Creatine and/or creatinine compositions and related methods

Abstract

The disclosure is directed to methods related to creatinine, for example, a method for its safe administration to a human, a method for using creatinine to improve the bioavailability of creatine, and a metn.

Claims

1. A method of improving the oral bioavailability of creatine, the method comprising co-administering to a human subject an effective amount of creatine, creatinine, and nitrate (NO.sub.3.sup.−), wherein the effective amount of creatine is at least about 500 mg, the effective amount of creatinine is at least about 500 mg, and the effective amount of nitrate (NO.sub.3.sup.−) is at least about 50 mg nitrate (NO.sub.3.sup.−).

2. The method of claim 1, wherein the effective amount of nitrate (NO.sub.3.sup.−) is between about 50 mg and about 1200 mg nitrate (NO.sub.3.sup.−).

3. The method of claim 1, wherein the effective amount of nitrate (NO.sub.3.sup.−) is between about 50 mg and about 1500 mg nitrate (NO.sub.3.sup.−).

4. The method of claim 1, wherein the effective amount of nitrate (NO.sub.3.sup.−) is between about 60 mg and about 1500 mg nitrate (NO.sub.3.sup.−).

5. The method of claim 1, wherein the effective amount of nitrate (NO.sub.3.sup.−) is between about 60 mg and about 1200 mg nitrate (NO.sub.3.sup.−).

6. The method of claim 1, wherein the effective amount of creatine is at least about 1000 mg, the effective amount of creatinine is at least about 1000 mg, and the effective amount of nitrate (NO.sub.3.sup.−) is at least about 100 mg.

7. The method of claim 1, wherein the effective amount of creatine is at least about 2 g, the effective amount of creatinine is at least about 2 g, and the effective amount of nitrate (NO.sub.3.sup.−) is at least about 500 mg.

8. The method of claim 1, wherein the effective amount of creatine is at least about 5 g, the effective amount of creatinine is at least about 3 g, and the effective amount of nitrate (NO.sub.3.sup.−) is at least about 1000 mg.

9. A method of improving the oral bioavailability of creatine, the method comprising co-administering to a human subject an effective amount of creatine, creatinine, and nitrate (NO.sub.3.sup.−), wherein the molar ratio of the amount of nitrate (NO.sub.3.sup.−) to the amount of creatinine is between about 20:1 and about 1:9.

10. The method of claim 9, wherein the molar ratio of the amount of nitrate (NO.sub.3.sup.−) to the amount of creatinine is between about 10:1 and about 1:1.

11. The method of claim 9, wherein the molar ratio of the amount of nitrate (NO.sub.3.sup.−) to the amount of creatinine is between about 3:1 and about 1:3.

12. The method of claim 9, wherein the molar ratio of the amount of nitrate (NO.sub.3.sup.−) to the amount of creatinine is between about 2:1 and about 1:4.

13. The method of claim 9, wherein the molar ratio of the amount of creatine to the amount of nitrate (NO.sub.3.sup.−) is between about 20:1 and about 1:9.

14. The method of claim 9, wherein the molar ratio of the amount of creatine to the amount of nitrate (NO.sub.3.sup.−) is between about 10:1 and about 1:1.

15. The method of claim 9, wherein the molar ratio of the amount of creatine to the amount of nitrate (NO.sub.3.sup.−) is between about 3:1 and about 1:3.

16. The method of claim 9, wherein the molar ratio of the amount of creatine to the amount of nitrate (NO.sub.3.sup.−) is between about 2:1 and about 1:4.

17. The method of claim 1, wherein the oral bioavailability of the effective amount of creatine administered is greater than the oral bioavailability of creatine when administered alone.

18. A method of improving the oral bioavailability of creatine, the method comprising administering to a human subject a composition comprising an effective amount of creatine and creatinine, wherein the molar ratio of the effective amount of creatine and creatinine is 1:1 to 1:1.7 and the pH of the composition is 4.2 to 4.4.

19. The method of claim 18, wherein the method further comprises administering an effective amount of nitrate (NO.sub.3.sup.−), wherein the effective amount of nitrate (NO.sub.3.sup.−) and the effective amount of creatine are administered as creatine nitrate.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a graph tracking the change in the creatine and creatinine content of an exemplary liquid composition of the disclosure stored at room temperature (about 25° C.) over a period of 14 months.

(2) FIG. 2 is a graph tracking the change in the creatine and creatinine content of an exemplary liquid composition of the disclosure stored at room temperature (about 25° C.) over a period of a year.

(3) FIG. 3 is a graph tracking the change in pH and in the creatine, creatinine, and nitrate content of a liquid composition produced from dissolving 5 g creatine nitrate and 4 g creatinine with 500 ml water. The liquid composition was stored at room temperature (about 25° C.).

(4) FIG. 4 is a graph tracking the change in pH and in the creatine, creatinine, and nitrate content of a liquid composition produced from dissolving 5 g creatine nitrate and 4 g creatinine with 500 ml water. The liquid composition was stored in refrigeration (2-8° C.).

(5) FIG. 5 is a graph tracking the change in pH and in the creatine, creatinine, and nitrate content of a liquid composition produced from dissolving 5 g creatine nitrate and 5 g creatinine with 500 ml water. The liquid composition was stored at room temperature (about 25° C.).

(6) FIG. 6 is graph tracking the change in pH and in the creatine, creatinine, and nitrate content of a liquid composition produced from dissolving 1.5 g creatine nitrate and 1 g creatinine with 500 ml of a multicomponent energy drink. The liquid composition was stored at room temperature (about 25° C.). At day 60, the pH of the half of the solution was adjusted to 4.4 to study the effect of slightly less acidic pH on the levels of creatine and creatinine in the solution.

DETAILED DESCRIPTION

(7) Detailed aspects and applications of the disclosure are described below in the following drawings and detailed description of the technology. Unless specifically noted, it is intended that the words and phrases in the specification and the claims be given their plain, ordinary, and accustomed meaning to those of ordinary skill in the applicable arts.

(8) In the following description, and for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the various aspects of the disclosure. It will be understood, however, by those skilled in the relevant arts, that implementations of the technology disclosed herein may be practiced without these specific details. It should be noted that there are many different and alternative configurations, devices and technologies to which the disclosed technologies may be applied. The full scope of the technology disclosed herein is not limited to the examples that are described below.

(9) The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a step” includes reference to one or more of such steps.

(10) As used herein, the term “about” refers to a deviation up to but not more than 10% of the given value, for example a deviation of 10%, 7.5%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the given value.

(11) As used herein, the term “dietary supplement” refers to an addition to the human diet which is not a natural food, which has additional beneficial effects on the body unattainable by regular nutrition. In some aspects, a dietary supplement is manufactured to be used over time, allowing for precise dosing. In some aspects, a dietary supplement includes fortified food.

(12) As used herein, the term “nutraceutical” refers to a dietary supplement, a dietary ingredient, a food additive, or a fortified food that provides health benefits, including preventing, treating, or curing a physical or mental condition.

(13) As used herein, the term “dietary ingredient” refers to a dietary substance for use by man to supplement the diet by increasing total dietary intake.

(14) As used herein, the term “food additive” refers to a substance that is a component added to food.

(15) As used herein, the term “fortified food” refers to food where its nutritional and health value is increased (or fortified) by the additional of a dietary supplement, dietary ingredients, or food additive.

