WO2013175386A1 - Compositions and methods for increasing strength and muscle mass - Google Patents

Abstract

Phosphatidic acid, lyso-phosphatidic acid, glycerol-3-phosphate and/or phospholipase D can be administered to exercising mammals to increase muscle mass and strength. These actives can be administered orally to aging, bedridden or cachectic patients to improve nitrogen balance. A highly suitable form of phosphatidic acid for administration is phosphatidic acid-enriched lecithin. Creatine can be co-administered orally to increase muscle-building and strength-enhancing effects. Other additives can include nutritional and herbal supplements, micronutrients and hormones.

Description

COMPOSITIONS AND METHODS FOR INCREASING

STRENGTH AND MUSCLE MASS

Phosphatide acid, lyso-phosphatidic acid, glycerol-3-phosphate and/or phospholipase D can be administered to exercising mammals to increase muscle mass and strength. These actives can be administered orally to aging, bedridden or cachectic patients to improve nitrogen balance. A highly suitable form of phosphatidic acid for administration is phosphatidic acid-enriched lecithin. Creatine can be co-administered orally to increase muscle-building and strength- enhancing effects. Other additives can include nutritional and herbal supplements,

micronutrients and hormones.

BACKGROUND OF THE INVENTION

Muscles are the engines that move the body. Muscles are composed of the contractile proteins myosin and actin, which together form myofibrils. Contraction occurs when actin ratchets over myosin, shortening the length of myofibrils. Like all proteins, these contractile proteins begin with the genetic response, through the ribosomal synthetic apparatus. The resulting proteins are incorporated into existing myofibrils to increase the size of a muscle or to repair any damage that occurs during contraction. The increase in size of a muscle is called muscular hypertrophy. This system requires adequate nutrition to provide amino acids to form the protein, and the pathways are controlled by various activating factors.

Muscular hypertrophy can be is achieved by exercise, especially exercise vigorous enough to reach the anaerobic threshold. Within a short time of commencing such exercise, a mammal can achieve measurable increases in muscle mass and strength. The increased demand causes the synthetic machinery to be up regulated. The activating factors that can initiate up-regulation in response to demands include the "second messenger system", which is known to include phospho lipases, protein kinases and other enzymes.

During growth, pregnancy, and muscle development, the metabolism is in the anabolic phase, that is, more muscle is added than is broken down during the catabolic phase.

Understanding the complexities of anabolism and catabolism and particularly, shifting the balance toward anabolism, is an ongoing and active research area.

The anabolic/catabolic balance is an important factor in disease and disease management. Muscle wasting in patients on bed rest is a common and problematic clinical issue. Patients in intensive care units often become catabolic. In many cases muscle tissue begins to deteriorate almost immediately after confinement. Astronauts become catabolic in the weightless environment of space and also begin losing muscle tissue and strength almost immediately in that environment. Even exercise is not completely sufficient to keep up with the muscle lost through catabolism the weightless environment of space. Significant loss of muscle has also been shown even in healthy, young volunteers whose leg has been immobilized a cast for only two weeks (Hespel et al. J. Physiol.536:625-633, 2001). Extreme loss of muscle tissue leads cachexia, which is often seen in cancer, trauma and burn patients.

A shift toward catabolism may occur as a normal part of aging. Extraordinary measures are necessary to stave it off and shift the metabolism to a more anabolic state. Athletes also regularly seek to achieve a more anabolic state to enhance muscle development. In their training, especially in weight or cardiovascular training intense enough to reach the anaerobic threshold, they are regularly tearing down muscle fiber (catabolism) followed by rebuilding the fibers (anabolism). Muscle rebuilding is especially rapid during the first 90 minutes following vigorous exercise (the "anabolic window"). While daily training itself increases muscle mass and strength, the addition of certain elements, vitamins, and minerals to daily nutrition through supplementation helps increase muscle repair and growth.

Protein is the main nitrogen-containing compound in the human body. About 60%-70% of protein is found in muscle mass. A convenient measure of the anabolic/catabolic status is the nitrogen balance: the ratio between nitrogen ingested and nitrogen excreted. A positive nitrogen balance indicates net growth and an increase in muscle mass; equilibrium indicates a zero balance; while a negative nitrogen balance, if chronic, is an indication of bodily dysfunction that can lead to cachexia.

Accordingly, methods and compositions to up-regulate protein synthesis, in particular the synthesis of contractile proteins, to improve the anabolic/catabolic ratio and nitrogen balance in both athletes and other persons are needed in the art.

SUMMARY OF THE INVENTION

This invention provides compositions and methods for the administration of therapeutically effective amounts of naturally occurring, isolated compounds that are biologically active in increasing muscle mass and strength by stimulation of anabolic metabolism. In one embodiment the invention therefore provides for the oral use of phosphatidic acid (PA) for the enhancement of muscle mass and/or strength in mammals such as humans, equines and canines. The invention further provides a particularly a novel form of PA from soy lecithin called PA-enriched lecithin, and methods for the administration of PA and PA-enriched lecithin. The methods are also directed to reversing muscle catabolism that leads to sarcopenia, for example, in bedridden, aging or cachectic subjects, or those in a weightless environment.

Compositions having a therapeutically effective amount of PA or PA-enriched lecithin sufficient to affect intracellular and extracellular concentrations of PA in a mammal can shift the metabolism from a catabolic state to an anabolic state. This shift counteracts the decrease in muscle tissue that occurs with normal aging, and in more extreme cases such as bed rest, cachexia, and weightlessness. The compositions and methods can also increase exercise capacity in normal healthy mammals where increased muscle mass and strength is desired.

The administration of PA should be combined with as much exercise as the subject is able to perform on a regular basis, for example, according to an intense personal training plan. The administration of PA is preferably combined with the exercise within the anabolic window, when the effect of ingesting PA is more pronounced. This cycle of rebuilding can benefit from protein ingestion up to approximately 90 minutes before exercise and the cycle of rebuilding is especially rapid and intense during the 45-90 minutes following exercise. An easily digested protein supplement such as whey protein increases the effect (for example, about 10 to about 50 grams of whey protein). Another recommended protein is partially hydrolyzed collagen, in similar amounts. The protein should be taken approximately 90 minutes before until 90 minutes after exercise, for example, in two or more separate quantities, for best effect.

An additional composition to increase muscle mass is the combination of creatine and PA. Creatine is stored mainly in muscle tissue, where it is phosphorylated to creatine phosphate by ATP. The high energy phosphate bond of creatine phosphate is readily transferred to adenosine diphosphate by the enzyme creatine kinase forming ATP, which is available for muscle contraction and relaxation. Thus creatine phosphate may be considered a reservoir of muscle energy.

Creatine is readily available in the market place. However, about 30% of humans are creatine non-responders. In these subjects, no creatine is found in the tissues after creatine supplementation. PA has been found to switch creatine non-responders to creatine responders by a yet unknown mechanism. Creatine is generally administered at a dosage of about 3 to about 20 grams per day.

Accordingly, the invention provides a composition that includes one or more of essentially pure phosphatidic acid, phosphatidic acid-enriched lecithin, lyso-phosphatidic acid,

glycerol-3-phosphate, and phospholipase D, optionally in combination with creatine. The essentially pure phosphatidic acid or phosphatidic acid-enriched lecithin can be prepared from soybeans, peanuts, wheat, oats, safflower, fish, milk, bovine liver, eggs or egg yolks. The creatine can be present in a ratio of about 5 to about 3 with respect to the phosphatidic acid, phosphatidic acid-enriched lecithin, or lyso-phosphatidic acid.

The composition can include about 50 mg to about 1 gram of phospholipase D (PLD). The amount of essentially pure phosphatidic acid, phosphatidic acid-enriched lecithin, or lyso-phosphatidic acid can be 0.1 grams to about 40 grams. The amount of creatine can be, for example, about 3 grams to about 10 grams. Each of these amounts can be administered one to about three times per day.

