 1.8 38.3 1.7 O.7%. 37.4 1.O 38.91.1 4.2% 37.5 - 1.4 38.6 1.3 2.9% O42 MCV, fL 884 - 1.5 88.5 - 1.4 O.1% 88.1 - 1.5 886 - 1.5 O.6% 884 - 1.5 89.3 - 1.4 1.0% O.21 MCH, pg 30.6 OS 30.7, 0.6 O.2%. 30.6 - 0.6 30.5 + 0.6 -0.3% 3O4. OS 3 O.S. O.S O.3% O.S2 MCHC, g/dL 34.7 O2 34.7, 0.2 O.O%. 34.8 O2 34.5 O2 -O.8% 34.5 O2 34.5 + 0.2 O.1% O.14 RDW, 9% 13.4 O2 13.40.1 -0.1% 13.30.2 13.2 O2 -0.5% 13.3 O2 13.40.2 O.7% O.66 Platelets, x10/L 228.0 17.6 235.9 10.9 3.5%. 229.O. 12.9 242.4 - 12.2 5.8%. 233.8 - 14.2 2443 - 17.5 4.5% O.92 Neutrophils,% 44.93.8 45.6 3.3 1.7% 45.6 2.7 46.0 - 4.4 O.8% 45.35.2 42-43.5 -6.4% O.90 Lymphocytes, 96 44-83.2 42.5 3.5 -5.0%. 43.6 2.4 43.33.9 -0.9% 43.1 + 4.2 45.6 3.4 5.8% O.68 Monocytes, % 6.9 O.9 8.5 - 1.5 23.6% 7.5 - 1.1 6.9 O.8 -8.3% 8.3 - 1.0 8.4 O.9 1.5% O.24 Eosinophils, % 2.9 O.S 2.60.4 -8.7% 2.8 0.5 3.0 - O.S 9.1% 25 O.S 3.30.5 30.0% 0.57 Basophils,% O.6 O2 O.8 0.2 20.0% O.S. O.2 O.90.3 75.0% O.6 O2 O4 0.2 -40.0% O.36 Neutrophils, x10/L 3.303 3.0 - 0.2 -9.9% 2.90.2 3.30.4 10.6% 3.30.5 2.90.3 - 12.1% O49 Lymphocytes, x10/L 3.303 2.80.3 - 13.5% 2.8 0.2 3.10.3 9.4% 3.00.3 3.10.3 S.0% O.O6 Monocytes, x10/L O.S. O.1 O.S. O.1 S.O% O.S. O.1 O.S. O.04 2.6% O60.05 O60.05 O.0% O.71 Eosinophils, x10/L O2 (0.04 O.2 0.03 - 12.5% O.2 0.03 O2 (0.04 13.3% O2 (0.04 O.2 0.03 35.7% O42 Basophils, x10/L O.O6 O.O2 OO6 O.O2 O.0% O.OS, O.O2 0.09 O.O3 75.0% O.O6 O.O2 O.O4 O.O -40.0% O.29 'Meant Standard Error of the Mean. *P value for treatment effect of the difference in starting and ending values indicated by 96 change for each treatment,

Example 2 increase in leakage of the enzyme creatine phosphokinase (CPK) from muscle cells (21:31). Human studies have shown 0068. In this example the effect of administration of that muscle damage following intense exercise, measured by HMB-acidgelis compared to that of calcium HMB on muscle elevated plasma CPK is reduced with calcium HMB supple damage after an eccentric bout of exercise. As shown in mentation (14; 22:26:33). A study on muscle damage after a Example 1, peak plasma HMB levels and HMB clearance rate prolonged 20km run on a collegiate cross country course with are increased with HMB free acid gel administration com both inclines and declines also showed chronic calcium HMB pared with CaHMB (13). This example shows that the quicker administration is effective in decreasing the rise in plasma response of HMB administered as free acid gel prior to and CPK over a 4 day period following the run (23). Nissen et al. following about of extreme exercise protects the muscle from (26) demonstrated a dosage effect of calcium HMB (with 3.0 damage better than HMB administered as the calcium HMB gram per day being more effective than 1.5 grams per day) in salt. decreasing CPK as well as resulting in significant reductions 0069 Effects of HMB and exercise on markers of muscle in urinary 3-methylhistidine (3-MH, a well established indi damage and inflammatory factors: Strenuous exercise. Such cator of myofibrillar protein degradation (39)). Gallagher et as resistance training or maximal effort exercise, causes an al. supplemented dosages of HMB to 37 male college stu US 2012/0053240 A1 Mar. 1, 2012