(16) As used herein, the term “room temperature” encompasses of a range of temperatures between about 15° C. and about 27° C., for example, between about 15° C. and about 25° C., between about 18° C. and about 22° C., or about 20° C.

(17) As used herein, the time period of “a day” refers to a period of between 18 and 30 hours, for example, between 22 and 26 hours or about 24 hours.

(18) As used herein, the term “effective amount” refers to an amount that induces a measurable or observable physiological change in a human. For example, in certain embodiments, an effective amount of creatinine refers to an amount of creatinine that increases the bioavailability of creatine or an amount that counteracts the inhibitory effect of caffeine on the ergogenic effects of creatine.

(19) The present disclosure addresses the need for ensuring the stability of creatine in a solution, for example of water or other liquid or water-based formulations.

(20) Whereas solid creatine is stable, the presence of water leads to intramolecular cyclization that converts creatine to creatinine (see Scheme 1).

(21) ##STR00001##
The rate of creatine degradation in solution is not dependent on the concentration of creatine but on the pH of the solution. Generally, the lower the pH and higher the temperature, the faster creatine becomes creatinine in solution (see, for example, Edgar and Shiver, 1925; Cannon et al., 1990; Dash et al., 2002). While creatine was relatively stable in solution at neutral pH (7.5 or 6.5), lowering of pH resulted in an increased rate of degradation. After only three days of storage at 25° C., creatine degraded by 4% at pH 5.5, by 12% at pH 4.5, and by 21% at pH 3.5. Similarly, creatine monohydrate in solution stored at room temperature degraded into creatinine within several days, while refrigerating creatine monohydrate in solution slowed the rate of degradation (Ganguly et al., 2003). Accordingly, the rapid degradation of creatine in solution precludes the manufacture of shelf-stable beverages containing efficacious amounts of the ingredient.

(22) Another issue with creatine supplementation has been its limited bioavailability and finding methods to improve its bioavailability as well as its overall plasma levels. In the past it was erroneously assumed that creatine possesses a bioavailability of near 100% because of a 2007 article that assumed that since no creatine or creatinine was detected in feces, the bioavailability of creatine should be around 100% (Deldicque et al., 2008). However recent data from a radiokinetic bioavailability study (a standard for pharmacokinetic studies that produces more detailed and accurate data than merely measuring the contents of an orally administered compound excreted) indicated that this hypothesis is highly erroneous; in fact, creatine bioavailability was shown to be sharply reduced with increasing doses (Alraddadi et al., 2018). This finding validated the hypothesis by McCall and Persky that creatine bioavailability is less than 100% because of bacterial flora degradation in the gastrointestinal tract, gastric degradation, site dependent intestinal absorption and incomplete dissolution of creatine solid dosage forms (McCall and Persky, 2007).

(23) It was surprisingly discovered by the inventors that the presence of creatinine in a solution comprising creatine reduces the rate of creatine degradation, and in some cases, creatine degradation is eliminated. Therefore, the present disclosure is directed in part to a liquid composition (for example, a liquid food product or liquid dietary supplement formulation) containing a creatine compound and a creatinine compound wherein the creatine is stable for at least one month when stored at room temperature or near room temperature. The liquid also possesses increased stability, for example of creatine, during refrigerated storage.

(24) The disclosure also relates to methods of stabilizing creatine in a liquid wherein the creatine content of the liquid composition after a month, two months, or three or more months or over a year of storage at about room temperature or no greater than room temperature is at least 70% of the amount of creatine nitrate provided thus enabling the preparation of a liquid dietary supplement formulation comprising stable creatine. In some aspects, the methods of stabilizing creatine in a liquid results the amount of creatine in the liquid composition being at least 90% or at least 95% of the amount of creatine nitrate provided after a month, two months, or three or more months or over a year of storage at about room temperature or no greater than room temperature. The methods comprise providing an amount of creatine (for example, provided as a creatine compound selected from the group consisting of anhydrous creatine and a salt or hydrate or solvate of creatine); providing an amount of creatinine (for example, provided as a creatinine compound selected from the group consisting of anhydrous creatinine and a salt or hydrate or solvate of creatinine); and dissolving the amount of creatine and/or the amount of creatinine in water or a water-based composition. In some aspects, the water-based composition is a ready-to-drink food product, dietary supplement, vegetable juice, or fruit juice.

(25) In some implementations, the amount of creatine is first dissolved in water or water-based composition to produce a liquid composition and the amount of creatinine is then added to the liquid composition. In a preferred implementation, the amount of creatinine is added to the liquid composition, preferably no more than a day after creatine is dissolved in water or water-based composition. However, creatinine may be added to the liquid composition formed from dissolving creatine in a liquid more than a day after the creatine is dissolved. In such implementations, precise formulation and labeling for the resulting composition is difficult as creatine may have degraded a significant amount according to labeling regulations.

(26) In other implementations, the amount of creatine and the amount of creatinine are both dissolved in water or water-based composition to produce a liquid composition, which can also be the liquid food product or liquid dietary supplement formulation.

(27) In still other implementations, the amount of creatinine is first dissolved in water or water-based composition to produce a liquid composition, and the amount of creatine is then dissolved in the liquid composition at a later time. In such implementations, the timing of when creatine is dissolved in the liquid composition is not important, as the dissolved creatinine does not lose its ability to stabilize creatine in water over time.

(28) In some implementations of the methods, the weight of the amount of creatinine or salt or hydrate thereof is 5%-800% or 50-200% the weight of the amount of creatine compound. In other implementations, the molar ratio of the amount of the creatine compound to the amount of the creatinine or salt or hydrate thereof is between about 23:1 and 1:9, for example, between 20:1 and 1:9, between 2:1 and 1:3, between 3:1 and 1:3, 1:1, or 1:1.7. In some embodiments, the creatine compound provides at least 500 mg creatine, at least 1000 mg creatine, at least 2 g creatine, or at least 5 g creatine, while the amount of creatinine provided is at least 500 mg, at least 1000 mg, at least 2 g, or at least 3 g. In certain implementations, the amount of creatine nitrate is 5 g and the amount of anhydrous creatinine is 4 g. In another implementation, the amount of the creatine compound consists of 1.5 g creatine nitrate and 3.5 g anhydrous creatine while the amount of creatinine compound consists of 5 g anhydrous creatinine. In another implementation, the amount of the creatine compound consists of 3 g creatine nitrate and 2 g anhydrous creatine while the amount of creatinine compound consists of 5 g creatinine.

(29) In some implementations of the methods for increasing the stability of creatine in solution, the methods further comprise providing at least one source of nitrate (NO.sub.3.sup.−), wherein the at least one source of nitrate (NO.sub.3.sup.−) is dissolved with the creatine compound and/or the creatinine compound in water or water-based composition. In such implementations, the source of nitrate (NO.sub.3.sup.−) enhances the conversion of creatinine to creatine in the solution. Thus, such methods increase the creatine-stabilizing effect of creatinine in an aqueous solution and enhances conversion of creatinine to creatine in an aqueous solution. Accordingly, in some aspects, the disclosure is also directed to methods of increasing the creatine-stabilizing effect of creatinine in an aqueous solution and to methods of enhancing the conversion of creatinine to creatine in an aqueous solution.