The compositions can further include one or more nutritional supplements. Examples of such supplements include, but are not limited to, protein, one or more amino acids such as leucine or L-aspartic acid, beta alanine, leucine peptide, oc-lipoic acid, P-hydroxy-P-methyl butyrate, glycine propionyl L-carnitine, carnitine, Russian tarragon, gymnema sylvestre, bitter melon, cissus quadrangularis, cinnamon and fenugreek, CLA, tribulus, mulberry, ribose, caffeine, ZMA, betaine, carnosine, or a combination thereof. Additional additives can include Co-Ql 0, chromium, magnesium, vanadium, or a combination thereof. The composition can be in a form for oral administration, such as a tablet,

The invention also provides methods for increasing muscle mass and strength in mammals comprising orally administering an effective amount of a composition as described herein. The composition can include, for example, an effective amount of essentially pure phosphatidic acid, phosphatidic acid-enriched lecithin, or lyso-phosphatidic acid. The effective amount can be 0.1 grams to about 40 grams, administered orally one to three times daily. For example, in a human, the effective amount can be 0.5 to about 5 grams. In a canine, the effective amount can be 0.1 to about 3 grams, and in an equine, the effective amount can be about 10 to about 40 grams

The active or combination of actives can be administered to an exercising or previously exercised subject during the anabolic window. The method can also include the ingestion of 20 to 100 grams of protein during the anabolic window. The protein can be a complete protein containing all the essential amino acids for humans, horses, or dogs. The protein can be whey or partially hydrolyzed collagen protein.

The invention also provides methods for improving the nitrogen balance of a mammal, such as human, canine or equine, and, in particular, such as an aging, bedridden or cachectic human or an aging or cachectic canine or equine, comprising the administration of a therapeutically effective amount of essentially pure phosphatidic acid, phosphatidic acid-enriched lecithin, lyso- phosphatidic acid, or a combination thereof, or a composition as described herein. The effective amount can be 0.1 grams to 4 grams, given orally one to four times daily. The effective amount can be administered orally or by parenteral infusion. The method can further include the administration of an effective amount of creatine, such as about 3 to about 20 grams.

The invention further provides methods for increasing the response to the administration of creatine. The methods can include administering an effective amount of essentially pure phosphatidic acid, phosphatidic acid-enriched lecithin, lyso-phosphatidic acid, or a combination thereof, concomitant with the co -administration of an effective amount of creatine, to a human subject 1 to 3 times daily. The effective amount of can be 0.5 to about 4 grams, and the effective amount of creatine can be about 3 to about 10 grams.

The invention yet further provides methods for increasing muscle mass and strength in mammals comprising the administration of an effective amount of a composition as described herein, as well as methods for increasing muscle mass and strength in mammals comprising administering an effective amount of a composition as described herein, and a hormone. The hormone can be, for example, testosterone, human growth hormone, insulin, or insulin-like growth factor.

DETAILED DESCRIPTION OF THE INVENTION

Phospholipids occur widely throughout the plant and animal kingdoms. For example, the human spinal cord contains 6- 10% and the human brain 4-6% lecithin by weight (w/w).

Soybeans are the most important and economical source of commercial lecithin, which has many applications in foods and industrial processes. Although the following examples use lecithin (about 1.5 to about 3.1% w/w) and PA-enriched lecithin from soybeans (10% to 60% w/w), various embodiments of the invention can include lecithin, essentially pure PA, and/or

PA-enriched lecithin from any source, including but not limited to peanuts (1.11% w/w), calf liver (0.85% w/w), wheat (0.61% w/w), oatmeal (0.65% w/w), or eggs (0.39% w/w). Among refined substances, especially concentrated sources of lecithin include dehydrated egg yolk (14-20% w/w), natural egg yolk (7-10% w/w), wheat germ (2.82% w/w), soy oil (1.8% w/w) and butterfat (1.4% w/w).

Lecithin has been generally recognized as safe (GRAS) by the US FDA since 1979. Lecithin supplementation has been tested by numerous studies in healthy young athletes with no severe side effects (Jager et al, 2007 J. Internal Soc. of Sports Nutrition, 4:5). The effect of lecithin on lowering cholesterol levels (Cobb, 1980 Nutr. Metab. 24:228-237) has been studied. The daily consumption of lecithin in those studies, i.e., 22.5 grams per day for four weeks, contained from 0.4 to 0.7 grams of PA, compared to the 1.6 grams of PA or PA-enriched lecithin per day for four to eight weeks, as is described in the Example below. Interestingly, Cobb reports that no PA was found in plasma after 21 days of supplementation, verifying the ephemeral nature of PA as a result of normal metabolism (Cobb, page 232; Table III). At high lecithin levels, undesirable side effects of lecithin may include gastrointestinal distress, nausea and increased salivation.

The biological importance of phosphatidic acid is becoming recognized. PA is a common phospholipid and is a constituent of all cell membranes. The administration of PA has been suggested to improve membrane stability. However, the cell membrane portion is a minor component of the total phospholipid pool. PA is the smallest of the phospholipids on a molecular weight basis, but is important because it acts as a major precursor to the other phospholipids, many of which are crucial for membrane health. A further role of PA has been found to be as a key and crucial second messenger in muscular contraction, muscle cell growth and development.

Although it is found in the food supply and is a natural component formed during digestion, its existence is ephemeral due to further degradation and entry into the phospholipid synthetic cycle. Before this invention, it was unknown whether oral PA could raise systemic PA levels.

In addition to its structural role, PA is an important controller of protein synthesis. The pathways that regulate PA concentration in response to mechanical demand are as yet not fully defined, especially in the intact body. Under normal conditions, the concentration of PA depends on phospholipase D (PLD) enzyme activity, which causes the hydrolysis of phosphatidylcholine, a major membrane component, to PA and choline. PA then binds the FKBP 12-rapamycin binding (FRB) domain of the protein mTOR and activates p70S6K, which is one of the key ribosomes of the protein translation phase of protein synthesis. Blocking mTOR with the antibiotic rapamycin has been shown to block protein translation and stop up-regulation in response to mechanical stimulation, thereby inhibiting muscle growth.

In vitro studies with skeletal muscle stretch models, cell lysates, or intact cell lines have pointed to PA's role in muscle metabolism. Signaling by the mammalian target of rapamycin (mTOR) is reported to be one aspect necessary for mechanical load-induced growth of skeletal muscle, muscular hypertrophy. The exact mechanisms for the mechanical activation of mTOR are not known, however, several studies indicate that both phospholipase D (PLD) and PA acting as a second messenger play important roles in the activation of mTOR signaling (see, for example, Hornberger, et al. Cell Cycle, 5, 1391-1396, 2006; and Foster, Cancer Research, 67, 1-4, 2007).

The mechanism of mTOR activation is understood to proceed as follows. PA binds to the FKBP12-rapamycin binding (FRB) domain of the protein mTOR and activates p70S6K, a ribosomal dual pathway signaling kinase, which is a key ribosome of the translation phase of protein synthesis. It has been shown that the PA role is critical to the synthesis of protein, particularly muscle proteins. In this in vitro study, an elevation in PA concentration was sufficient for the activation of mTOR signaling. Second, mechanical stimulation results in muscle growth. Such mechanical stimulation can include events such as weight lifting-induced PLD activation, PA accumulation, or mTOR signaling. Finally, when PLD was blocked, PA did not accumulate and mTOR signaling was prevented.

Interestingly, further studies have indicated that PA binds to and activates p70S6K directly even in the absence of mTOR (Lehman et al. FASEB J. vol. 21 , 1075-1087, 2007). This suggests that PA can have an anabolic potential at other times of the day regardless of whether mechanical activation takes place. This finding is of importance in the case of the cachectic, bedridden, or elderly patient who is unable to perform sufficient exercise to induce mechanical activation.

Recent research has also revealed that adenosine monophosphate-activated protein kinase

(AMPK) can inhibit mTOR signaling through the phosphorylation of TSC2, an upstream regulator of mTOR (Inoki et al. 2003 Genes Dev. 17: 1829-1834). PA has been shown to increase AMPK activity, which can result in the inhibition of mTOR activity (Kimball 2007 Biochem. Soc. Trans.

35, part 5: 1298-1301). Moreover, AMPK activation has been linked to the reduction of p70S6 kinase activity. Therefore, because AMPK inhibits protein synthesis via a number of different pathways, it is likely that AMPK is a key regulator of cardiac hypertrophy. These results are contrary to the earlier findings and suggest that PA could actually decrease protein synthesis.

These in vitro studies can provide theoretical bases for the administration of PA to increase muscle protein synthesis. As stated above, because of the well-known gastro-intestinal degradation and entry into the phospholipid synthetic cycle upon uptake into the vascular system, only actual in vivo experimentation can resolve this question. Previous to this invention, oral administration of PA has not been studied. Muscle growth is very complex, with many factors contributing to the optimal compositions and methods for promoting growth. In addition to signaling at the gene level as discussed above, good muscle health begins with adequate nutrition. The diet can be optimized to provide the best combination of nutrients for each individual if the correct combination of nutrients is known. Supplements are available to increase the metabolism of carbohydrates, proteins and fats. For example, whey protein is a complete protein, containing the proper balance of essential amino acids, and is easily digested. Partially hydrolyzed collagen is another complete protein and is even more easily digested. An athlete in the muscle building phase can require 20 to 120 grams of protein daily. Protein supplement of an easily digestible protein such as whey or collagen is even more beneficial for the aging, cachectic or bedridden person.