dents, performing resistance exercise training, corresponding tion lasted about 2 seconds and 12 seconds were allowed to 3.0 (38 mg/kg body weight-d') and 6.0 g (76 mg/kg body between contractions. This protocol was performed on the weight-d') of calcium HMB per day. Both doses had similar right leg followed by the left leg. The subjects received 2 more effects on improving lean mass and strength gains, however, daily dosages of treatment (placebo or HMB) with instruc the higher dosage resulted in significant improvements in tions to take the dosages at lunch and dinner. For the next 4 minimizing CPK leakage Suggesting significant amelioration days the subjects returned to the laboratory fasted each morn in muscle damage following exercise. These observations ing, had blood taken and urine collected and then consumed indicate that higher plasma levels of HMB appear to more the morning dosage of their supplement. The Subjects were protective of muscle damage following exercise. again given the 2 remaining daily Supplement dosages with 0070. As shown in Example 1, HMB as a free acid (in a instructions to take them at lunch and dinner. gel) is absorbed faster than calcium HMB, results in higher 0075 Serum and Urine Samples: A fasting blood sample plasma levels of HMB, and is cleared more readily by muscle. was taken from a Superficial forearm vein each morning when the subjects reported to the laboratory for testing. Addition Methods: ally a clean catch urine sample was collected at this time. 0071. Subjects: We conducted a study at the Iowa State Serum CPK was analyzed by a commercial laboratory (Quest University Health and Human Performance Laboratory; this Diagnostics, Madison N.J.). Urine 3-Methylhistidine (3 MH) study was approved by the Iowa State University Institutional was analyzed by a previously published GC/MS method (9). Review Board, and was registered at ClinicalTrials.gov Urinary creatinine was analyzed by colorimetric assay (Cay (NCT01 150526). We recruited 12 men and 13 women man Chemical Company, Ann Arbor, Mich.). The urinary 3 between the ages of 20 and 36 from the Iowa State University MH data was normalized to urinary creatinine and expressed community and Surrounding area. as 3 MH:creatinine ratio, umol:mg. 0072 Treatments: The experimental design is depicted in 0076 Statistics: Data were analyzed using the mixed FIG 8. model procedure in Statistical Analysis System for Windows Five treatments were administered as follows: (Release 9.1.3, SAS Institute, Cary, N.C.). The change in each Treatment 1: Placebo. This group received a placebo capsule variable was analyzed at each measurement time point. The and a placebo Syringe dosage at each dosage administration. model included the baseline or time Ovalue as a covariate and Treatment 2: CaHMB pre-exercise. This group received a included gender and treatment as main effects. CPK data CaHMB capsule and a placebo syringe dosage 30 min prior to were transformed using the Rank procedure in SAS before the acute exercise bout. The remaining additional dosages analysis. Contrasts were used to compare the HMB-acid gel during the study consisted of one placebo capsule and one pre- and post exercise mean to the other treatment means. placebo Syringe dosage. 0.077 Results: Treatment 3: HMB-acidgel pre-exercise. This group received 0078. The subjects’ demographics are shown in Table 8. a placebo capsule and a syringe dosage of HMB-acid gel 30 The age, height, and weight of the Subjects by treatment were min prior to the acute exercise bout. The remaining additional similar. dosages during the study consisted of one placebo capsule and one placebo Syringe dosage. TABLE 8 Treatment 4: CaHMB pre- and post-exercise. This group received a CaFIMB capsule and a placebo Syringe dosage at Subiect Characteristics all administration times during the study. Treatment Treatment Treatment Treatment Treatment Treatment 5: HMB-acid gel pre- and post-exercise. This 1 2 3 4 5 group received a syringe dosage of HMB-acid gel and one N 5 4 5 5 6 placebo capsule at all administration times during the study. MF 3.2 2.2 2.3 2.3 3.3 0073. To blind the treatments to both the researchers and Age (y) 25.2 2.5 21.O.O.4 24.O. 14 25.82.6 22.2 10 Subjects, each Subject took