(30) In some implementations, the amount of nitrate (NO.sub.3.sup.−) provided by the at least one source of nitrate (NO.sub.3.sup.−) results in a molar ratio with the amount of creatinine of between about 20:1 and about 1:9, between about 10:1 and about 1:1, between about 3:1 and about 1:3, or between about 2:1 and about 1:4. In some implementations, the amount of nitrate (NO.sub.3.sup.−) provided by the at least one source of nitrate (NO.sub.3.sup.−) results in a molar ratio with the amount of creatine of between about 20:1 and about 1:9, between about 10:1 and about 1:1, between about 3:1 and about 1:3, or between about 2:1 and about 1:4. In certain embodiments, the molar ratio of the amount of creatine to the amount of nitrate (NO.sub.3.sup.−) provided by the at least one source of nitrate (NO.sub.3.sup.−) is 1:1. In such embodiments, the source of nitrate (NO.sub.3.sup.−) may be creatine nitrate. In some aspects, the at least one source of nitrate (NO.sub.3.sup.−) provides at least 50 mg nitrate (NO.sub.3.sup.−), at least 100 mg nitrate (NO.sub.3.sup.−), at least 250 mg nitrate (NO.sub.3.sup.−), at least 500 mg nitrate (NO.sub.3.sup.−), at least 100 mg nitrate (NO.sub.3.sup.−), between 50 mg and 1200 mg nitrate (NO.sub.3.sup.−), between 60 mg and 1200 mg nitrate (NO.sub.3.sup.−), between 50 mg and 1500 mg nitrate (NO.sub.3.sup.−), or between 60 mg and 1500 mg nitrate (NO.sub.3.sup.−).

(31) In some embodiments, the method further comprises adjusting the pH of the liquid composition (after the creatine compound and the creatinine compound are dissolved) to 4.4 or less, for example, between about 4.2 and about 4.4. The pH can be adjusted using any acceptable pH buffer, for example, sodium hydroxide.

(32) In some aspects, the disclosure also relates to a liquid composition comprising creatine and creatinine, for example a drink fortified with creatine and creatinine, wherein the liquid composition comprises a stable amount of creatine. The stable liquid creatine formulation is produced by combining a creatine compound and creatinine compound into a composition and then dissolving the composition in water or a water-based composition. In other implementations, either the creatine compound or the creatinine compound is dissolved in water or water-based composition before the other compound is dissolved. The order of which of the creatine compound or the creatinine compound is dissolved first in water or the water-based composition is not critical, though dissolved creatine should not be allowed to remain in water or water-based composition alone for more than an hour. For example, creatine monohydrate can first be dissolved in 500 ml of water, and an hour later, creatinine is dissolved in the same solution. If the creatinine compound is dissolved in water or water-based composition first, there is no similar urgency for when the creatine compound is dissolved in the resulting solution. In some preferred implementations, the creatinine compound is first dissolved in water or water-based composition. In some implementations, the liquid composition is produced by first mixing the creatine compound and the creatinine compound separately in water to produce two separate solutions and then mixing the two solutions.

(33) In some embodiments, the stable liquid creatine formulation further comprises reducing the water content of the composition or thickening the composition. Accordingly, in some aspects, the stable liquid creatine formulation is semisolid, for example, in the form of an emulsion, a pudding, or a gel.

(34) In another implementation, the creatine compound and the creatinine compound are dissolved in water or water-based composition separately to produce a creatine solution and a creatinine solution before the two solutions are combined to produce a liquid composition described herein. To ensure no significant degradation of creatine takes place (for example, more than 90% of the creatine provided is degraded), the two solutions are combined within a day, or preferably within one hour dissolving the creatine compound. In some aspects, the method further comprises thickening or reducing the moisture content of the creatine solution and/or the creatinine solution, wherein combining the two solutions produces a semisolid composition or semiliquid composition, for example, a gel or pudding. In other aspects, the method further comprises thickening or reducing the liquid composition to produce a semisolid composition, for example, a gel or pudding.

(35) In some embodiments, the stable liquid creatine formulation has a pH of 4.4 or less, for example, between 4.2 and 4.4 or preferably about 4.4. Accordingly, in some implementations, the method of producing the stable liquid creatine formulation further comprises buffering the solution containing the dissolved creatine compound and the dissolved creatinine compound to a pH of 4.4 or less, for example, between 4.2 and 4.4 or about 4.4.

(36) In some aspects, the invention is also directed to solid compositions comprising a creatine compound and creatinine compound. In some aspects, the weight of the creatinine compound is 5%-800% or 50-200% the weight of the creatine compound. In a preferred embodiment, the weight of the creatinine provided by the creatinine compound is 5%-800% or 50% to 200% the weight of creatine provided by the creatine compound. In other implementations, the molar ratio of the creatine compound to the creatinine compound is between about 23:1 and 1:9, for example, between 20:1 and 1:9, between 2:1 and 1:3, between 3:1 and 1:3, 1:1, or 1:1.7. In some embodiments, the creatine compound provides at least 500 mg creatine, at least 1000 mg creatine, at least 2 g creatine, or at least 5 g creatine, while the amount of creatinine provided is at least 500 mg, at least 1000 mg, at least 2 g, or at least 3 g. In one embodiment, the creatine compound in the composition is 5 g creatine nitrate and the creatinine compound is 4 g anhydrous creatinine. In another embodiment, the amount of the creatine compound in the composition consists of 1.5 g creatine nitrate and 3.5 g anhydrous creatine, while the amount of creatinine compound in the composition consists of 5 g anhydrous creatinine. In still another embodiment, the amount of the creatine compound in the composition consists of 3 g creatine nitrate and 2 g anhydrous creatine, while the amount of creatinine compound in the composition consists of 5 g creatinine.

(37) In certain embodiments, the compositions including creatine and creatinine are dietary supplements, for example, to increase the amount of creatine in one's diet. As such, in some aspects, the disclosure is also directed to the use of creatinine as a dietary ingredient or as a food additive.

(38) Prior to the present disclosure, creatinine was primarily considered a waste product from the normal breakdown of muscle tissue. As creatinine is produced, it is filtered through the kidneys and excreted in urine. To this day, no beneficial biological role for creatinine has been established. In contrast, creatinine is believed to be a toxic compound which can impair human performance and health. Tambaru et al. and Gangopadhya et al. both describe creatinine as a compound which can cause kidney damage. In view of high levels of creatinine being correlated with a bad health prognosis, such as high creatinine levels in the urine indicating kidney failure, it would be unethical to study the biological effects caused by extremely high levels of creatinine in humans. While it would be unethical to administer extremely high levels of creatinine in a human subject, animal studies have supported the avoidance of supplementing creatinine or taking action that results in high creatinine levels in the blood, tissues, or urine. In mice, administration of creatinine had a sedating or stupefying effect from an injection of creatinine (see Lis and Bijan, 1970). A similar effect was seen in dogs (Giovannetti et al., 1969). In addition to their observations of the animal's aberrant behavior, Giovannetti et al. further concluded that creatinine was also responsible for a significant decrease in the animal's erythrocyte survival time. In human blood cells, the addition of creatinine initiated a significant increase in spontaneous hemolysis. This same red cell lysing pattern was observed in normal human volunteers whom had ingested creatinine (Giovannetti et al., 1969). Barsotti's research in 1975 gave further evidence of this potential membrane-associated molecular blockade by showing that creatinine was able to effectively inhibit glucose utilization by erythrocytes (Barsotti et al., 1975).