Recent emphasis on the oc-lipoic acids has indicated an additional benefit. Some herbal products such as Russian tarragon, Cissus quadruangularis or Gymnema sylvestre can be beneficial. Other beneficial products include amino acids, creatine, L-carnitine, glycine propionyl L-carnitine, bitter melon, cissus quadrangularis , cinnamon and fenugreek,

creatinol-o-phosphate, leucine peptide, leucine, CLA, tribulus, ribose, caffeine, beta alanine, ZMA, betaine, L-aspartic acid and carnosine, alone or in combination. Each of these

supplements, acting at a different level of metabolism, can enhance the effect of PA-enriched lecithin administration.

A leading recommended nutritional supplement is creatine. Creatine is phosphorylated by creatine kinase (CK) to form an energy reservoir, especially in muscle tissue, for the resynthesis of ATP expended during exercise. Numerous studies have shown that an increase in

intramuscular creatine levels with creatine supplementation is variable, with mammals falling into "responder" or "non-responder" groups. Much of this variability can lie within the regulation and activity of the creatine transporter. In one study the observation was that approximately 20% to 30% of participants following a creatine loading regime did not respond with an increase in intracellular creatine (Greenhaff et al. 1994 Amer. J. Physiol. 266 (5Pt 1): E725-30). Another study conducted a descriptive profile of the characteristics of individuals portraying Greenhaff s classification of responders versus non-responders. Because mTOR has been shown to stimulate the creatine transporter SLC6A8 through mechanisms at least partially shared by the serum and glucocorticoid-induced kinase SGK1 , creatine supplementation combined with PA may show a synergistic effect, not only in extending the benefits of creatine supplementation to creatine non-responders, but also to increase the creatine effect in responders. Therefore a composition of creatine with PA, PA-enriched lecithin is recommended or lyso-PA. Creatine is available in several forms such as creatine salt, creatine ester, creatine ether, creatinol, creatinol ether, creatinol salt, all of which can be used with the compositions and methods described herein. Various forms of creatine that may be used are also described in U.S. Patent Nos. 7,772,428 (Heuer et al.) and 7,476,749 (Heuer et al.)

Beyond nutritional supplements, hormones such as testosterone, human growth hormone, insulin, and insulin-like growth hormones can also play a role in promoting anabolism. These hormones may be especially efficacious for cachectic patients. Other "micronutrients" such as chromium, vanadium and Coenzyme Q10 may also be added to the diet or the compositions described herein.

It can be concluded that signalling through mTOR is necessary for mechanically induced growth of skeletal muscle and that mTOR signalling requires a certain concentration of PA. Until this disclosure, it was unknown whether PA could be sufficiently raised by ingestion by an intact mammal to affect and significantly amplify the growth signalling cascade. Surprisingly, it has been found that PA or lyso-PA amplifies the growth signalling cascade, even in the absence of mechanical induction. Thus, PA and lyso-PA are important in shifting the metabolism from the catabolic state to the anabolic state and improving nitrogen balance. The shift in metabolism from the catabolic state to the anabolic state can be readily measured by determining the nitrogen balance, the ratio between nitrogen consumed as protein and nitrogen excretion as urea.

Alternatively, the urinary excretion of creatinine may be analyzed to determine the nitrogen balance. A positive nitrogen balance is critical for preventing, reducing, or inhibiting cachexia and other forms of muscle deterioration.

Definitions

As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley 's Condensed Chemical Dictionary 14^th Edition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001. References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or use of a "negative" limitation.

The term "and/or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrase "one or more" is readily understood by one of skill in the art, particularly when read in context of its usage. For example, one or more components in a formulation can refer to one to five, or one to four, or one to three.

The term "about" can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and/or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment.

As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements. As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percents or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for weights of components, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, as used in an explicit negative limitation.

An "effective amount" refers to an amount effective to treat a disease, disorder, and/or condition, or to bring about a recited effect. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptoms being treated.

Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art, especially in light of the detailed disclosure provided herein. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a condition or disorder, or to treat the symptoms of the condition or disorder, in a host. Thus, an "effective amount" generally means an amount that provides the desired effect.

In some embodiments, "an effective amount" can refer to an amount effective for enhancing lean muscle stimulus, growth, strength and recovery, increasing nutrient delivery and/ or promoting increased vascular response, e.g., blood flow and circulation, in a subject, for example, when administered over a period of week(s). An effective amount of the actives (e.g., creatine, essentially pure phosphatidic acid, phosphatidic acid-enriched lecithin, lyso-phosphatidic acid, or phospho lipase D) can be from about 0.1 g to about 20 g of a nutritional composition per serving. In various embodiments, an effective amount of the composition comprises from about 1 g to about 10 g of the nutritional composition per serving.

The terms "treating", "treat" and "treatment" include (i) preventing a disease, pathologic or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting the disease, pathologic or medical condition or arresting its development; (iii) relieving the disease, pathologic or medical condition; and/or (iv) diminishing symptoms associated with the disease, pathologic or medical condition. Thus, the terms "treat", "treatment", and "treating" can extend to prophylaxis and can include prevent, prevention, preventing, lowering, stopping or reversing the progression or severity of the condition or symptoms being treated. As such, the term "treatment" can include medical, therapeutic, and/or prophylactic administration, as appropriate.

Lecithin is the commercial term for a naturally occurring mixture of phospholipids (also called phosphatides or phosphoglycerides). The most common phospholipids in lecithin are phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylcholine (PC),

phosphatidylserine (PS), and phosphatidylinositol (PI). The "head" of a phospholipid is hydrophilic, while the hydrophobic "tails" are repelled by water and form aggregates in aqueous compositions. As a result of this configuration, phospholipids form natural barriers, segregating or insulating structures. The hydrophilic head contains the negatively charged phosphate group, and may contain other polar groups such as choline. The hydrophobic tail consists of long fatty acid hydrocarbon chains.

Lecithin is found in many natural products including but not limited to soybeans, peanuts, eggs, grains, liver, fish, legumes, safflower, and milk. A typical lecithin used in the compositions and methods described herein is soy lecithin. Lecithin from any source may be isolated to an essentially pure phosphatidic acid (PA) (, for example, at least 98% pure) by enzymatic conversion, a method well known in the art. However, a suitable composition can be prepared from soy lecithin to contain at least about 40% PA to provide PA-enriched lecithin. Minor components of PA-enriched lecithin can be, for example, 5-15% phosphatidyl choline, 1-5% lyso-phosphatidylcholine, and/or 1 -5% N-acyl phosphatidylethanolamine. These components neither increase nor interfere with the PA content and activity. Lecithin is meant to include chemically or enzymatically altered derivatives, such as DHA-soy lecithin.

The term "enriched" refers to a partially purified extract or composition from which undesirable impurities have been removed and/or by certain actions, the concentration of a particular component is increase. The undesirable impurities may be a single compound or multiple compounds. In some embodiments, at least 25%, at least 50%, or at least 75%, of the undesirable impurities have been removed.

Phosphatidic acid (PA) is a diacyl-glycerophospholipid and a major constituent of cell membranes. The structure of PA is:

where Cx is the alkyl or alkenyl moiety of a fatty acid. Typically, an unsaturated fatty acid bonded to carbon-2 and a saturated fatty acid is bonded to carbon-3. PA can also be used in the compositions described herein as its phosphate salt.

Essentially pure PA refers to a composition of phosphatidic acid (PA) that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5%, pure PA, by weight. PA-enriched lecithin includes at least about 10%, at least 40%, at least 50%, at least 60%, or at least 70%, PA.

Lysophosphatidic acid (lyso-PA or LP A) is a phospholipid having the structure:

HaOaPO^^j^OR¹

OR² (lyso-PA)

where one R¹ is the alkanoyl or alkenoyl moiety of a fatty acid and R² is H, or vice versa. When

1 2

R is H, the compound is 1 -lyso-PA; when R is H, the compound is 2-lyso-PA. Examples of lyso-PA fatty acid moieties (e.g., OR¹ or OR²) include (Z)-octadec-9-enoate or octadecanoate.

