WO2008025114A1

WO2008025114A1 - Increasing atp availability by inhibition of creatine kinase (ck) leakage resulting from high-intensity exercise

Info

Publication number: WO2008025114A1
Application number: PCT/CA2006/001427
Authority: WO
Inventors: James D. Ramsbottom; Jason R. Peters; Shan Chaudhuri
Original assignee: Aplodan Formulations Ltd.
Priority date: 2006-08-30
Filing date: 2006-08-30
Publication date: 2008-03-06
Also published as: AU2006347819A1; EP2061473A1; EP2061473A4

Abstract

The present invention relates to a method of increasing Adenosine Triphosphate levels in a mammal wherein 2-(carbamimidoyl-methyl-amino)ethoxyphosphonic acid inhibits the leakage of the Adenosine Triphosphate-dependant enzyme Creatine Kinase. Via the inhibition of Creatine Kinase leakage, Adenosine Triphosphate is not utilized in the conversion of creatine to phosphocreatine, thereby making it available for use in muscular contractions. The increase in availability leads to longer endurance and more forceful muscular contractions.

Description

INCREASING ATP AVAILABILITY BY INHIBITION OF CREATINE KINASE (CK) LEAKAGE RESULTING FROM HIGH-INTENSITY EXERCISE

Field of the Invention

The present invention relates to a method of increasing the cellular concentration of Adenosine Tπphosphate, hereinafter referred to as ATP via the inhibition of the ATP-dependant enzyme Creatine Kinase, hereinafter referred to as CK.

Background

The energy requirements of contracting muscles involved m high-intensity exercise may increase 100-fold relative to resting muscles, thereby exceeding the aerobic energy production capacity of the cells (Westerblad H, et al News Physiol Sci. 2002 Feb; 17: 17-21). In this case, anaerobic metabolism is required to provide additional energy. However, high-intensity exercise results in an eventual reduced capacity for muscular contractile function, or commonly known as fatigue. Thus, there is seemingly a link between anaerobic metabolism and fatigue.

In a 2000 review on the role of creatine in skeletal muscle, Casey and Greenhaff provide a thorough overview of energy supply and its utilization in muscle (Casey A, et al Am J CIm Nutr. 2000 Aug;72(2 Suppl):607S-17S). ATP is the direct energy source for contracting muscle as the energy needed for muscular contraction is released by the dephosphorylation of ATP to yield the low-energy metabolite Adenosine Diphosphate, hereinafter referred to as ADP and inorganic phosphate (Pi) according to the following reaction: ATP + H₂O → ADP + Pi + H⁺ + energy (reaction 1)

Therefore, it is naturally observed that the function of muscle is largely dependent upon the availability of ATP. However, the concentration of ATP available m muscle at rest prior to the start of exercise, is only sufficient to supply about 1-2 seconds of the energy required for intense activity. ATP, however, can be readily and rapidly regenerated through the anaerobic dephosphorylation of available phosphocreatme. However, like that of ATP, the concentration of phosphocreatme in muscle is low and only sufficient to sustain muscular activity for an additional 6 seconds. After repeated bouts of contraction, muscle phosphocreatme levels become nearly depleted (Greenhaff PL, et al. J Physiol. 1993 Jan;460:443-53). Naturally, fatigue, although likely multifaceted in terms of biochemical events, is the point at which the energy required by contracting muscle exceeds the level available either from the stored supply of ATP or the indirect synthesis of high-energy ATP through phosphocreatine dephosphorylation. CK is an ATP-dependent enzyme, which, using magnesium, hereinafter referred to as Mg²⁺, as a co-factor, phosphoylates ADP to ATP. CK is normally found within cells however, when muscle cells are damaged the cells often rupture and the normally cell-bound proteins leak out in to the surrounding serum. Since the phosphoylation reaction is ATP-dependent, an elevated presence of CK outside the cells ultimately leads to the consumption of the high-energy molecule ATP. The consumption of ATP by CK in the serum is detrimental to cells, which requires ATP as an energy source for energetic properties in relation to muscular contractions.