(39) Often creatinine is found in creatine supplements, but due to the evidence suggesting extra creatinine would have deleterious effects, creatinine is considered an impurity in such compositions. Accordingly, strict regulations exist to limit the amount of creatinine in commercial creatine powders, for example, Health Canada allows the import of creatine monohydrate powders that contain a maximum of 100 ppm creatinine (0.01% or less by weight).

(40) It was also surprisingly discovered that, in contrast to prior art describing creatinine as toxic, a waste product, useless, and harmful to human performance (athletic, mental, and otherwise), concomitant administration of creatine and creatinine to human subjects actually yield beneficial effects. As shown in Example 7, concomitant administration of creatine and creatinine resulted in improved creatine bioavailability, improved creatine maximum concentration, and improved creatine body utilization. Instead of hindering performance, creatinine actually increased the ergogenic effects of creatine without producing any toxic or performance inhibiting effects.

(41) In some aspects, the disclosure relates to methods of increasing the bioavailability of creatine, the method comprising administering a creatine compound in combination with a creatinine compound. The method also results in greater serum concentration of creatine, greater muscle utilization of creatine, or overall beneficial effect of creatine. As demonstrated in Example 7, no negative effects are associated with co-administration of a creatine compound with a creatinine compound. Accordingly, the disclosure also relates to methods of safely administering creatinine to a subject in need thereof. In some implementations, an amount of between 0.5 and 20 g creatine is administered by the administration of the creatine compound and an amount of between 0.5 and 20 g creatinine is administered by the administration of the creatinine compound. For example, at least 1.5 g creatine, for example at least 2 g creatine is administered through the administration of the creatine compound. In some implementations, at least 1.5 g creatinine is administered through the administration of the creatinine compound, for example when the amount of creatine administered is at least 2 g.

(42) In some aspects, the method of increasing the bioavailability of creatine further comprises administering an effective amount of a source of nitrate (NO.sub.3.sup.−). Such methods also safely administered creatinine to a subject in need thereof. In some embodiments, the creatine compound provides at least 500 mg creatine, at least 1000 mg creatine, at least 2 g creatine, or at least 5 g creatine; the amount of creatinine provided is at least 500 mg, at least 1000 mg, at least 2 g, or at least 3 g; and the amount of nitrate (NO.sub.3.sup.−) provided by the source of nitrate (NO.sub.3.sup.−) is at least 50 mg, at least 100 mg, at least 250 mg, at least 500 mg, or at least 1000 mg. In certain embodiments, the amount of nitrate (NO.sub.3.sup.−) provided by the source of nitrate (NO.sub.3.sup.−) is between between 50 mg and 1200 mg nitrate (NO.sub.3.sup.−), between 60 mg and 1200 mg nitrate (NO.sub.3.sup.−), between 50 mg and 1500 mg nitrate (NO.sub.3.sup.−), or between 60 mg and 1500 mg nitrate (NO.sub.3.sup.−).

(43) As demonstrated in Example 9, Applicants also surprisingly discovered that solubility of creatine in water at standard temperature and ambient pressure can be increased without the need to reduce the pH of the solution with the presence of creatinine. Accordingly, this disclosure also relates to a method of increasing the solubility of creatine in water. The method comprises adding a creatine compound to a water-based composition comprising creatinine to create a creatine solution. A benefit of this method is that the resulting composition comprising dissolved creatine can have a pH of between about 7 and about 8, which makes the composition suitable for parenteral administration, such as intravenous administration. In some aspect, the water-based composition comprising creatinine is produced by dissolving a creatinine compound in water or a water-based composition. In some implementations, the method further comprises adjusting the pH of the creatine solution to a pH of between about 7 and about 8. In some aspects, methods are also directed to method of producing a composition for parenteral or intravenous administration of creatine to humans. In one implementation, the weight of the creatine in the water-based composition is 50 to 500% the weight of the creatine provided by the creatine compound.

(44) It was also surprisingly discovered that co-administration of a creatinine compound with a creatine compound counteracted caffeine's neutralizing effect on the ergogenic actions of creatine (see Example 8). Thus, in some aspects, this disclosure also relates to a method of neutralizing caffeine's negative effect on the ergogenic actions of creatine, where the method includes administering to a subject consuming caffeine an effective amount of creatinine or a combination of an effective amount of a creatine compound and an effective amount of a creatinine compound. For a subject consuming between 60 to 1200 mg caffeine per day, the effective amount of creatine and creatinine administered to ensure the effectiveness of dietary supplementation of creatine is between 1-30 g creatine per day and between 1-30 g creatinine per day, for example, about 20 g creatine and about 20 g creatinine per day. In certain implementations, the daily amount of creatine and creatinine is administered in multiple doses in a day, for example split across two, three, or four doses.

(45) In some aspects, the creatinine compound and/or the creatine compound is/are administered to the subject consuming caffeine within a day of the consumption of caffeine. In some implementations, the creatine compound and the creatinine compound are administered separately. For example, the creatinine compound is administered to the subject within a day, about 24 hours, or about 2 hours of the administration of the creatine compound. In other implementations, the creatine compound and the creatinine compound are administered in a dietary supplement composition comprising an effective amount of the creatine compound and an effective amount of the creatinine compound.

(46) Thus, Applicants discovered that creatinine is suitable as a dietary ingredient or food additive. In view of creatinine's beneficial supportive role in creatine supplementation, the disclosure also relates to the use of creatinine in producing a food fortified with creatine. As such, the disclosure also relates to dietary supplements and fortified foods comprising creatine.

(47) In some implementations of the methods for neutralizing caffeine's prohibitive effect on the ergogenic actions of creatine, the methods further comprises administering to the subject consuming caffeine at least one source of nitrate (NO.sub.3.sup.−). In some aspects, the at least one source of nitrate (NO.sub.3.sup.−) provides at least 50 mg, at least 100 mg, at least 250 mg, at least 500 mg, or at least 1000 mg. In certain embodiments, the amount of nitrate (NO) provided by the source of nitrate (NO.sub.3.sup.−) is between 50 mg and 1200 mg nitrate (NO.sub.3.sup.−), between 60 mg and 1200 mg nitrate (NO.sub.3.sup.−), between 50 mg and 1500 mg nitrate (NO.sub.3.sup.−), or between 60 mg and 1500 mg nitrate (NO.sub.3.sup.−).

(48) In some implementations of using creatinine as a dietary ingredient or food product, the dietary supplement or food product comprises at least one source of nitrate (NO.sub.3.sup.−). In some aspects, the at least one source of nitrate (NO.sub.3.sup.−) provides at least 50 mg, at least 100 mg, at least 250 mg, at least 500 mg, or at least 1000 mg. In certain embodiments, the amount of nitrate (NO.sub.3.sup.−) provided by the source of nitrate (NO.sub.3.sup.−) is between 50 mg and 1200 mg nitrate (NO.sub.3.sup.−), between 60 mg and 1200 mg nitrate (NO.sub.3.sup.−), between 50 mg and 1500 mg nitrate (NO.sub.3.sup.−), or between 60 mg and 1500 mg nitrate (NO.sub.3.sup.−).