Examples of fatty acids that can form esters with the glycerol backbone of PA or lyso-PA include, but are not limited to, decanoic acid (10:0), undecanoic acid (1 1 :0), 10-undecanoic acid (11 : 1), lauric acid (12:0), cz^'s-5-dodecanoic acid (12: 1), tridecanoic acid (13:0), myristic acid (14:0), myristoleic acid (cz^'s-9-tetradecenoic acid, 14: 1), pentadecanoic acid (15:0), palmitic acid (16:0), palmitoleic acid (cz^'s-9-hexadecenoic acid, 16: 1), heptadecanoic acid (17: 1), stearic acid (18:0), elaidic acid (irara-9-octadecenoic acid, 18: 1), oleic acid (cz^'s-9-octadecanoic acid, 18: 1), nonadecanoic acid (19:0), eicosanoic acid (20:0), cis-l 1-eicosenoic acid (20: 1),

11 ,14-eicosadienoic acid (20:2), heneicosanoic acid (21 :0), docosanoic acid (22:0), erucic acid

(cz^'s-13-docosenoic acid, 22: 1), tricosanoic acid (23:0), tetracosanoic acid (24:0), nervonic acid

(24: 1), pentacosanoic acid (25:0), hexacosanoic acid (26:0), heptacosanoic acid (27:0), octacosanoic acid (28:0), nonacosanoic acid (29:0), triacosanoic acid (30:0), trans vaccenic acid

(trans- 11-octadecenoic acid, 18: 1), tariric acid (octadec-6-ynoic acid, 18: 1), and ricinoleic acid

(12-hydroxyoctadec-cz^'s-9-enoic acid, 18: 1) and ω3, ω6, and ω9 fatty acyl residues such as

9,12,15-octadecatrienoic acid (a-linolenic acid) [18:3, ω3]; 6,9,12,15-octadecatetraenoic acid

(stearidonic acid) [18:4, ω3]; 11 ,14,17-eicosatrienoic acid (dihomo-.alpha.-linolenic acid) [20:3, ω3]; 8,11 ,14,17-eicosatetraenoic acid [20:4, ω3], 5,8,11,14,17-eicosapentaenoic acid [20:5, ω3];

7,10,13,16,19-docosapentaenoic acid [22:5, ω3]; 4,7,10,13,16,19-docosahexaenoic acid [22:6, ω3] ; 9, 12-octadecadienoic acid (linoleic acid) [18:2, ω6] ; 6,9, 12-octadecatrienoic acid (γ-linolenic acid) [18:3, ω6]; 8,1 1,14-eicosatrienoic acid (dihomo-Y-linolenic acid) [20:3 ω6];

5,8,11 ,14-eicosatetraenoic acid (arachidonic acid) [20:4, ω6]; 7,10,13,16-docosatetraenoic acid

[22:4, ω6]; 4,7,10,13,16-docosapentaenoic acid [22:5, ω6]; 6,9-octadecadienoic acid [18:2, ω9];

8,1 1-eicosadienoic acid [20:2, ω9]; 5,8,1 1-eicosatrienoic acid (Mead acid) [20:3, ω9];

trans-\0,cis-\2 octadecadienoic acid; cis-\0,trans-\2 octadecadienoic acid; cis-9 ,trans-\ 1 octadecadienoic acid; trans-9, cis-l 1 octadecadienoic acid, as well as "omega-3 fatty acids" such as

Δ-5, 8,1 1,14,17-eicosapentaenoic acid (EPA), A-4,7,10,13,16,19-docosahexanoic acid (DHA) and

A-7,10,13,16,19-docosapentanoic acid (n-3 DP A). The acyl residues of a fatty acid moiety can also be conjugated alkenes, hydroxylated, epoxidized, and/or hydroxyepoxidized acyl residues.

Lyso-PA can act as a signalling molecule. Lyso-PA can be used in the compositions described herein as its phosphate salt. Lyso-PA can be substituted for PA in any embodiment of this invention, or it can be added to a composition or method that uses PA to supplement the composition's activity.

Phospholipase D (PLD) is an enzyme that catalyzes the hydrolysis of phosphatidylcholine to form phosphatidic acid (PA), releasing the soluble choline headgroup into the cytosol. PLD is often located in the plasma membrane of cells. The two mammalian isoforms of phospholipase D are PLD1 and PLD2. One specific example of PLD is autotaxin, also known as ecto nucleotide pyrophosphatase/phosphodiesterase family member 2 (E-NPP 2). Autotaxin has

lysophospholipase D activity that converts lysophosphatidylcholine into lyso-PA. PLD can be substituted for PA in any embodiment of this invention, or it can be added to a composition or method that uses PA to supplement the composition's activity. The amount of PLD used can be about 50 mg to about 1 gram, about 100 mg to about 800 mg, about 200 mg to about 750 mg, about 100 mg to about 400 mg, about 400 mg to about 800 mg, or 200 mg, 400 mg, 500 mg, 750 mg, or 1 gram.

Creatine refers to the chemical compound N-methyl-N-guanyl glycine (CAS Registry No. 57-00-1), also known as (oc-methyl guanido)acetic acid, N-(aminoiminomethyl)-N-glycine, methylglycocyamine, methylguanidoacetic acid, andN-methyl-N-guanylglycine, whose chemical structure and zwitterionic form are shown below.

As used herein, "creatine" also includes derivatives of creatine such as esters, and amides, and salts, as well as other derivatives, including derivatives that become active upon metabolism. Creatinol (CAS Registry No. 6903-79-3), also known as creatine-O-phosphate,

N-methyl-N-(beta-hydroxyethyl)guanidine O-phosphate, Aplodan, or

2-(carbamimidoyl-methyl-amino)ethoxyphosphonic acid, is also a creatine derivative that can be used in the compositions and methods described herein.

Creatine and creatine derivatives are widely available from a number of commercial sources. Commercially available creatine derivatives include creatine phosphate, creatine citrate, magnesium creatine, alkaline creatine, creatine pyruvate, creatine hydrates (including, but not limited to creatine monohydrate), and creatine malate. Glycocyamine, an in vivo precursor of creatine, is also commercially available and suitable in the practice of the invention described herein. In some embodiments, the compositions include creatine malate or creatine monohydrate.

The "anabolic window" or the "metabolic window" refers to the first 90 minutes following vigorous exercise, when the body is typically in a catabolic state as a result of the exercise.

During this window the metabolism of protein can be extremely rapid. Catabolism of protein can be even more enhanced in the first 45 minutes following vigorous exercise. However, proper consumption of protein and other nutrients can shift the body from a catabolic state to an anabolic state during the anabolic window, thereby enhancing muscle building, strength gains, and muscle recovery.

Formulation Components

The compounds and active agents described herein can be used to prepare therapeutic compositions. The compounds may be added to the compositions in the form of a salt or solvate. For example, in cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, oc-ketoglutarate, and oc-glycerophosphate.

Suitable inorganic salts may also be formed, including hydrochloride, halide, sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable ionic compound. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example, calcium) salts of carboxylic acids can also be prepared by analogous methods.

The actives described herein can be formulated as therapeutic compositions and administered to a mammalian host, such as a human patient, in a variety of forms. The forms can be specifically adapted to a chosen route of administration, e.g., oral or or subcutaneous routes. The actives may be systemically administered in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. For oral administration, compounds can be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the food of a patient's diet. Compounds may also be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations typically contain at least 1% of active compound. In some embodiments, the percentage of the

compositions and preparations can vary and may conveniently be from about 2% to about 90% of the weight of a given unit dosage form. The amount of active in such therapeutically useful compositions is such that an effective dosage level can be obtained.

The tablets, troches, pills, capsules, and the like may also contain one or more of the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; and a lubricant such as magnesium stearate. A sweetening agent such as sucrose, fructose, lactose or aspartame; or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring, may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol.

Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and flavoring such as cherry or orange flavor. Any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active may be incorporated into sustained-release preparations and devices.

The active may also be administered as a solution or dispersion. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can be prepared in glycerol, liquid polyethylene glycols, triacetin, or mixtures thereof, or in a pharmaceutically acceptable oil. Under ordinary conditions of storage and use, preparations may optionally contain a preservative to prevent the growth of microorganisms.

Dosage

Useful dosages of the actives described herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Patent

No. 4,938,949 (Borch et al.). The amount of an active, an active salt or derivative thereof, or a combination of actives, can vary not only with the particular compound or salt selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and can be ultimately at the discretion of an attendant physician, clinician, nutritional advisor.

The dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more doses or sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced

administrations.