Summary of the Invention

The foregoing needs and other needs and objectives that will become apparent for the following description are achieved in the present invention, which comprises, a method of increasing the intracellular, intercellular, extracellular and intra-tissue levels of ATP in a mammal through the inhibition of leakage of the ATP-dependant enzyme CK. In certain embodiments, the mammal is a human.

Detailed Description of the Invention

In the following description, for the purposes of explanations, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced without these specific details.

The present invention is directed towards a method of increasing the amount of ATP available in a muscle or cell via the inhibition of leakage of the ATP-dependant enzyme CK. Moreover, the present invention provides a method for the inhibition of leakage of CK from both normal cells and cells undergoing necrosis stemming from intense training or hypoxic damage wherein the result of said inhibition of CK leakage is an increase in ATP level. By way of increasing ATP levels, longer muscular endurance and more forceful muscular contractions are achieved.

2-(carbamimidoyl-methyl-amino)ethoxyphosphonic acid

2-(carbamimidoyl-methyl-amino)ethoxyphosphonic acid is a phosphoric ester derivative of creatine. 2-(carbamimidoyl-methyl-amino)ethoxyphosphonic acid has been shown to be well tolerated and without side effects (Melloni GF et al Arzneimittelforschung. 1979, 29(9a): 1447-9). Early studies of 2-(carbamimidoyl -methyl -amino)ethoxyphosphonic acid explored its use as a treatment for heart lesions and to restore reduced cardiac contractile function, particularly following hypoxia. (Godfraind T, et al Arzneimittelforschung. 1984;34(9):968-72). Futhermore, 2-

(carbamimidoyl-methyl-amino)ethoxyphosphonic acid has been successfully used to improve cardiac parameters in patients with inadequate coronary blood flow (Barlattani M, et al. Arzneimittelforschung. 1979;29(9a): 1483-5).

Clinical trials have shown that 2-(carbamimidoyl-methyl-amino)ethoxyphosphonic acid has effects related to skeletal muscle performance similar to those established for creatine. 2-

(carbamimidoyl-methyl-amino)ethoxyphosphonic acid has been shown to improve muscular development as well as increase the capacity to perform physical activity, hi one study, hand-grip strength was improved via the administration of 2-(carbamimidoyl-methyl-amino)ethoxyphosphonic acid whereas hand-grip strength was unaffected in the placebo group (Nicaise J. Curr Ther Res Clin Exp. 1975, 17(6):531-4). Additionally, in another study conducted in elderly subjects, it was found that 2-(carbamimidoyl-methyl-amino)ethoxyphosphonic acid improved muscular performance

(Cavalieri U, et al. Clin Ther. 1974, 69: 215-223). Creatine Kinase

Creatine Kinase (CK) is an enzyme which catalyzes the following reaction: ATP + creatine «→ ADP + phosphocreatme + H⁺ (reaction 2)

Reaction 2 is reversible depending on the energy state of the cell. In fast-twitch skeletal muscles, a large pool of phosphocreatme is available for immediate regeneration of ATP hydrolyzed during short periods of intense muscle contraction. Due to high CK activity in these muscles, the CK reaction remains m a near-eqmlibπum state, keeping the concentration of [ADP] and [ATP] almost constant over several seconds at the expense of phosphocreatme. CK, with its involvement m ATP formation, has an impact on the onset of fatigue. Usmg Mg²⁺ as a co-factor, CK phosphoylates ADP to ATP Thus, since the aforementioned reaction is ATP-dependent, an elevated presence of CK m the serum ultimately leads to the consumption of the high-energy molecule ATP

A number of parameters are associated with potential or realized damage to muscle cells and are used as diagnostic indicators, particularly for cardiac muscle cells e.g. myocardial infarction, but also for skeletal muscle cells e.g high-intensity exercise. The measurement of CK levels in the blood is used as an assessment of muscle damage since CK is normally found within cells, specifically m the cytoplasm and in the mitochondria. When muscle cells are damaged as they are as a result of heart attacks or intense exercise, the cells often rupture and allow normally cell-bound proteins to leak out.