(49) In some implementations of methods of producing a food fortified with creatine, the methods further comprise adding to the food fortified with creatine at least one source of nitrate (NO.sub.3.sup.−). In other aspects, the food fortified with creatine comprises at least one source of nitrate (NO.sub.3.sup.−). In some aspects, the at least one source of nitrate (NO.sub.3.sup.−) provides at least 50 mg, at least 100 mg, at least 250 mg at least 500 mg, or at least 1000 mg. In certain embodiments, the amount of nitrate (NO.sub.3.sup.−) provided by the source of nitrate (NO.sub.3.sup.−) is between 50 mg and 1200 mg nitrate (NO.sub.3.sup.−), between 60 mg and 1200 mg nitrate (NO.sub.3.sup.−), between 50 mg and 1500 mg nitrate (NO.sub.3.sup.−), or between 60 mg and 1500 mg nitrate (NO.sub.3.sup.−).

(50) In some implementations of the methods of increasing the solubility of creatine in water, the methods further comprise adding at least one source of nitrate (NO.sub.3.sup.−) to the water-based composition comprising creatinine. In some aspects, the at least one source of nitrate (NO.sub.3.sup.−) provides at least 50 mg, at least 100 mg, at least 250 mg, at least 500 mg, or at least 1000 mg. In certain embodiments, the amount of nitrate (NO.sub.3.sup.−) provided by the source of nitrate (NO.sub.3.sup.−) is between 50 mg and 1200 mg nitrate (NO.sub.3.sup.−), between 60 mg and 1200 mg nitrate (NO.sub.3.sup.−), between 50 mg and 1500 mg nitrate (NO.sub.3.sup.−), or between 60 mg and 1500 mg nitrate (NO.sub.3.sup.−).

(51) The amount of creatine compound in the compositions of the invention (for both the solid composition and the liquid composition) is variable depending on the desired supplemental amount of creatine. Generally, a dose of creatine for supplementation includes amounts between 500 mg to 25 g creatine per dose. However, the molar ratio of the creatine compound and creatinine compound in the compositions of the invention may be between about 23:1 and about 1:9, for example, between about 20:1 and about 1:3, between about 10:1 and about 1:1, between about 3:1 and about 1:3, between about 2:1 and about 1:1, about 1:1 or about 1:1.7. In some aspects, the amount of creatinine compound is between 5% and 800% (for example between 50% and 200%) the weight of creatine compound. In certain embodiments, for example, where the dietary ingredients of the dietary supplement consist of a creatine compound and a creatinine compound, the ratio by weight of creatine (from the creatine compound) to creatinine (from the creatinine compound) is preferably 5.5-7 weight parts creatine to 8 weight parts creatinine. It is preferred that only minimal amount of the creatinine compound (lowest amount possible to produce the desired effect, such as increased solubility or bioavailability of creatine or increased stability of creatine in solution) is included in the compositions of the invention. For example, in certain embodiments, the molar ratio of the creatine compound to the creatinine compound is about 1:1.1 or about 1:1.7.

(52) One exemplifying composition comprises about 5 g creatine nitrate (which corresponds to the composition providing about 3.34 g creatine) and about 4 g creatinine. Another exemplifying composition comprises about 5 g creatine nitrate and about 5 g anhydrous creatinine. Still another exemplifying composition comprises about 4 g creatine anhydrous and about 5 g creatinine nitrate. In some aspects, the composition comprises about 4 g creatine and between about 4 g and about 5 g creatinine. In some aspects, the amount of creatine in the solid composition is provided as a composition consisting of 1.5 g creatine nitrate and 3.5 g creatine monohydrate. In such composition, the amount of creatinine is 5 g anhydrous creatinine. In another embodiment, the composition comprises 5 g creatine nitrate is 5 g and 4 g anhydrous creatinine. In another implementation, the composition comprises 3 g creatine nitrate, 2 g anhydrous creatine, and 5 g creatinine.

(53) The corresponding liquid composition (for example, liquid food product or liquid dietary supplement formulation) would further comprise water or some other water-based composition or liquid, such as a commercial sports drink formulation, to dissolve the creatine compound and the creatinine compound. In some aspects, the amount of water or some other water-based composition or liquid is about 500 ml, about 475 ml, about 16 fluid oz, or about 240 ml. In some embodiments, the liquid composition further comprises a pH buffer, wherein the pH buffer adjusts the pH of the liquid composition to 4.4 or less, for example, between about 4.2 and about 4.4. In certain embodiments, the pH of the liquid composition is about 4.4.

(54) The concentration of creatine from the creatine compound in the liquid composition of the disclosure does not fall below 70%, preferably 90% or 95%, of the original concentration of creatine during storage, for example, at or around room temperature for at least a month, three months, six months, or a year. In some embodiments, the concentration of creatine in the liquid composition of the disclosure remains steady. In particular, the concentration of creatine after 30 days of storage at room temperature remains the same or higher than the concentration of creatine on day 1. In some aspects, the concentration of creatine after 30 days is higher than the concentration of creatine after 1 day.

(55) Compositions and/or formulations of the present invention may be in any form for administration, whether solid or liquid. For example, the composition and/or formulation is in the form of a capsule, a cachet, a pill, a tablet, a powder, a granule, a pellet, a bead, a particle, a troche, a lozenge, a pastille, a solution, an elixir, a syrup, a tincture, a suspension, an emulsion, a mouthwash, a spray, a drop, an ointment, a cream, a gel, a paste, a transdermal patch, a suppository, a pessary, cream, a gel, a paste, a foam, or combinations thereof for example. It is convenient to have an efficacious dose of creatine in a good-tasting, already mixed drink. Thus, liquid compositions where the creatine is stable at a greater than 95% amount over a long time (for example, 30 days, a month, three months, six months, or a year) without requiring refrigeration are preferred.

(56) Compositions and/or formulations of the present invention may also include at least one additional ingredient.

(57) In one aspect, the additional ingredient produces a composition with intermediate rigidity and/or intermediate fluidity properties between solid and liquid, which is described interchangeably herein as a semisolid composition, a semiliquid composition, or a quasi-solid composition. In such embodiments, the additional ingredient includes but is not limited to a semi-solid lipophilic vehicle, a paste, a solubilizer, thickener, or a gelling agent. In some aspects, the additional ingredient in a solid composition produces a semiliquid composition. In other aspects, the additional ingredient in a liquid composition produces a semisolid composition.

(58) In some aspects, the at least one additional ingredient comprises an acceptable additive for human consumption. Accordingly, the at least one additional ingredient is at least one additive selected from the group consisting of: a solubilizer, an enzyme inhibiting agent, an anticoagulant, an antifoaming agent, an antioxidant, a coloring agent, a coolant, a cryoprotectant, a hydrogen bonding agent, a flavoring agent, a plasticizer, a preservative, a sweetener, and a thickener. These additives may be solids or liquids, and the type of additive may be generally chosen based on the type of administration being used. Those of ordinary skill in the art will be able to readily select suitable additives from the disclosure in this document. In particular implementations, the acceptable additive is a pharmaceutically acceptable. For example, pharmaceutically acceptable additives include, calcium phosphate, cellulose, stearic acid, croscarmellose cellulose, magnesium stearate, and silicon dioxide. In another aspect, the at least one additional ingredient comprises an acceptable carrier for human consumption. Accordingly, the at least one additional ingredient is at least one carrier selected from the group consisting of: an excipient, a lubricant, a binder, a disintegrator, a diluent, an extender, a solvent, a suspending agent, a dissolution aid, an isotonization agent, a buffering agent, a soothing agent, and an amphipathic lipid delivery system. In some aspects, the at least one additional ingredient is selected from the group consisting of: a flavoring agent, a colorant, a viscosity modifier, a preservative, a fragrance, an amino acid, a salt of an amino acid, a vitamin, a mineral, a fatty acid, an enzyme, a co-enzyme, a mono-glyceride, a di-glyceride, a tri-glyceride ester oils emulsifiers, a hydrolyzed protein, whey protein, a stabilizer, a flow modifier, a chelating agent, an antioxidant, an anti-microbial, a benzoate, an alcohol, an ester of para-hydroxybenzoic acid, a propionate, and a surfactant.