A dosage of 0.1 grams to about 40 grams of PA-enriched lecithin can be administered to a mammal orally one to three times daily, preferably 90 minutes before to 90 minutes after exercise, e.g., during the anabolic window. When the mammal is a human, 0.5 to four grams is a recommended single dosage. When the mammal is a horse, 10 to 40 grams is a recommended dosage. When the mammal is a whippet, 0.1 to 0.3 grams is a recommended dosage. When the mammal is a greyhound, 0.2 to 0.4 grams is a recommended dosage.

A gastric acid secretion inhibitory coating may be applied to the dose in a manner that protects the PA from degradation by gastric juices. Examples of such enteric coatings include polymers such as cellulose. Enteric coated PA can be incorporated in the manufacture of foods, drugs, and dietary supplements of complex formulations and various dosage forms including capsules, tablets, caplets, lozenges, liquids, solid foods, powders and other dosage forms that may be developed, without the need to impart enteric protection to the entire mixture, any other part of the mixture, or finished products.

A variety of methods for delivery and/or administration of PA can be carried out, for example and not by way of limitation, by tablet, capsule, powder, granule, microgranule, pellet, soft gel, controlled release form, liquid, solution, elixir, syrup, suspension, emulsion, magma, gel, cream ointment, lotion, transdermal, sublingual, ophthalmic, nasal, otic, aerosol, inhalation, spray, parenteral, suppository and the like. In suitable cases, PA may be administered by intravenous or intraarterial infusion. Compositions of the invention may also be administered in nutraceutical or functional foods. In addition, the effective amount of PA may be combined with amino acids, botanicals, functional foods, herbals, nucleotides, nutraceuticals, pharmaceuticals, proteins, minerals, and/or vitamins in an effort to enhance the targeted activity. Vitamins, amino acids, and other additives are described by, for example, Remington: The Science and Practice of Pharmacy, 22^th edition (Lippincott Williams & Wilkins, 2000).

The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention. Thus, the following experiments were carried out to show more clearly the effect of PA administration in increasing muscle hypertrophy and strength. These examples are given in detail in order to more clearly explain how to make and use the invention and do not limit the scope of the appended claims . Human subj ects were used, but the results are readily obtained with other mammals such as the horse and the dog, with adjustments in dosage appropriate to body weight.

Example 1. Enrollment criteria

A double-blinded study was planned to test the effect of PA on muscle strength. The inclusion criteria were: participation in a resistance training program on a regular basis at recreational level or higher; no physical limitations as determined by health and activity questionnaire; between the ages of 18 and 29. Subjects were excluded if they had allergy to soy, dairy, egg and wheat ingredients, peanuts, seeds and tree nuts. Those taking any other nutritional supplement or performance enhancing drugs were excluded. Finally, subjects were excluded if it was determined they were unable or unwilling to perform the physical exercise to be performed for the study.

Example 2. Recommended supplements Essentially pure PA and PA-enriched lecithin were prepared from soy lecithin by enzymatic conversion. The PA-enriched lecithin product produced by Chemi Nutra, Inc. (White Bear Lake, MN) contains 50-60% phosphatidic acid, 5-15% phosphatidylcholine, 1-5% lyso-phosphatidylcholine and 1-5% N-acyl phosphatidyl ethanolamine. The PA-enriched lecithin was given in four 400 milligram capsules to provide 1.6 grams of PA-enriched lecithin. The placebo was rice flour in a capsule identical in weight and color to the PA-enriched lecithin capsule.

30 The methods as described below included a protein snack. Any easily digested protein may be given. The protein used was partially hydro lyzed and termed "collagen protein" having the components described in Table 1. Proline and hydroxyproline comprised about one quarter of the amino acids and the leucine content was low. Collagen protein with low leucine content was chosen because leucine can have an effect on muscle and for clarity, that effect was minimized by choice of protein.

Table 1. Collagen Protein Content.

Example 3. Resistance training schedule

A. Four recreationally trained, young, healthy men, with at least one year of resistance training experience who met the enrolment criteria, were recruited for this study. All subjects performed the same training program, four days each week, split routine program as described below in Table 2. The four-day a week workout that was recommended to each subject included core exercises (denoted with an asterisk) which were a requirement of the study. Other exercises, assistance exercises, could be substituted for the core exercises only with the investigator's approval. However, all sets and repetitions were required to be the same in number. Subjects were allowed a 90 second rest period between each set. No additional sets or exercises were allowed as this would change the training volume, defined as the total work load (reps times weight.).

Table 2. Eight Week Resistance Training Program.

Monday/Thursday Tuesday/Friday

denotes required exercise

The subjects were randomly divided into two groups, the test group and the control group. The test group received 4 capsules of 400 mg equaling 1.6 grams per day of PA-enriched lecithin (Mediator^®, Chemi Nutra, Inc., White Bear Lake, MN). The control subjects received 4 capsules of 400 mg equalling 1.6 grams per day of rice flour. Subjects consumed either the test supplement or the placebo 15 minutes prior to workout. At the end of each workout, subjects were provided with a collagen protein drink consisting of 36 grams of collagen peptides mixed with 500 mL of water. On days of no workout, subjects consumed the respective capsules at approximately the same time of day that they worked out. During these non-workout days, subjects did not receive the protein drink.

At weeks 1 and 7 (pre-and post-study) subjects performed a 1 -repetition maximum (1RM) strength test on the squat and bench press exercises. Each subject performed a warm-up set using a resistance that is approximately 40-60% of his perceived maximum and then performed three to four subsequent attempts to determine the 1RM. Subjects were allowed 3 to 5 minutes of rest between each lift. Results are summarized in Table 3. Table 3. Strength Change Data.

StrenPth flR!Vn Bench Press

PA: average increase: +20 kg (plus 10.5%) Placebo: unchanged (0%)

PA supplementation resulted in an increase in strength in the bench press of 10.5% and an increase in strength in the squat of 21 %. Supplementation with the placebo resulted in no increase in strength in either exercise.

B. The effects of PA supplementation on muscle mass and training volume were determined in two recreationally trained, young, healthy men, with at least one year of resistance training experience. As above, these subjects received either 1.6 grams per day of Mediator^® PA-enriched lecithin (Chemi Nutra, White Bear Lake, MN) or 1.6 grams of rice flour for 6 weeks. Changes in muscle mass were measured by analyzing the muscle thickness of the vastus lateralis, the large lateral muscle on the thigh, using a GE Logiq PS Premium BT09 (Wauwatosa, WI). Training volume was calculated as weight lifted times repetitions performed.

The two subjects performed the same 6-week training program, consisting of a 2 day per week lower body resistance program (squats, lunge/front squat, leg curl, knee extension, calf raises, seated row, EZ bar curls, dumbbell curls.) There was a 90 second rest period between each set. The addition of any additional sets or exercises was prohibited as it would change the training volume.

PA supplementation resulted in an increase of 13% in training volume (pre: 49,640; post: 56,000), whereas placebo had no effect on training volume (pre: 92,800; post: 92,800). PA supplementation also resulted in a greater increase in muscle thickness compared to the placebo. In summary, PA supplementation resulted in greater increase in muscle mass, as demonstrated in this study, resulting in an increase of 9% more between a PA supplemented subject and a non-supplemented subject. In addition, PA supplementation resulted in greater increase in training volumes (13%).

Example 4. Creatine responders

As discussed above, creatine is a known muscle building substance, but about 30% of any population does not respond to creatine administration.

A. PvJ, a 42-year old male, 198 cm tall, a known non-responder to creatine

supplementation, followed a two-week strength training program while testing whether supplementation with PA-enriched- lecithin improves creatine response. Total starting body weight and strength were measured on day one, followed by the training program, consisting of concentric and eccentric isotonic lifting exercises that worked the upper and lower body muscle groups. Either free weights or weight machines were used once or twice weekly. Strength training was performed three times per week with at least one day of rest between sessions, which alternated between lower and upper body exercises. During the two-week period, a total of six training sessions (three upper and three lower) were performed. Each exercise included two sets of ten repetitions at 30% and 60% 1RM, followed by two sets of 3 to 5 repetitions at 90% 1RM.

The lower body exercise included seven different exercises: seated leg press, leg curls, standing calf raises, leg extensions, inclined leg lift, inverted sit-ups (back extension) and 45° inclined sit-ups . The upper body exercise consisted of seven different exercises: bench press, latissimus pulldown, triceps pulldown, inclined dumbbell curls, seated preacher curls, seated rows, and CyBec Pec Fly.

Total upper body weight and lower body weight lifted were calculated as the average weight lifted during the last three sets multiplied by the average repetition in each set. Total strength was determined as the combined lower and upper total weight lifted. Total body weight was determined after day 8 and day 15. Strength was measured on days 1 and 15.