a capsule and Syringe dosage of Height (cm) 173 + 2.7 173 + 5.5 170+ 6.0 173 + 6.0 171 + 3.1 Supplement at each administration. The capsules contained Weight (kg) 67.0 + 2.3 68.9 + 9.8 68.1 + 5.8 69.7 + 6.9 65.2 + 5.1 either one gram of calcium lactate (Placebo) or one gram of Data are presented as Mean+ SEM. calcium B-hydroxy-f-methylbutryate (CaHMB). The Syringe dosages were formulated to be similar in taste and 0079 Treatments were: (Treatment 1): placebo capsule appearance and contained either 0.8g of corn syrup (Placebo) and placebo syringe dosage; (Treatment 2) Calcium HMB or 0.8 g of B-hydroxy-3-methylbutryate free acid, the same capsule and placebo Syringe dosage pre-exercise followed by amount of HMB as in the capsule dosage. The 3 daily doses placebo capsule and placebo Syringe dosage post exercise; provided the same total HMB dosage (2.4 g as calcium HMB (Treatment 3): Placebo capsule and HMB-acid gel syringe in the capsules or 2.4 g as HMB free acid in the Syringes). dosage given pre-exercise followed by placebo capsule and 0074 Eccentric Exercise Bout: Subjects refrained from placebo Syringe dosage post exercise; (Treatment 4): Calcium vigorous exercise for three days before reporting to the labo HMB capsule and placebo Syringe dosage pre- and post exer ratory. All subjects were studied after an overnight fast. Sub cise; and (Treatment 5): Placebo capsule and HMB-acid gel jects had a fasting blood sample taken and a spot urine sample Syringe dosage given pre- and post exercise. Treatments were was collected. Subjects then consumed their assigned Supple administered 3 times daily, 30 min before the morning testing ment and 30 minutes later the eccentric exercise session was and then again at approximately noon and 6 PM. performed. The exercise consisted of 50 maximal effort con 0080 Serum CPK and urinary 3 MH:urinary creatinine tractions of knee extensors while attempting to resist the ratios are shown in Table 9. There were no differences in Biodex lever arm as it moves the knee joint from full exten baseline (time 0) values. The eccentric exercise caused up to sion to 90 degrees flexion; this is similar to letting a heavy a four-fold increase in serum CPK, indicating muscle mem weight down slowly while in a seated position. Each contrac brane damage. However, HMB-acid gel administered both US 2012/0053240 A1 Mar. 1, 2012

pre- and post exercise (Treatment 5), blunted this increase by REFERENCE LIST up to 64% (P<0.03) at 24 h post exercise and 86% 48 h post I0083 1. The Body Fluid Compartments: Extracellular and exercise (P<0.005). Continuing at 72 h the increase in CPK Intracellular Fluids; Interstitial Fluid and Edema. In: Text still tended to be less with the HMB-acid gel treatment. FIG. book of Medical Physiology, edited by Guyton A C and 9 illustrates the rise and fall in CPK values over the course of Hall J. E. Philadelphia: W B Saunders Company, 2007. the study. Also shown in Table 2 is the effect of the treatments I0084 2. Adamson L. F and Greenberg D. M. The signifi on 3 MH:Cr ratio, a measure of protein degradation in muscle. cance of certain carboxylic acids as intermediates in the Although these data only are indicative of a trend, the data do biosynthesis of cholesterol. Biochim Biophy's Acta 23: 472 show no increase and even a decrease in protein degradation 479, 1957. in the HMB-acid gel pre- and post exercise treatment. This 0085 3. Bachhawat B K, Robinson W G and Coon M J. would indicate a decrease in protein degradation resulting in The enzymatic cleavage of beta-hydroxy-beta-methylglu more favorable protein turnover and protein accretion in taryl coenzyme a to aceto-acetate and acetyl coenzyme A. muscle. J Biol Chem 216: 727-736, 1955.