(59) In particular embodiment, the compositions and/or formulations of the present invention further comprise at least one source of nitrate (NO.sub.3.sup.−). In some aspects, a source of nitrate is an inorganic nitrate salt (for example, sodium nitrate or potassium nitrate). In other aspects, a source of nitrate is a nitrate salt of an amino acid or a nitrate salt of an amino acid derivative, for example, the nitrate salt of arginine, agmatine, beta alanine, betaine, carnitine, creatine, citrulline, glutamine, L-histidine, isoleucine, leucine, norvaline, ornithine, valine, aspartic acid, cysteine, glycine, lysine, methionine, phenylalanine, proline, taurine, or tyrosine. Where the creatine compound of the composition is creatine nitrate, the at least one source of nitrate in the composition does not include creatine nitrate. In still other aspects, a source of nitrate is a botanical source, for example juice, extract, powder, or other derivative product from cabbage, spinach, beet leaf, beetroot, artichoke, asparagus, broad bean, eggplant, garlic, onion, green bean, mushroom, pea, pepper, potato, summer squash, sweet potato, tomato, watermelon, broccoli, carrot, cauliflower, cucumber, pumpkin, chicory, dill, turnip, savoy cabbage, celeriac, Chinese cabbage, endive, fennel, kohlrabi, leek, parsley, celery, cress, chervil, lettuce, rocket (rucola), and other vegetables or fruits known to containing high levels of nitrate. In preferred embodiments, the botanical source of nitrate is beet juice.

(60) In certain embodiments, the at least one source of nitrate (NO.sub.3.sup.−) provides between about 50 mg and about 2000 mg nitrate (NO.sub.3.sup.−), for example, between about 60 mg and 1200 mg nitrate (NO.sub.3.sup.−).

(61) Creatine Compound

(62) The creatine compound includes anhydrous creatine or a salt, solvate, or hydrate of creatine. While the creatine compound may be any salt of creatine, it is preferable the creatine compound is creatine nitrate. Other creatine compounds for use in the disclosed compositions include single administration physiologically active salts, creatine's tautomeric, polymeric and/or isomeric forms, creatine's analog forms, or creatine's derivative forms. It should be noted that as disclosed herein, the creatine compound does not include creatine esters and peptides, such as creatine ethyl ester and creatinyl-L-leucine. Creatine esters and peptides are unsuitable for the compositions described herein although they are generally stable in an acidic environment. Creatine esters and peptides are not actual sources of creatine, because cleavage of the peptide bond results in the formation of creatinine instead of creatine. Also in many cases creatine esters and peptides may be excreted unchanged to at least some degree.

(63) As a non-limiting example, the creatine compound may be selected from the group consisting of: creatine nitrate, creatine anhydrous, creatine monohydrate, creatine hydrochloride, creatine acetate, creatine malate, creatine ascorbate, creatine phosphate, creatine adipate, creatine aspartate, creatine caproate, creatine cinammate, creatine formate, creatine formic acid solvate, creatine fumarate, creatine gluconate, creatine glucuronate, creatine glycerophosphate, creatine glycolate, creatine lactate, creatine hydrobromide, creatine malonate, creatine methanesulfonate, creatine oleate, creatine orotate, creatine nicotinate, creatine pyroglutamate, creatine pyruvate, creatine stearate, creatine tartrate, creatine succinate, creatine citrate, creatine ferulate, and creatine toluenesulfonate.

(64) Creatine nitrate has been synthesized and patented by the applicants. The applicants found that creatine nitrate is more stable in aqueous compositions than creatine monohydrate and buffered creatine (kre-alkalyn). In preferred embodiments, the creatine compound is creatine nitrate.

(65) The chemical stability of creatine nitrate (CN), creatine monohydrate (CM), and buffered creatine (BC) were examined under two different storage conditions: (1) 37° C. in pH 2.5 buffer and (2) 40° C. in pH 6.8 buffer. A concentration of about 10 mg/ml of CN, CM and BC were prepared in both pH 2.5 and pH 6.8 buffer and stored in stability chambers in screw capped bottles at 37° C. and 40° C., respectively. The degradation rate constants for CN, CM and BC at 37° C. in pH 2.5 buffer were 0.075±0.001, 0.119±0.011, and 0.108±0.002 per day, respectively, while the degradation rate constants at 40° C. in pH 6.8 buffer were 0.115±0.001, 0.015±0.001, and 0.013±0.002 per day, respectively. The pH of CN samples at 40° C. in pH 6.8 buffer changed from 2.83±0.01 to 4.31±0.01 within a period of 12 days. The pH changes noticed at 37° C. in pH 2.5 buffer samples over the same period of time for CM, and BC were 3.08±0.01 to 4.12±0.01 and 3.11±0.01 to 4.16±0.01, respectively. No significant change in pH was observed for the rest of the samples. No change in the color and the clarity was noticed over 12 days.

(66) All the creatine samples followed first order degradation kinetics under both these experimental conditions. The degradation rate constants for CN was found to be higher at 40° C. in pH 6.8 buffer as compared to at 37° C. in pH 2.5 buffer. However, both CM and BC showed a faster rate of degradation at 37° C. in pH 2.5 buffer than at 40° C. in pH 6.8 buffer. The major degradation product detected was creatinine. For CN the increase in pH was higher at 40° C. in pH 6.8 buffer as compared to 37° C. in pH 2.5 buffer. However, opposite effect was noticed for both CM and BC.

(67) When creatine nitrate is combined with creatinine before dissolving into a solution, the concentration of creatine in the solution remains constant even after storage at around 25° C. for a long period of time, for example, at least a month (see Examples 1 and 2).

(68) Creatinine Compound

(69) The creatinine compound of the compositions of the disclosure is selected from any form of creatinine, including single administration physiologically active salts, solvates, or hydrates, creatinine's tautomeric, polymeric and/or isomeric forms, creatinine's analog forms, or creatinine's derivative forms. The creatinine compound includes anhydrous creatinine or a salt or hydrate of creatinine. The specific kind of creatinine compound used in the composition of the invention affects the stability of creatine. The salts of creatinine for use in the composition include salts of creatinine formed using either an organic acid or an inorganic acid, although the stability of creatine nitrate could be affected with every different creatinine salt chosen. Such salts include, but are not limited to: creatinine nitrate, creatinine hydrochloride, creatinine acetate, creatinine malate, creatinine ascorbate, creatinine phosphate, creatinine adipate, creatinine aspartate, creatinine caproate, creatinine cinammate, creatinine formate, creatinine fumarate, creatinine gluconate, creatinine glucuronate, creatinine glycerophosphate, creatinine glycolate, creatinine lactate, creatinine hydrobromide, creatinine malonate, creatinine methanesulfonate, creatinine oleate, creatinine orotate, creatinine nicotinate, creatinine pyroglutamate, creatinine pyruvate, creatinine ferulate, creatinine citrate, creatinine stearate, creatinine tartrate, creatinine succinate, and creatinine toluenesulfonate, creatinine pyruvate.