A four-week rest period followed the first two-week training program. The same program was repeated twice, the control was supplementation of creatine monohydrate (Creapure, Alzchem, Germany) 4 times 5 grams per day for five days of loading, followed by nine days of five grams creatine monohydrate . During the second program, creatine monohydrate was given as for the control program with the addition of PA-enriched lecithin (Chemi Nutra, White Bear Lake, MN) which was about 50% PA. The results are shown in Table 4. Table 4. Changes in Body Mass.

As noted in Table 4, creatine loading alone was not effective in preventing a slight weight loss, while creatine plus PA-enriched lecithin reversed the weight loss and allowed a slight weight gain, presumably due to an increased muscular creatine concentration with concomitant muscle weight gain, as expected from the known non-responder status of RJ. Looking at total strength substantiated this theory: during the two-week baseline period, strength increased 6%, the same as during the creatine supplement period, which showed a similar, 5% strength increase., verifying that RJ was a creatine non-responder. The supplementation with both creatine and PA-enriched lecithin showed a gain in strength of 13.4%, more than double that of exercise alone or supplementation with creatine plus exercise.

B. MP, a 43-year old male, 185 cm tall, also a known non-responder to creatine supplementation, followed a three-week strength training program while testing whether supplementation with PA-enriched-lecithin improves creatine response. Total starting total body weight and strength were measured on day one, followed by the training program, consisting of concentric and eccentric isotonic lifting exercises that worked the upper and lower body muscle groups with either free weights or weight machines used once or twice weekly. Strength training was performed three times per week with at least one day of rest between sessions, which alternated between lower and upper body exercises. During the three-week period, a total of 10 training sessions (five upper body and five lower body) were performed. Each exercise included two sets of ten repetitions at 40% and 65% IRM, followed by two sets of 3 to 5 repetitions at 90% IRM.

The lower body exercise included seven different exercises: seated leg press, leg curls, standing calf raises, leg extension, inclined leg lift, inverted sit-ups (back extension) and 45° inclined sit-ups The upper body exercise consisted of seven different exercises: bench press, latissimus pull-down, triceps pull-down, inclined dumbbell curls, seated preacher curls, seated rows, and CyBec Pec Fly. Total upper body and lower body weight lifted were calculated as the average weight lifted during the last three sets multiplied by the average repetition in each set. Total strength was determined as the combined lower and upper total weight lifted. Total body weight was determined after day 8 and day 22. Strength was measured on days 1 and 22.

A four-week rest period followed the first three-week training program. The same program was repeated twice, the control was supplemented with creatine monohydrate

(Creapure®, Alzchem, Germany), 5 grams per day of five grams creatine monohydrate for 21 days. During the second program, creatine monohydrate was given as for the control program with the addition of 1 gram per day PA-enriched lecithin (Chemi Nutra, White Bear Lake, MN), which contained about 50% PA. The results are shown in Table 5.

Table 5. Changes in Body Mass.

As noted in Table 5, creatine loading alone was not effective in preventing a slight weight loss, while creatine plus PA-enriched lecithin reversed the weight loss and allowed a slight weight gain, presumably due to an increased muscular creatine concentration with concomitant muscle weight gain, as expected from the known non-responder status of MP. Looking at total strength substantiated this theory: during the two-week baseline period, strength increased 5%, the same as during the creatine supplement period, which showed a similar 5% strength increase, verifying that MP was a creatine non-responder. The supplementation with both creatine and PA-enriched lecithin showed a gain in strength of 1 1.5%, more than double that of exercise alone or supplementation with creatine plus exercise.

Example 5. PA-enriched lecithin for the improvement of nitrogen balance

Preliminary studies show that those unable to exercise, such as the bedridden or patients with diseases causing cachexia, can improve their condition with a shift of metabolism from catabolic to anabolic. The primary aspect of cachexia is the loss of protein as a result of muscle breakdown. Because about 60% to 70% of bodily protein is found in muscle and the nitrogen is excreted as urea, measurement of 24-hour urea outcome versus protein nitrogen intake provides the nitrogen balance. When the excretion of nitrogen is greater than the ingestion of protein nitrogen, the patient is said to be in negative nitrogen balance, which can lead to sarcopenia. There are several ways of determining nitrogen balance. First, a diary of foods eaten can be kept and protein intake recorded and compared with a 24 hour collection of urinary nitrogen. This direct measurement is often standard care in hospitals and nursing homes for these patients.

Another indicium is 24 hour urinary creatinine. Creatinine is the metabolite of creatine, as noted above, an important compound in muscle. Creatinine is excreted without reabsorption from the kidney tubules and can be determined as an estimate of renal function. Creatinine recovery varies greatly from patient to patient and is affected by such things as degree of hydration.

However, once a baseline is established, variations from the patient's idiosyncratic "normal" creatinine excretion are indicative of muscle breakdown. It is also a secondary indicium of nitrogen balance.

The patients will be given 0.5 to four grams of PA-enriched lecithin three times a day. While oral administration is preferred, for those patients unable to ingest or who are on intravenous or intraarterial therapies, PA or PA-enriched lecithin may be infused. The results will show an improvement in nitrogen balance.

Example 6. Increase of muscle mass and strength in the elderly

Even healthy older subjects may lose muscle to the point of sarcopenia. It may be considered inevitable and irreversible. However, it has been shown that 70-year old adults show a response to the known muscle stimulant P-hydroxy-P-methyl butyrate similar to that of young adults (Vukovich, et al. 2001 Am. Soc. Nutr. Sci. 2049-2053). Therefore, the compositions and methods of this invention will improve the muscle mass and strength of older subjects. It is especially recommended to combine ingestion of about 5 grams of creatine and 3 grams of an amino acid such as leucine or glutamine, taken 1 to 3 times daily in their exercise regimen.

Example 7. Phosphatidic Acid Increases Lean Body Mass and Strength in Resistance Trained Men

Phosphatidic acid (PA) is a natural phospholipid compound derived from lecithin, which is commonly found in egg yolk, grains, fish, soybeans, peanuts and yeast. PA is involved in several intracellular processes associated with muscle hypertrophy. Specifically, PA has been reported to activate protein synthesis through the mammalian target of rapamycin (mTOR) signalling pathway and thereby can enhance the anabolic effects of resistance training. To our knowledge, no one has examined the effect of PA supplementation in humans while undergoing a progressive resistance training program.

This example examined the effect of PA supplementation on lean soft tissue mass (LM) and strength after 8 weeks of resistance training.

Fourteen resistance-trained men (mean ± SD; age 22.7 ± 3.3 years; height: 1.78 ± 0.10 m; weight: 89.3 ± 16.3 kg) volunteered to participate in this randomized, double-blind,

placebo-controlled, repeated measures study. The participants were assigned to a PA group (750 mg/day; Mediator^®, Chemi Nutra, MN; n=7) or placebo group (PL; rice flower; n=7), delivered in capsule form that was identical in size, shape and color. Participants were tested for 1RM strength in the bench press (BP) and squat (SQ) exercise. Lean soft tissue mass (LM) was measured using dual-energy X-ray absorptiometry (DEXA). After base line testing, the participants began supplementing PA or PL for 8 weeks during a progressive resistance training program intended for muscular hypertrophy. Data was analyzed using magnitude-based inferences on mean changes for BP, SQ and LM. Furthermore, the magnitudes of the inter-relationships between changes in total training volume and LM were interpreted using

Pearson correlation coefficients, which had uncertainty (90% confidence limits) of approximately

±0.25.

In the PA group, the relationship between changes in training volume and LM was large (r=0.69, ±0.27; 90% CL). However, in the PL group, the relationship was small (r=0.21 , ±0.44; 90% CL). Changes in strength and LM in PA and PL groups, and qualitative inferences about the effects are recorded in Table 6.

Table 6.

In conclusion, PA supplementation was determined to beneficial at improving SQ and LM over PL by 26% and 64%, respectively. The strong relationship between changes in total training volume and LM in the PA group suggest that greater training volume can lead to the greater changes in LM. No such relationship was found with PL group. For the BP data, the PA group resulted in a 42% greater increase in strength over PL, although the effect is still under evaluation. While more research is needed to elucidate the mechanism of action, the current findings suggest that in experienced resistance trained men, supplementing 750 mg PA per day for 8 weeks provides greater changes in muscle mass and strength compared with resistance training alone.