TABLE 9 Serum Creatine Phosphokinase (CPK) and Urinary 3-Methylhistidine to Urinary Creatinine Ratio (3MH:Cr) After an Acute Bout of Exercise

Treatment 1 Treatment 2 Treatment 3 Treatment 4 Treatment 5 P-value CPK, U/mL

Baseline 1323S 95 - 12 10717 12738 146 - 43 at 24h 356 108 375 - 68 434 126 416 - 157 264 75 O.O3 at 48 h. 2S3 73 246 38 27569 22873 17O 41 O.OOS at 72h 19748 23159 209-54 223 S2 18O34 O.09 at 96 h 16936 281 105 18133 214 - 52 187 46 O.S1 3MH:Cr, Imol/mg

Baseline O-233 OO66 0.164 O.O37 (0.173 0.028 O2O5 O.O25 0.225 O.O34 at 24h O.211 O.O35 0.190 O.O27 0.213 O.O37 O2O3 + O.O39 O.213 O.O25 O.88 at 48 h. O.238 O.O.33 O.240 0.052 (0.190 O.O14 O.22O, O.O27 0.2O8 OO12 O.62 at 72h O.251 0.046 0.204 0.058 (0.172 O.O13 O.231 O.O37 0.175 OO13 O.14 at 96 h O.231 O.O44. O.187 O.O29 O.2O2 O.O2O O.269 O.O38 0.1860.043 0.27 LData are presented as Mean+ SEM. Treatments were: (Treatment 1): placebo capsule and placebo syringe dosage; (Treatment 2) Calcium HMB capsule and placebo syringe dosage pre-exercise followed by placebo capsule and placebo syringe dosage post exercise; (Treatment3):Placebo capsule and HMB free acidgel syringe dosage given pre-exercise followed by placebo capsule and placebo syringe dosage post exercise; (Treatment 4); Calcium HMB capsule and placebo syringe dosage pre- and post exercise; and (Treatment 5): Placebo capsule and HMB free acid gel syringe dosage given pre- and post exercise, Treatments were administered 3 times daily, 30 min before the morning testing and then again at approximately noon and 6PM. P-value HMB Free acid gel (Treatment 5) contrasted with the other treatments.

Implications: I0086 4. Bloch K, Clark LC and Haray I. Utilization of 0081 Based upon the observations in Example 1, muscle branched chain acids in cholesterol synthesis. J Biol Chem tissue was exposed to much higher levels of serum HMB 211: 687-699, 1954. when HMB free acid was administered. Additionally, in I0087 5. Clark RH, Feleke G. Din M, Yasmin T. Singh G, Example 1 it was shown that clearance of HMB from serum Khan F and Rathmacher J. A. Nutritional treatment for to muscle and tissues was also much greater when HMB-acid acquired immunodeficiency virus-associated wasting was administered compared with CaHMB administration. using B-hydroxy-3-methylbutyrate, glutamine and argin Thus, in Example 2 it is shown that this additional utilization ine: A randomized, double-blind, placebo-controlled of HMB by muscle in the free acid form is more protective of study.JPENJ Parenter Enteral Nutr 24(3): 133-139, 2000. the muscle tissue after an acute bout of exercise than HMB I0088 6. Coon M.J. Enzymatic synthesis of branched chain administered in the calcium form. acids from amino acids. Fed ProC 14: 762-764, 1955. 0082. The foregoing description and drawings comprise I0089. 7. Dreyer HC, Drummond MJ, Pennings B, Fujita illustrative embodiments of the present invention. The fore S. Glynn E. L., Chinkes D L, Dhanani S, Volpi E and Ras going embodiments and the methods described herein may mussen B. B. Leucine-enriched essential amino acid and vary based on the ability, experience, and preference of those carbohydrate ingestion following resistance exercise skilled in the art. Merely listing the steps of the method in a enhances mTOR signaling and protein synthesis in human certain order does not constitute any limitation on the order of muscle. Am J Physiol Endocrinol Metab 294: E392-E400, the steps of the method. The foregoing description and draw 2008. ings merely explain and illustrate the invention, and the (0090 8. Dreyer HC, Fujita S, Cadenas JG, Chinkes D L, invention is not limited thereto. Those skilled in the art who Volpi E and Rasmussen B. B. Resistance exercise increases have the disclosure before them will be able to make modifi AMPK activity and reduces 4E-BP1 phosphorylation and cations and variations therein without departing from the protein synthesis in human skeletal muscle.JPhysiol 576: Scope of the invention. Administration of the composition of 613-624, 2006. the present invention will be in an amount sufficient to (0091 9. Eley HL, Russell ST, Baxter J. H. Mukherji Pand achieve a desired effect as recognized by one of ordinary skill Tisdale M J. Signaling pathways initiated by B-hydroxy in the art. B-methylbutyrate to attenuate the depression of protein US 2012/0053240 A1 Mar. 1, 2012