2-(carbamimidoyl-methyl-amino)ethoxyphosphomc acid has been shown in rats to reduce serum CK levels increased by administration of isoprenalme, a pharmacological agent known to increase cardiac activity by causing the release of calcium. As a result of this property, it is often used to induce cardiac damage in animal models of myocardial damage (Marzo A et al Arzneimittelforschung. 1979;29(9a): 1471-3). Further to this, 2-(carbarmmidoyl-methyl- ammo)ethoxyphosphomc acid has been shown to afford cardioprotection (Godfraind T, et al Arzneimittelforschung. 1984;34(9)-968-72) and has been shown to reduce the leakage of CK as well as several other enzymes in human patients suffering from acute myocardial infarction (Knippel M, et al Arzneimittelforschung. 1979;29(9a): 1480-2). Therefore, at the tissue level i.e. muscle, through its protective effect, 2-(carbamimidoyl- methyl-amino)ethoxyphosphonic acid prevents the increase in CK levels occurring during muscle damage e.g. myocardial infarction or high-intensity exercise, which reflects the leakage of CK from the cytoplasm of damage cells into the blood stream. In tissue that has been subjected to damaging conditions e.g. myocardial infarction or high-intensity exercise, the number of intact and functioning cells is been reduced. This reduced cell number results in reduced CK activity at the tissue level. In turn, this reduced CK activity equates to reduced capacity to regenerate phosphocreatine, and by extension, the ability to regenerate ATP. Thus by preventing cell damage, 2-(carbamimidoyl- methyl-amino)ethoxyphosphonic acid inhibits the reduction of CK activity in muscle tissue i.e. a single, or all muscles, and thereby acts to maintain high ATP levels in highly active muscles.

Since 2-(carbamimidoyl-methyl-amino)ethoxyphosphonic acid counteracts the increase of CK, an ATP dependant enzyme in the serum, it is henceforth understood that administration of 2- (carbamimidoyl-methyl-amino)ethoxyphosphonic acid increases the amount of ATP intracellularly, intercellularly, extracellularly as well as within tissues. In resting muscle, 2-(carbamimidoyl- methyl-amino)ethoxyphosphonic acid, via its inhibition of CK leakage allows for an increase in

ATP levels. In order for creatine to be transformed into phosphocreatine, ATP is required. During rest this will, therefore deplete the stock of ATP which could be used during periods where muscular contraction is required. Therefore, administration of 2-(carbamimidoyl-methyl- amino)ethoxyphosphonic acid to a mammal acts in manner to increase ATP levels within a muscle by inhibiting the leakage of the ATP dependant enzyme CK.

Oral administration in test animals has revealed that 2-(carbamimidoyl-methyl- amino)ethoxyphosphonic acid is optimally absorbed by the intestine up to about 60% at 48 hours (Marzo A, et al. Clin Ter. 1972 Sep 15;62(5):419-30). Moreover, 2-(carbamimidoyl-methyl- amino)ethoxyphosphonic acid was found to be stable in both alkaline and acidic solutions, an important characteristic for orally administered treatments. Additionally, in vitro testing has suggested that 2-(carbamimidoyl-methyl-amino)ethoxyphosphonic acid is dephosphorylated to creatine to some degree in the kidney, intestine and liver, and less so in the blood and muscle suggesting that mtact 2-(carbamimidoyl-methyl-ammo)ethoxyphosphonic acid is received by the cell in addition to supplying creatine.

Thus, the beneficial effects afforded by 2-(carbamimidoyl-methyl-amino)ethoxyphosphonic acid includes, in one aspect 2-(carbamimidoyl-methyl-amino)ethoxyphosphonic acid serving as a source of creatine through a dephosphorylated fraction; and in a second aspect protecting the cell membrane from leaking as a result of damaging conditions e.g. high-intensity exercise.