Example 8. Lyso-phosphatidic acid supplement increases lean body mass, muscle hypertrophy, power and strength comparable to whey protein following resistance exercise

Lyso-phosphatidic acid (LP A) activates protein synthesis through the mTOR signaling pathway. Supplementing with LP A along with resistance training enhances lean body mass. Three highly resistance trained subjects with an average lean body mass of 66.9 ± 3.6 kg trained for 4 weeks as a part of a daily undulating periodized resistance-training program centered around the following standard compound movements: the squat, the deadlift and the bench press. Subjects were supplemented with a 500 mg LPA formulation administered prior to their workouts and again after their exercise. Dual X-ray absorptiometry was used to determine changes in lean body mass. All 3 subjects increased in lean body mass an average of 3.1 kg. Individual subject data and average changes are illustrated in Figure 1.

Example 9. Effects of G3P and Chemi Nutra PA on mTOR signaling in C2C12 myoblasts

20 P16 C2C12 myoblasts were plated at apx 20% confluence and grown for 36 hour in 10 % FBS High Glucose DMEM. Cells were switched to 2mL / well serum free High Glucose DMEM (no antibiotics) for 16 hours prior to the experiment. Cells were 80% confluent at the time of the experiment.

Cells were then stimulated with 30 μΜ of C8 PA, 300 μΜ of Egg PA, 30 - 300 μΜ of Glycerol 3-Phosphate or 30 - 300 μΜ of Chemi Nutra PA. This was added to the serum free media as detailed on the next page and the cells were then incubated for 20 min and collected in 150 μί WIK buffer + inhibitors.

The assays were performed as follows (see Figure 2).

Day 1. Plate cells on 6 well dishes at apx 20% and grow in 10% FBS-DMEM + antibiotics.

Day 2. Prep all tubes for sample collection and stimulations. Serum starve with DMEM - no antibiotic media. Plate 1 at 8:30PM, Plate 2 at 8:40PM, Plate 3 at 9: 10PM, Plate 4 at 9:20PM.

Day 3. Prep G3P stock stimulant solutions, setup PBS solutions. Thaw inhibitors, Bring C8, Egg PA, Chemi Nutra PA up to room temp. Set up for collection, set up for Cell Culture Conditions (pipettes, tips, stimulant solutions, etc.).

Dry and Prep 2 x 1 OOuM Chemi PA and 1 x 300uM Egg PA for Plate 1. Stimulate Plate 1.

Dry and Prep 2 x 300uM Chemi PA and 1 x 30uM C8 PA for Plate 2. Stimulate Plate 2

Prep Fresh WIK Buffer with Inhibitors (Enough for 2 Plates )

124.8 μ∑ of ^-Glycerophosphate (600 mM stock)

75 μ∑ ofNaF (1 M stock)

15 μ∑ ofPMSF (0.2 M stock)

3 μ∑ ofLeupeptin (10 mg/mL stock)

3 μ∑ ofNa₃V0₄ (1 M stock)

2.8 mL of WIK Buffer stock (ICE COLD)

Collect Plate 1 in 150ul WIK + Inhibitors / well.

Collect Plate 2 in 150ul WIK + Inhibitors / well.

Dry and Prep 2 x 300uM Chemi PA and 1 x 30uM C8 PA for Plate 3. Stimulate Plate 3.

Dry and Prep 30um, 1 OOum and 300uM Chemi PA for Plate 4. Stimulate Plate 4.

Prep Fresh WIK Buffer with Inhibitors (Enough for 2 Plates ) as described above.

Collect Plate 3 in 150ul WIK + Inhibitors / well.

Collect Plate 4 in 150ul WIK + Inhibitors / well.

Stimulant Prep Instructions.

Vehicle Control. All stimulant conditions will use 100 μ L of PBS as the vehicle, so for all control wells incubate with 100 of PBS only (aliquot taken from same bottle used in stimulant preps).

Chemi Nutra PA. The original stock powder was diluted at 20 mg/mL in chloroform with the purity listed at 50% (hence will assume an apx. 10 mg/ml of PA).

Chemi Nutra PA 300 μΜ. Stimulate with 300 μΜ Chemi Nutra PA using 100μ L of solution. The media volume is 2mL, so add 0.6 μπιοΐ of PA to the 2ml of media (300 μπιοΙ/L = 0.300 μηιοΙ/mL). Make 125 ΐ, of solution, thus use 0.75 μηιοΐ of PA in 125 ΐ, of PBS.

Chemi Nutra PA MW = 696.92g/mol = 696.92μg/μmol * 0.75 μπιοΐ = 533.7 μg = 0.5337 mg (stock = lOmg/mL, so need 53.37 μ L of stock). Dry the stock under nitrogen and re-suspend in 125 μ L of dPBS with 3 min of sonication and repeated vortexing. Immediately use for the cell stimulation.

Chemi Nutra PA 100 μΜ. Stimulate with 100 μΜ Chemi Nutra PA using 100μ L of solution. The media volume is 2mL, so add 0.2 μηιοΐ of PA to the 2 mL of media (100 μηιοΙ/L = 0.100 μιηοΙ/mL). Make 125 μΐ, of solution, thus use 0.25 μιηοΐ of PA in 125 μΐ, of PBS.

Chemi Nutra PA MW = 696.92g/mol = 696.92μg/μmol * 0.25 μιηοΐ = 177.9 μg = 0.1779 mg (stock = 10 mg/mL, so need 17.79 μΐ_^ of stock). Dry the stock under nitrogen and re-suspend in 125 μL· of dPBS with 3 min of sonication and repeated vortexing. Immediately use for the cell stimulation.

Chemi Nutra PA 30 μΜ. Stimulate with 30 μΜ Chemi Nutra PA using 1 ΟΟμί of solution. The media volume is 2mL, so add 0.06 μπιοΐ of PA to the 2 mL of media (30 μπιο1/ί = 0.030 μηιοΙ/mL). Make 125 μΐ_^ of solution, thus prepare 0.075 μηιοΐ of PA in 125 μΐ_^ of PBS.

Chemi Nutra PA MW = 696.92g/mol =

* 0.075 μιηοΐ = 52.26 μg = 0.0523 mg (stock = 10 mg/mL, so prepare 5.23 μί

the stock under nitrogen and re-suspend in 125 μί of dPBS with 3 min of sonication and repeated vortexing. Immediately use for the cell stimulation.

Egg PA 300 μΜ. Stimulate with 300 μMEgg PA using ΙΟΟμί of solution. The media volume is 2mL, so add 0.6 μπιοΐ of PA to the 2 mL of media (300 μπιοι/L = 0.300 μπιοι/mL). Make 125 μί of solution, thus use 0.75 μπιοΐ of PA in 125 μί of PBS.

Egg PA MW = 696.92g/mol = 696.92μ§ μιηο1 * 0.75 μιηοΐ = 533.7 μg = 0.5337 mg (stock = lOmg/mL, so prepare 53.37 μί of stock). Dry the stock under nitrogen and re-suspend in 125 μί of dPBS with 3 min of sonication and repeated vortexing. Immediately use for the cell stimulation.

C8 PA. Stimulate with 30 μΜ C8 PA using ΙΟΟμί of solution. The media volume is 2mL, so add 0.06 μηιοΐ of PA to the 2 mL of media (30 μηιοΙ/L = 0.030 μηιοΐ/ηιυ). Make 125 μΐ_^ of solution, thus prepare 0.075 μηιοΐ of PA in 125 μΐ_^ of PBS.

C8 PA MW = 446.45g/mol =446.45μg/μmol *0.075 μmol = 33.48μ g = 0.03348 mg (stock = lOmg/mL, so prepare 3.35μί οί stock). Dry the stock under nitrogen and re-suspend in 125 μί of dPBS with 3 min of sonication and repeated vortexing. Immediately use for the cell stimulation.

-Glycerophosphate (G3P). The stock powder was obtained from TCI (catalog #G0096) and was used to make a 6 mM stock solution in PBS. G3P MW = 216.04g/mol. Make 45ml of a 6 mmol/L solution = 6 μπιοΐ/ml * 45 ml = 270 μπιοΐ. 216.04 ug/μπιοΐ * 270 μπιοΐ = 58320μ g = 58.3 mg dissolved in 45 mL PBS. This stock solution was made fresh on the day of the experiment. Ο3Ρ 300 Μ. Stimulate with 300 μΜϋ3Ρ using ΙΟΟμΙ. of solution. The media volume is 2mL, so add 0.6 μmol of G3P to the 2 mL of media (300 μηιοΙ/L = 0.300 μιηοι/mL). Prepare stock solution with 6 mmol/L = 6 μιηοι/mL. Adding 0.1 mL of the 6 mM stock solution to the media is equivalent to adding 0.6 μηιοΐ.