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Effects off-hydroxy-3-methylbutyrate on Induced by Tumor Necrosis Factor Alpha and Angiotensin muscle damage following a prolonged run. JAppl Physiol II by beta-Hydroxy-beta-methylbutyrate. Am J Physiol 89(4): 1340-1344, 2000. Endocrinol Metab 295: E1417-E 1426, 2008. 0106. 24. Krebs HA and Lund P. Aspects of the regulation 0094) 12. Eubanks May P. Barber A, Hourihane A, of the metabolism of branched-chain amino acids. Advan D'Olimpio J.T and Abumrad N. N. Reversal of cancer Enzyme Regul 15:375-394, 1977. related wasting using oral Supplementation with a combi 0107 25. Nissen S. Panton L, Sharp R L. Vukovich M, nation of B-hydroxy-3-methylbutyrate, arginine, and Trappe S W and Fuller J C, Jr. B-Hydroxy-3-methylbu glutamine. Am J Surg 183: 471-479, 2002. tyrate (HMB) Supplementation in humans is safe and may 0095 13. Fuller, J.C., Jr., Sharp, R. L., Angus, H. F., Baier, decrease cardiovascular risk factors. J Nutr 130: 1937 S. M., and Rathmacher, J. A. Free acid gel form of B-hy 1945, 2000. droxy-3-methylbutyrate (HMB) improves HMB clearance 0108. 26. Nissen S, Sharp R, Ray M, Rathmacher JA, Rice from plasma in humans compared to the calcium HMB J. Fuller J C, Jr., Connelly AS and Abumrad N N. Effect of salt. Br. J. Nutr. 2010. the leucine metabolite B-hydroxy B-methylbutyrate on muscle metabolism during resistance-exercise training. J Ref Type: In Press Appl Physiol 81(5): 2095-2104, 1996. 0096) 14. Gallagher P M, Carrithers J A Godard M. P. 0109. 27. Nissen S, Van Koevering Mand Webb D. Analy Schulze K E and Trappe S. W. B-Hydroxy-3-methylbu sis of B-Hydroxy-f-methyl Butyrate in Plasma by Gas tyrate ingestion, Part I: Effects on strength and fat free Chromatography and Mass Spectrometry. Anal Biochem mass. MedSci Sports Exerc32(12): 2109-2115, 2000. 188(1): 17-19, 1990. 0097 15. Gallagher P M, Carrithers J A Godard M. P. 0110 28. Nissen S, Van Koevering Mand Webb D. Analy Schutze K E and Trappe S. W. 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Nutritional supplementation of the leucine metabolite malian target of rapamycin complex 1 (mTORC1) signal B-hydroxy B-methylbutyrate (HMB) during resistance ing via a phosphatidylinositol-3-kinase (PI3K)-dependent training. Nutr 16(9): 734-739, 2000. pathway. FASEB J. 2011. 0116. 34. Phillips SM, Tipton KD, Aarsland A, Wolf S E RefType: Abstract and Wolfe RR. Mixed muscle protein synthesis and break down after resistance exercise in humans. Am J Physiol 0102 20. Isler, O. Ruegg, R., Wursch, J., Gey, K. F., and 273: E99-107, 1997. Pletsher, A. Biosynthesis of cholesterol from B.t-dihy 0117 35. Plaut G W E and Lardy H. A. Enzymatic incor droxy-3-methylvaleric acid. Helvetica Chim. Acta 40, poration of C14-bicarbonate into acetoacetate in the pres 2369. 1957. ence of various substrates. J Biol Chem 192: 435-445, 1951. RefType: Abstract 0118. 36. Rabinowitz, J. L., Dituri F, Cobey F and Gurin S. (0103). 21. 