EXAMPLES

(70) The disclosure is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application are incorporated herein by reference in their entirety for all purposes.

(71) 1. Stability of Creatine in a Liquid Composition Comprising Creatine Nitrate and Creatinine Over a Period of 14 Months: Storage at Room Temperature (25° C.)

(72) Creatine nitrate (5 g, equaling 25.5 mmol or 3.34 g creatine) was combined with creatinine (4 g equaling 35.4 mmol creatinine) and then dissolved in 500 ml of water. The solution was left at room temperature (about 25° C.). Over the period of 14 months, the amount of creatine and creatinine in ppm were measured (see Table 1 and FIG. 1).

(73) TABLE-US-00001 TABLE 1 Analysis Date Creatinine (ppm) Creatine (ppm) 2017 Oct. 27 10014.99 5244.27 2017 Nov. 3 10086.3 5417.66 2017 Nov. 10 9909.82 5342.83 2017 Nov. 14 9880.29 5443.84 2017 Nov. 28 9644.02 5422.60 2017 Dec. 31 8969.06 6108.93 2018 Jan. 30 8723.39 6041.02 2018 Mar. 2 8282.80 6127.22 2018 Mar. 27 8183.38 6075.87 2018 Apr. 28 8557.34 6830.62 2018 Jun. 1 8161.07 6647.78 2018 Jun. 30 8422.65 7029.07 2018 Sep. 8 8401.57 5611.32 2018 Oct. 11 8522.94 5319.45 2018 Dec. 6 8015.59 5559.72 2019 Jan. 26 7812.69 5602.11

(74) Contrary to the observations in the prior art regarding the various forms of creatine converting to creatinine over time, the creatine content in the liquid creatine nitrate-creatinine composition has not reduced over time in the liquid formulation of the invention, thereby creating a unique stable creatine solution that may be used in foods, dietary supplements, and pharmaceutical preparations for example. The amount of creatine at day 30 of the current invention is at a minimum the same concentration, if not a higher concentration of creatine than the amount of creatine at day 1. In fact, in the surprising results of the original experiment, the creatine content in the liquid formulation of the invention actually increased from the initial creatine concentration, as the solution comprising creatine nitrate as the creatine compound and creatinine is stored for longer than a month at room temperature.

(75) 2. Stability of Creatine in a Liquid Composition Comprising Creatine Nitrate and Creatinine Over a Period of 12 Months: Storage at Room Temperature (25° C.)

(76) Creatine nitrate (5 g, equaling 25.5 mmol creatine or 3.34 g creatine) was combined with creatinine (4 g equaling 35.4 mmol creatinine) and then dissolved in 500 ml of water. The solution was left at room temperature (about 25° C.). Surprisingly, the creatine content in the liquid increased from the initial creatine concentration as the liquid was stored at room temperature for longer than a month (see Table 2 and FIG. 2).

(77) TABLE-US-00002 TABLE 2 Analysis Date Creatinine (ppm) Creatine (ppm) 2018 Jan. 18 8837.51 5130.22 2018 Jan. 19 8710.34 5057.29 2018 Jan. 20 8804.51 5147.82 2018 Jan. 24 8818.74 5231.74 2018 Jan. 30 8638.97 5198.99 2018 Mar. 2 8493.77 6065.45 2018 Mar. 27 8521.45 5768.23 2018 Apr. 28 8906.06 6562.30 2018 Jun. 1 8326.96 6914.92 2018 Jun. 30 8633.51 6039.40 2018 Sep. 8 8666.33 6106.06 2018 Oct. 11 8640.62 5564.04 2018 Dec. 6 8724.83 5579.68 2019 Jan. 26 7926.65 5121.64
3. Stability of Creatine in a Liquid Composition Comprising Creatine Nitrate and Creatinine Over a Period of 7 Months: Storage at Room Temperature (25° C.)

(78) In 500 ml of water at room temperature, 5 g creatine nitrate and 4 g creatinine were added and creatine, creatinine, nitrate and pH levels were assessed at the time point intervals indicated in the table below. Creatine content did not degrade, but actually increased, after 210 days of storage (see Table 3 and FIG. 3).

(79) TABLE-US-00003 TABLE 3 Analysis Day Creatine (ppm) Creatinine (ppm) Nitrate (ppm) pH 0 6673 8182 3153 4.39 1 6356 7951 3134 4.35 7 6357 8066 3142 4.28 30 5770 8080 3142 4.28 60 6119 8293 3177 4.31 90 5420 8260 3060 4.35 120 6180 8130 3170 4.45 150 6430 7970 3180 4.58 210 6590 7920 3220 4.46
4. Stability of Creatine in a Liquid Composition Comprising Creatine Nitrate and Creatinine Over a Period of 7 Months: Storage in Refrigeration (2-8° C.)

(80) In 500 ml of water at a temperature of between 2-8° C., 5 g creatine nitrate and 4 g creatinine were added. The solution was stored in refrigeration (2-8° C.). Creatine, creatinine, nitrate and pH levels were assessed at the time point intervals indicated in Table 4 and FIG. 4.

(81) TABLE-US-00004 TABLE 4 Analysis Day Creatine (ppm) Creatinine (ppm) Nitrate (ppm) pH 0 6647 8151 3189 4.39 1 6358 8097 3128 4.34 7 7024 8241 3304 4.28 30 6389 7799 3156 4.22 60 6748 7903 3157 4.17 90 6110 8070 3100 4.21 120 6560 7940 3140 4.34 150 6530 7940 3190 4.41 210 6780 7810 3180 4.35

(82) Similar to the previous example, creatine levels remained stable during the whole 210 days regardless of refrigeration. This is very important. Previous approaches to the problem of creatine degradation tried to use refrigeration to slowdown creatine degradation, but it was surprisingly discovered that refrigeration, which carries a lot of drawbacks like need of a refrigerator, or other cooling device, increased costs, hurdles in transportation, etc., is not required to ensuring the stability of creatine a liquid composition in the invention disclosed herein.

(83) 5. Stability of Creatine in a Liquid Composition Comprising Creatine Nitrate and Creatinine Over a Period of 7 Months: Storage in Refrigeration (2-8° C.)

(84) In 500 ml of water at room temperature, 5 g creatine nitrate and 5 g creatinine were added and creatine, creatinine, nitrate and pH levels were assessed at the time point intervals indicated in Table 5 and FIG. 5.

(85) TABLE-US-00005 TABLE 5 Analysis Day Creatine (ppm) Creatinine (ppm) Nitrate (ppm) PH 0 6630 10075 3179 4.63 1 6468 10010 3148 4.61 7 6852 9791 3194 4.51 30 6774 9251 2984 4.45 60 7240 9242 3148 4.41 90 6840 9370 3540 4.37 120 7740 9250 3240 4.49 150 7420 8910 2870 4.53 210 7920 8630 3200 4.50

(86) As can be seen in the table and in FIG. 5, creatine levels actually increased while creatinine levels decreased. This is unprecedented: in an acidic environment of 4.4, which is well known to favor the degradation of creatine to creatinine, the opposite occurred. Not only was creatine not degraded, the total creatine content in the composition increased. The increased creatine content may be due to the conversion of creatinine to creatine.