G3P 100 μΜ. Stimulate with 100 ,MM G3P using ΙΟΟμί of solution. The media volume is 2mL, so add 0.2 μηιοΐ of G3P to the 2 mL of media (100 μηιοΙ/L = 0.100 μιηοι/mL). Prepare stock solution with 6 mmol/L = 6 μ mol/mL. Dilute the stock solution to 2 mM by combining 333 uL of 6mM solution plus 667 uL of PBS. Adding 0.1 mL of the 2 mM stock solution to the media is equivalent to adding 0.2 μηιοΐ.

Ο3Ρ 30 Μ. Stimulate with 30 μΜ G3P using ΙΟΟμί of solution. The media volume is 2mL, so add 0.06 μηιοΐ of PA to the 2 mL of media (30 μιηοι/L = 0.030 μιηοΙ/mL). Stock solution with 6 mmol/L = 6 μιηοι/mL. Dilute the stock solution to 0.6 mM by combining 100 uL of 6 mM solution plus 900 uL of PBS. Adding 0.1 mL of the 0.6 mM stock solution to the media is equivalent to adding 0.06 μηιοΐ.

Figure 3. CHEMI Nutra PA activates mTOR signaling. C2C12 myoblasts were stimulated with various dose of glucose-3-phosphoate (G3P), CHEMI Nutra PA (CN PA) or the vehicle (control) for 20 minutes as described on the previous slides. Stimulations with C8 PA or Egg PA were used as positive controls. A. Western blot of p70 phosphorylated on the threonine 389 residue [P- p70(389)] was compared to total p70 and used as a marker of mTOR signaling. B. Graphical representation of the P-p70(389) to total p70 ratio expressed as a percent of the control values. * P<0.05 compared to control.

Example 10. Dosage Forms

The following formulations illustrate representative dosage forms that may be used for the therapeutic or prophylactic administration of a compound (e.g., PA) described herein, a compound or composition specifically disclosed herein (e.g., a composition that includes some type of PA), or a pharmaceutically acceptable salt or solvate thereof (hereinafter referred to as 'Composition

X'):

(i) Tablet 1 mg/tablet

'Composition X' 100.0 Lactose 77.5 Povidone 15.0

Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesium stearate 3.0

300.0

(ii)Tablet 2 mg/tablet

'Composition X' 20.0 Microcrystalline cellulose 410.0 Starch 50.0

Sodium starch glycolate 15.0 Magnesium stearate 5

500.0

(iii) Capsule mg/capsule

'Composition X 10.0

Colloidal silicon dioxide 1.5

Lactose 465.5

Pregelatinized starch 120.0

Magnesium stearate 3.0

600.0

(iv) Injection 1 (1 mg/mL) mg/mL

'Composition X' (free acid form) 1.0

Dibasic sodium phosphate 12.0

Monobasic sodium phosphate 0.7

Sodium chloride 4.5

1.0 N Sodium hydroxide solution q.s. (pH adjustment to 7.0-7.5)

Water for injection q.s. ad 1 mL

(v) Injection 2 (10 mg/mL) mg/mL

'Composition X' (free acid form) 10.0

Monobasic sodium phosphate 0.3

Dibasic sodium phosphate 1.1

Polyethylene glycol 400 200.0

0.1 N Sodium hydroxide solution q.s. (pH adjustment to 7.0-7.5)

Water for injection q.s. ad 1 mL

(vi) Aerosol mg/can

'Composition X 20

Oleic acid 10 Trichloromonofluoromethane 5,000

Dichlorodifluoromethane 10,000

Dichlorotetrafluoroethane 5,000

(vii) Tablet 3 mg/tablet

'Composition X' 400

Rice flour 400

Magnesium stearate 50

850

These formulations may be prepared by conventional procedures well known in the

pharmaceutical art. It will be appreciated that the above compositions may be varied according to well-known techniques to accommodate differing amounts and types of active ingredient 'Composition X' (e.g., PA and/or other actives described herein). Aerosol formulation (vi) may be used in conjunction with a standard, metered dose aerosol dispenser. Additionally, the specific ingredients and proportions are for illustrative purposes. Ingredients may be exchanged for suitable equivalents and proportions may be varied, according to the desired properties of the dosage form of interest.

While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

Claims

1. Phosphatide acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use in improving the nitrogen balance of a mammal.

2. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 1 wherein said mammal is human, equine or canine.

3. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 2 wherein said mammal is an aging, bedridden or cachectic human.

4. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 2 wherein said mammal is an aging or cachectic equine or canine.

5. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 1 wherein the administered amount is 0.1 grams to 4 grams, given one to four times daily.

6. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 1 wherein it is administered by parenteral infusion.

7. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 1 wherein it is administered orally.

8. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to any one of the preceding claims wherein it further includes the administration of creatine.

9. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a about 3 to about 20 grams daily.

10. Phosphatide acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use in increasing the response to creatine.

11. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 10 wherein it is administered to a human.

12. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 10 wherein the administered amount is about 0.5 to about 4 grams.

13. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 10 wherein it is administered concomitant with creatine.

14. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 13 wherein the amount of creatine is about 3 to about 10 grams.

15. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 10 wherein it is administered 1 to 3 times daily.

16. A composition comprising creatine and one or more of phosphatidic acid,

lyso-phosphatidic acid, glycerol-3-phosphate, and phospholipase D.

17. The composition of claim 16 wherein phosphatidic acid is prepared from soybeans, peanuts, wheat, oats, safflower, fish, milk, bovine liver, eggs or egg yolks.

18. The composition of claim 16 or 17 wherein the creatine is present in a ratio of about 5 to about 3 with respect to the phosphatidic acid or lyso-phosphatidic acid.

19. The composition of claim 16 wherein it comprises phospholipase D, glycerol-3-

20. The composition of any one of claims 16-19 wherein the amount of phosphatidic acid or lyso-phosphatidic acid is 0.1 grams to about 40 grams.

21. The composition of any one of claims 16-20 wherein the amount of creatine is about 3 grams to about 10 grams.

22. The composition of any one of claims 16-21 wherein it further comprises one or more nutritional supplements.

23. The composition of claim 22 wherein the nutritional supplement is protein, one or more amino acids such as leucine or L-aspartic acid, beta alanine, leucine peptide, oc-lipoic acid, β- hydroxy-P-methylbutyrate₅ glycine propionyl L-carnitine, carnitine, Russian tarragon, gymnema sylvestre, bitter melon, cissus quadrangularis, cinnamon and fenugreek, CLA, tribulus, mulberry, ribose, caffeine, ZMA, betaine, carnosine, or a combination thereof.

24. The composition of claim 22 or 23 further comprising Co-QlO, chromium,

magnesium, vanadium, or a combination thereof.

25. The composition of any one of claims 16-24 wherein it is in a form for oral

administration.

26. The composition of any one of claims 16-25 for use in increasing muscle mass and strength in mammals.

27. The composition for use according to claim 26 wherein the composition is

administered together with a hormone.

28. The composition for use according to claim 27 wherein the hormone comprises testosterone, human growth hormone, insulin, or insulin-like growth factor.

29. The composition of any one of claims 16-25 for use in improving the nitrogen balance of a mammal.

30. The composition for use according to claim 29 wherein said mammal is human, equine or canine.

31. The composition for use according to claim 30 wherein said mammal is an aging, bedridden or cachectic human.

32. The composition for use according to claim 30 wherein said mammal is an aging or cachectic canine or equine.

33. The composition of any one of claims 16-25 for use in increasing the response to creatine in mammals.

34. The composition for use according to any one of claims 26-34 wherein the composition is administered orally.

35. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use in increasing muscle mass and strength in mammals.

36. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 35 wherein the amount is 0.1 grams to about 40 grams, administered orally one to three times daily.

37. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 35 wherein the mammal is human and the amount is 0.5 to about 5 grams.

38. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 35 wherein the mammal is canine and the amount is 0.1 to about 3 grams.

39. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 35 wherein the mammal is equine and the amount is about 10 to about 40 grams.

40. Phosphatide acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 35 wherein it is administered to an exercising subject during the anabolic window.

41. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 40 further comprising the ingestion of 20 to

100 grams of protein during the anabolic window.

42. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 41 wherein the protein is a complete protein containing all the essential amino acids for humans.

43. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 41 wherein the protein is whey or partially hydrolyzed collagen protein.

44. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 35 further comprising the administration of creatine.

45. Phosphatidic acid, glycerol-3-phosphate, lyso-phosphatidic acid, phospholipase D or a combination thereof for use according to claim 44 wherein the amount of creatine is about 3 to about 20 grams.