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0119 37. Rathmacher J A Link G. A. Flakoll P J and 0.135 53. Vukovich MD, Stubbs N B and Bohlken RM. Nissen S L. Gas chromatographic-mass spectrometric Body composition in 70-year old adults responds to dietary analysis of stable isotopes of 3-methylhistidine in biologi B-hydroxy-3-methylbutyrate (HMB) similar to that of cal fluids: application to plasma kinetics in vivo. Biol Mass young adults. J Nutr 131(7): 2049-2052, 2001. Spectrom 21:560-566, 1992. (0.136 54. Zabin I and Bloch K. The utilization of butyric 0120 38. Rathmacher JA, Nissen S. Panton L. Clark RH, acid for the synthesis of cholesterol and fatty acids. J Biol Eubanks MP, BarberAE. D'Olimpio J and Abumrad NN. Chem 192:261-266, 1951. Supplementation with a combination of beta-hydroxy 1. A method of administering beta-hydroxy-beta-methyl beta-methylbutyrate (HMB), arginine, and glutamine is butyric acid (HMB) comprising administering a composition safe and could improve hematological parameters.JPENJ comprising between about 0.5 grams of HMB-acid to about Parenter Enteral Nutr 28: 65-75, 2004. 30 grams of HMB-acid to result in a greater effectiveness of 0121 39. Rathmacher J A and Nissen S L. Development the composition than administration of a similar dosage of a and application of a compartmental model of 3-methylhis calcium salt HMB composition. tidine metabolism in humans and domestic animals. Adv 2. The method of administering HMB of claim 1, wherein Exp Med Biol 445: 303-324, 1998. the greater effectiveness resulting from administration of the 0122 40. Rennie M. J. Exercise- and nutrient-controlled composition is selected from the group consisting of improv mechanisms involved in maintenance of the musculoskel ing nitrogen retention, improving protein sparing, improving etal mass. Biochem Soc Trans 35: 1302-1305, 2007. lean body mass, improving muscle function, improving (0123 41. Robinson W. G. Bachhawat B K and Coon M. J. muscle performance, decreasing muscle damage in muscle Enzymatic carbon dioxide fixation by Senecioyl coenzyme Subjected to stress, decreasing muscle damage in muscle Sub A. Fed Proc. 13: 281, 1954. jected to damage, improving a person's immune response, 0.124 42. Rudney H. The synthesis of B-hydroxy-3-meth decreasing disease-associated wasting, improving a person's ylglutaric acid in ratliver homogenates. JAm ChemSoc. 76: lipid profile, and improving a person’s emotional state. 2595, 1954. 3. The method of administering HMB of claim 1, wherein 0125 43. Rudney H and Farkas T G. Biosynthesis of the HMB-acid is administered as a gel. branched chain acids. Fed Proc September: 757-759, 1955. 4. The method of administering HMB of claim 1, wherein 0.126 44. Russell S T and Tisdale M. J. Mechanism of the HMB-acid is administered sublingually. attenuation by beta-hydroxy-beta-methylbutyrate of 5. The method of administering HMB of claim 1, wherein muscle protein degradation induced by lipopolysaccha the HMB-acid is Swallowed. 6. The method of administering HMB of claim 1, wherein ride. Mol Cell Biochem 330(1-2): 171-179, 2009. the HMB-acid is administered one time per twenty-four hour 0127 45. SAS Institute Inc. SAS User's Guide: Statistics. period. Cary, N.C.: SAS Institute Inc., 1985. 0128 46. Slater G, Jenkins D. Logan P. Lee H. Vukovich 7. A method of administering beta-hydroxy-beta-methyl MD. Rathmacher J A and Hahn A. G. b-hydroxy b-meth butyric acid (HMB) comprising administering a composition ylbutyrate (HMB) supplementation does not affect comprising between about 0.5 grams of HMB-acid to about changes in strength or body composition during resistance 30 grams of HMB-acid to result in an increase of the effec tiveness of HMB by at least 10% than administration of a training in trained men. Int J Sport Nutr Exerc Metab 11: similar dosage of a calcium salt HMB composition. 384-396, 2001. 8. The method of administering HMB of claim 7, wherein 0129. 