(87) 6. Stability of Creatine Added to a Commercial Energy Drink

(88) Creatine nitrate and creatinine were dissolved in a multicomponent energy drink (1.5 g creatine nitrate and 1 g creatinine added to 500 ml of the energy drink), and the changes in pH and creatine and creatinine content were measured (See Table 6 and FIG. 6). After the addition of creatine nitrate and creatinine, the drink had a resulting pH of 3.71. Creatine continued to degrade through day 60, where 62% of the beginning creatine content was seemingly lost. On day 60, the liquid was split in half to examine the influence of the pH in the stability of the creatine-creatinine composition. In one half, the pH was adjusted to 4.4 using a pH buffer. Increasing the pH resulted in increased creatine content despite the pH remaining at an acidic level. At day 210, 82% of the original creatine content was restored in the half of the solution with adjusted pH. Thus, maintaining the pH to about 4.4 is important for creatine's stability even in the presence of creatinine.

(89) TABLE-US-00006 TABLE 6 Analysis Day Creatine (ppm) Creatinine (ppm) Nitrate (ppm) PH Day 0 1972 2306 951 3.71 Day 1 1911 1965 878 3.71 Day 7 1597 2689 996 3.69 Day 30 2240 2770 891 3.8 Day 60 769 3150 946 3.8 Day 90 632 3740 984 3.88 Day 90, 713 3220 4.52 pH adjusted Day 120 708 2990 950 3.86 Day 120, 1300 2480 4.45 pH adjusted Day 150 726 3230 915 3.86 Day 150, 1350 2590 4.36 pH adjusted Day 210 845 3170 944 3.82 Day 210, 1620 2760 4.35 pH adjusted
7. Human Study Comparing the Effects of Administering Creatine Monohydrate, Creatine Nitrate, and the Disclosed Composition

(90) A human study was designed to evaluate the effects of combining creatine and creatinine for bioavailability and performance. Ten healthy human volunteers (aged 20-25 years) were used to evaluate and compare the effects of administering 3 g creatine monohydrate (CrM), 3 g creatine nitrate (CN, providing about 2 g creatine) or a composition comprising 3 g creatine nitrate and 3 g creatinine (CN-CRN).

(91) Each human subject was administered CN, CrM, or CN-CRN with a glass of water with a washout period of 7 days among each experiment. Creatine serum levels were assessed at 0, 5, 30, 45, 60, 90, 120 minutes after administration of CN, CrM, or CN-CRN. The average peak serum creatine concentrations at 60-min sampling interval were significantly higher in CN-CRN group (183.7±15.5 μmol/L), as compared to CN group (163.8±12.9 μmol/L) and CrM group (118.6±12.9 μmol/L) (P<0.001). CN-CRN resulted in a more powerful rise in serum creatine levels comparing to either CN or CrM after single-dose intervention, as evaluated with the area under the concentration-time curve calculation (701.1±62.1 (μmol/L)×min vs. 622.7±62.9 (μmol/L)×min vs. 466.3±47.9 (μmol/L)×min; P<0.001). It is of great note that the much higher levels of serum creatine in the CN-CRN were achieved with 33% less creatine than the creatine monohydrate group. Accordingly, co-administration of creatine and creatinine significantly improves serum creatine concentration in human subjects.

(92) Based on muscle biopsies taken from the subject, higher creatine muscle levels were seen when the subjects were treated with CN-CRN.

(93) Nine of the subjects did not report any negative side effects as measured by a side effect reporting questionnaire. However, one subject reported gastrointestinal disturbances with all three treatments (CrM, CN, and CN-CRN).

(94) Liver and kidney function as measured by ALT AST remained unchanged while GFR estimation showed a slight less than 10% clinically insignificant reduction.

(95) 8. Creatinine Neutralizes Caffeine's Counteraction on the Ergogenic Actions of Creatine

(96) Vandenberghe et al. found that the ergogenic effect of creatine on muscle was completely eliminated by caffeine intake (Vandenberghe et al., 1996). As Hespel et al.'s experiments showed, this might be due to opposite effect of caffeine and creatine on muscle relaxation time. However, Applicants discovered that co-administration of creatine with creatinine eliminated the neutralizing effect of caffeine with respect of creatine's ergogenic actions on muscle.

(97) A 35-year-old male subject (weight of 240 lb) ingested creatine with creatinine supplement formulation for six days. During the period of supplementation, the subject was advised to abstain from creatine rich foods and caffeine sources. Specifically, the subject ingested a dose of 5 g creatine nitrate and 5 g creatinine four times a day (total daily supplementation of 20 g creatine nitrate and 20 g creatinine) for five days. On the fifth and sixth day, the subject also consumed 350 mg caffeine in the morning alongside the morning dose of creatine and creatinine.

(98) Prior to supplementation the maximum weight the subject could push for three knee extensions was 365 lb. On the morning of the sixth day of supplementation, the subject could push 380 lb for three knee extensions. Thus, an increase in strength and endurance was observed despite the co-administration of caffeine with creatine.

(99) 9. Creatinine Increases the Solubility of Creatine in Water

(100) A common problem with creatine in the production of liquid supplements is creatine's low solubility in water. Creatine has a solubility of 13.3 g/l in water, or 13.3 mg/ml, in 25° C. While one option of increasing the solubility of creatine in water is to reduce the pH of the solution, the cost of this approach is the reduced stability of creatine in solution. Applicants surprisingly found that creatinine, an alkaline substance, can increase creatine solubility of creatine even while it causes the pH of the solution to increase. Thus, in a solution of 10 g creatinine in one liter of water, the maximum solubility of creatine at 25° C. in water increased to 15.8 mg/ml or 15.8 g/L, which is an 18% increase of creatine's solubility in water. The increased water solubility of creatine in the presence of creatinine without the need to reduce the pH of the solution enables the manufacture of solutions with higher concentration of creatine for use as an injectable or intravenous solution, where the preferred pH range is between 7-8 (Lee et al., 2013).

REFERENCES CITED (WHICH ARE ALL INCORPORATED BY REFERENCE HEREIN IN THEIR ENTIRETIES)

(101) Alraddadi et al., Pharmaceutics., 2018, 10(1). pii: E31. Barsotti et al., Kidney Int, 1975 (7) Suppl: S299-S301. Cannon et al., Am J Physiol., 1990, 259(6 Pt 2):R1214-9. Dash et al., J Pharm Sci., 2002, 91(3):708-718. Dash and Sawhney, J Pharm Biomed Anal., 200229(5):939-45. Deldicque et al., Eur J Appl Physiol., 2008, 102(2):133-43. Edgar and Shiver, J Am Chem Soc., 1925 47:1179-1188. Gangopadhyay et al., World Academy of Science, Engineering and Technology International Journal of Physical and Mathematical Sciences, 2019, 13(2): 195. Ganguly et al., AAPS PharmSciTech, 2003, 4:119. Giovannetti et al., Clin. Sci., 1969, 36:445-452. Lee et al., International Journal of Pharmaceutics, 2003, 253: 111-119. Lis and Bijan, Physiol. Chem. & Physics. 1970, 2:293-299. McCall and Persky, Subcell Biochem. 2007, 46:261-73. Tambaru et al., AIP Conference Proceedings 1823, 020095 (2017). Vandenberghe et al., J Appl Physiol (1985), 1996, 80(2):452-7.