Additionally, as discussed above, it is herein understood that 2-(carbamimidoyl-methyl- amino)ethoxyphosphonic acid acts in vaπous ways to increase the available levels of ATP within a cell. 2-(carbamimidoyl-methyl-ammo)ethoxyphosphonic acid has been shown to be protective against cellular necrosis in models of myocardial infarction. As such, in protecting against necrosis, 2-(carbamimidoyl-methyl-ammo)ethoxyphosphonic acid provides a method decreasing CK in the serum through the prevention of necrosis under hypoxic conditions. Such conditions are also induced during period of intense exercise. Furthermore, it is well known in the art that exercise training can induce cellular necrosis and thus a leakage of the cellular contents including CK. Moreover, CK levels are also increased following cellular damage CK is then available m excess as substrate for ATP, thus CK uses ATP which but for the increased levels of CK would be stored for future use as an energy source, converting it to ADP which is devoid of energetic properties in relation to muscular contractions. Therefore, as an objective of this invention, it is ascertained that administration of 2-(carbamimidoyl-methyl-amino)ethoxyphosphonic acid provides a method by which an inhibition of CK leakage results, leading to an increase in ATP The increase in ATP can then be employed to improve muscular endurance and provide more forceful muscular contractions

In one embodiment of the invention, a portion of 2-(carbamimidoyl-methyl- ammo)ethoxyphosphomc acid is fine-milled. U.S. Provisional Application No. 60/776,325 entitled "Compositions and Method for Increasing Bioavailability of Compositions for Performance Improvement", which is herein fully incorporated by reference discloses a method of improving the absorption, palatabihty, taste, texture and bioavailability of compounds by increasing the solubility The increased bioavailability of a compound or ingredients is achieved via a reduction m particle size using a "fine-milling" technique. Any acceptable fine-milling technique will result in the fine- milled particles having an average particle size of between about 50 nm to about 2 nm. The reduction in size of the particle increases the surface area-to-volume ratio of each particle, thus increasing the rate of dissolution, thereby improving the rate of absorption. As used herein, the terms "fine-milled" and/or "fine-milling" refer the process of micronization. Micronization is a mechanical process which involves the application of force to a particle, thereby resulting in a reduction in the size of said particle.

As used herein, the term "particle size" refers to the diameter of the particle. The term "average particle size" means that at least 50% of the particles in a sample will have the specified particle size. Preferably, at least 80% of the particles in a given sample will have the specified particle size, and more preferably, at least 90% of the particles in a given sample will have the specified particle size

Although the preceding specification describes a how 2-(carbamimidoyl-methyl- amino)ethoxyphosphonic acid may be utilized as an method of increasing ATP via the prevention of CK leakage, it should not be construed as the only method by which 2-(carbamimidoyl-methyl- amino)ethoxyphosphonic acid may be employed to provide an energetic function related to muscular contractions. From consideration of the specification, those of skill in the art form may determine other methods wherein 2-(carbamimidoyl-methyl-amino)ethoxyphosphonic acid may be employed to enhance muscular contractions and prolong or increase muscular endurance.

Claims

ClaimsWhat is claimed:

1. A method of increasing Adenosine Triphosphate levels in a mammal comprising the step of administration of 2-(carbamimidoyl-methyl-amino)ethoxyphosphonic acid.

2. The method of claim 1 wherein 2-(carbamimidoyl -methyl -amino)ethoxyphosphonic acid inhibits a leakage of Creatine Kinase into serum and therefore reduces Creatine Kinase level in serum

3. The method of claim 1 wherein said increase in Adenosine Triphosphate occurs in somatic tissues of a mammal.

4. The method of claim 1 wherein said increase in Adenosine Triphosphate occurs in muscle cells and muscle fibers of a mammal.

5. The method of claim 1 wherein said mammal is a human.

6. The method of claim 1 wherein at least a portion of said 2-(carbamimidoyl-methyl- amino)ethoxyphosphonic acid is fine-milled.

7. The method according to claim 1 wherein said reduced serum Creatine Kinase level results in an increase in Adenosine Triphosphate.

8. A method according to claim 7 wherein muscular endurance is increased.

9. A method according to claim 7 wherein Adenosine Triphosphate levels in a mammal are maintained during resting periods via the inhibition of leakage of the Adenosine Triphosphate-dependant enzyme Creatine Kinase.

10. A method reducing muscle fatigue comprising the administration of 2-(carbamimidoyl- methyl-amino)ethoxyphosphonic acid or derivative thereof in order to maintain or increase Adenosine Triphosphate level in a mammal.

11. The method of claim 11 wherein the mammal is a human.