47. Smith HJ, Mukerji P and Tisdale MJ. Attenu the increase in effectiveness resulting from administration of ation of proteasome-induced proteolysis in skeletal muscle the composition is selected from the group consisting of by B-hydroxy-3-methylbutyrate in cancer-induced muscle improving nitrogen retention, improving protein sparing, loss. Cancer Res 6.5: 277-283, 2005. improving lean body mass, improving muscle function, 0130 48. Smith HJ, Wyke S M and Tisdale M.J. Mecha improving muscle performance, decreasing muscle damage nism of the attenuation of proteolysis-inducing factor in muscle Subjected to stress, decreasing muscle damage in stimulated protein degradation in muscle by beta-hydroxy muscle Subjected to damage, improving a person's immune beta-methylbutyrate. Cancer Res 64: 8731-8735, 2004. response, decreasing disease-associated wasting, improving 0131 49. Sousa MF, Abumrad NN, Martins C, Nissen S a person's lipid profile, and improving a person's emotional and Riella M. C. Calcium B-hydroxy-3-methylbutyrate. State. Potential role as a phosphate binder in uremia: In vitro 9. A method of administering beta-hydroxy-beta-methyl study. Nephron 72: 391-394, 1996. butyric acid (HMB) comprising administering a composition (0132 50. Thalhammer F. Schenk P. Burgmann H. El M, I, comprising between about 0.5 grams of HMB-acid to about Hollenstein UM, Rosenkranz, AR, Sunder-Plassmann G, 30 grams of HMB-acid to result in a greater increase in Breyer S and Ratheiser K. Single-dose pharmacokinetics plasma levels of HMB compared to administration of a simi of meropenem during continuous venovenous hemofiltra lar dosage of a calcium salt HMB composition. tion. Antimicrob Agents Chemother 42: 2417-2420, 1998. 10. A method of administering beta-hydroxy-beta-methyl 0133) 51. Urso R, Blardi Pand GiorgiG. A short introduc butyric acid (HMB) comprising administering a composition tion to pharmacokinetics. Eur Rev Med Pharmacol Sci 6: comprising between about 0.5 grams of HMB-acid to about 33-44, 2002. 30 grams of HMB-acid to result in a faster time to reach peak 013.4 52. Vukovich MD, Slater G. Macchi MB, turner M plasma levels of HMB relative to administration of a similar J. Fallon K, Boston T and Rathmacher J. B-Hydroxy-3- dosage of a calcium salt HMB composition. methylbutyrate (HMB) kinetics and the influence of glu 11. A method of administering beta-hydroxy-beta-methyl cose ingestion in humans. J Nutr Biochem 12: 631-639, butyric acid (HMB) comprising improving the effectiveness 2001. of HMB by administering HMB-acid. US 2012/0053240 A1 Mar. 1, 2012

12. The method of administering HMB of claim 11, 14. A method of administering beta-hydroxy-beta-methyl wherein the improvement in effectiveness of HMB resulting butyric acid (HMB) comprising administering a composition from the administration of HMB-acid is selected from the comprising HMB in amount being within the range of from group consisting of improving nitrogen retention, improving 0.01 to 0.2 grams of HMB per kilogram body weight per 24 protein sparing, improving lean body mass, improving hours based on HMB-acid to result in a greater effectiveness muscle function, improving muscle performance, decreasing of the composition than administration of a similar dosage of muscle damage in muscle Subjected to stress, decreasing a calcium salt HMB composition. muscle damage in muscle Subjected to damage, improving a 15. A method of increasing beta-hydroxy-beta-methylbu person's immune response, decreasing disease-associated tyric acid (HMB) utilization by tissue comprising adminis wasting, improving a person’s lipid profile, and improving a tering a composition comprising between about 0.5 grams of person's emotional state. HMB-acid to about 30 grams of HMB-acid to result increased 13. The method of administering HMB of claim 11, utilization of HMB by tissues resulting in improved meta wherein the improvement in effectiveness of HMB resulting bolic effects compared to the administration of a similar from the administration of HMB-acid is selected from the dosage of a calcium salt HMB composition. group consisting of a greater increase in plasma levels of HMB and a faster time to reach peak plasma levels of HMB. c c c c c