FORMULATIONS OF CREATINE AND CYCLODEXTRIN EXHIBITING IMPROVED BIOAVAILABILITY

Abstract

Formulations of creatine, preferably phosphocreatine and most preferably disodium phosphocreatine, combined with cyclodextrin exhibit improved uptake across digestive mucosa, including intestinal, esophageal, and stomach mucosa. In particular, the formulations of the present invention are designed for protection of the creatine as they come in contact with gastric juices so as to allow thereafter for unexpectedly improved site-specific intestinal release.

Claims

1. A method of manufacturing a creatine supplement comprising: co-granulating one or more creatine compounds, cyclodextrin, and a sodium source to form a granular composition having an average particle size of from 150 to 850 microns; and coating the granular composition with methacrylate copolymers to produce a coated creatine supplement.

2. The method of claim 1, wherein the one or more creatine compounds comprises phosphocreatine and the sodium source comprises trisodium citrate.

3. The method of claim 1, wherein the one or more creatine compounds comprises disodium phosphocreatine and the disodium moiety of the phosphocreatine constitutes all or part of the sodium source.

4. The method of claim 1, wherein the creatine compound is selected from the group consisting of creatine monohydrate, creatine HCl and phosphocreatine, and sodium of the sodium source is present in a molar ratio of sodium to creatine of from 1:1 to 3:1.

5. The method of claim 1, wherein the creatine supplement comprises disodium phosphocreatine and cyclodextrin selected from the group consisting of alpha, beta and gamma cyclodextrins.

6. The method of claim 1, wherein the creatine supplement comprises: (i) at least one of disodium phosphocreatine or phosphocreatine; (ii) polyethylene glycol; and (iii) cyclodextrin selected from the group consisting of alpha, beta and gamma cyclodextrins.

7. The method of claim 1, wherein the creatine supplement is coated with Eudraguard® as the methacrylate copolymer coating.

8. The method of claim 1, wherein the creatine supplement comprises 85% disodium phosphocreatine, 5% polyethylene glycol, 5% cyclodextrins and is coated with a 5% by weight of the formulation coating comprising methacrylate copolymer.

9. The method of claim 1, wherein the coating step comprises spray drying the granular compound utilizing a fluidized bed technique.

10. The method of claim 1, wherein the creatine supplement is used for treating a muscle wasting disorder.

11. The method of claim 1, wherein the creatine supplement is used to enhance body muscle mass for sports performance applications, sports injury recovery or for maintenance of normal muscle tone during the aging process of a mammal.

12. A method of manufacturing a creatine supplement comprising: co-granulating a plurality of the following: (i) one or more creatine compounds; (ii) cyclodextrin; or (iii) a sodium source; forming a granular composition having an average particle size of from 150 to 850 microns; and coating the granular composition with methacrylate copolymers to produce a coated creatine supplement.

13. The method of claim 12, further comprising: the one or more creatine compounds comprises disodium phosphocreatine; the cyclodextrin is selected from the group consisting of alpha, beta and gamma cyclodextrins; and the sodium source comprises trisodium citrate.

14. The method of claim 12, wherein the sodium source comprises trisodium citrate present in a molar ratio of sodium to creatine from the creatine compound of from 1:1 to 3:1.

15. The method of claim 12, wherein the cyclodextrin is present in a range of from 1% to 10% (w/w).

16. The method of claim 12, wherein the creatine supplement is coated with Eudraguard® as the methacrylate copolymer coating.

17. A method of manufacturing a creatine supplement comprising: co-granulating a plurality of the following: (i) creatine monohydrate, creatine HCL and/or phosphocreatine; (ii) cyclodextrins selected from the group consisting of alpha, beta and gamma cyclodextrins; or (iii) a sodium source; forming a granular composition having an average particle size of from 150 to 850 microns; and coating the granular composition with methacrylate copolymers to produce a coated creatine supplement.

18. The method of claim 17, further comprising: the phosphocreatine being disodium phosphocreatine; the cyclodextrins being a mixture selected from the group consisting of alpha, beta and gamma cyclodextrins; the sodium source comprising trisodium citrate; the methacrylate copolymers being Eudraguard®; and the addition of at least one of starch, maltodextrin, and polyethylene glycol to the co-granulation prior to coating.

19. The method of claim 18, further comprising: the disodium phosphocreatine present from 75% to 95% (w/w); the mixture of cyclodextrins present from 1% to 10% (w/w); the trisodium citrate present in a molar ratio of sodium to creatine from the creatine compound of from 1:1 to 3:1; the Eudraguard® present from 1% to 10% (w/w/); and the polyethylene glycol being PEG 3350 present from 1% to 10% (w/w).

20. The method of claim 18, further comprising: the disodium phosphocreatine present from 75% to 95% (w/w); the mixture of cyclodextrins present from 0.1% to 10% (w/w); the trisodium citrate present in a molar ratio of sodium to creatine from the creatine compound of from 1:20 to 20:1; the Eudraguard® present from 1% to 10% (w/w); and the polyethylene glycol being PEG 3350 present from 0.1% to 10% (w/w).

Description

DESCRIPTIONS OF THE FIGURES

[0023] FIG. 1 illustrates creatine permeation through a Caco-2 monolayer in simulated gastric and intestinal buffers, of formulations of creatine HCl with and without cyclodextrin compared to formulations containing disodium phosphocreatine with and without cyclodextrin.

[0024] FIG. 2 illustrates creatine release from coated and uncoated granules in simulated gastric fluid over time.

[0025] FIG. 3 illustrates creatine release from coated and uncoated granules in simulated intestinal fluid over time.

[0026] FIG. 4 illustrates creatinine levels from coated and uncoated disodium phosphocreatine (DSPC) in simulated intestinal fluid over time.

[0027] FIG. 5 illustrates coated (blue) and uncoated (white) granules in sealed teabags before (upper) and after (lower) gastric buffer.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0028] In looking for a formulation manifesting improved creatine uptake in the digestive tract, a CaCo2 cellular diffusion model substantially similar to that described by A. K. Dash, et al., “Evaluation of Creatine Transport Using Caco-2 Monolayers as an In Vitro Model for Intestinal Absorption” was used. Uptake of creatine HCl (“CrHCl”) was compared to that of disodium phosphocreatine with and without cyclodextrin.

[0029] The various compounds used in the experiments supporting the present invention include disodium creatine phosphate from Tiancheng International Inc., creatine HCl from Tiancheng International Inc., trisodium citrate from Cargill, creatine monohydrate from Sigma Life Science, C3630, creatinine from Sigma Aldrich, C4255, Water—Optima from Fisher Scientific, W 7-4, acetonitrile—Optima from Fisher Scientific, A995-4, Eudraguard® control from Evonik, Eudraguard® protect readymix from Evonik, ammonium sulfate, ACS from RICCA, RDCA0520-500B1, DMEM from Corning cellgro, 50-013-PC, Hanks' balanced salts (modified) from Sigma, H2387, and monobasic potassium phosphate from Spectrum, PO200.

[0030] To evaluate creatinine formation in simulated gastric and simulated intestinal buffers, a solution of 0.2N was prepared by dilution of 1 g NaOH QS to 125 mL with DI water. Simulated intestinal fluid without enzymes was prepared with 6.8 g monobasic potassium phosphate, 77 mL 0.2 N sodium hydroxide, pH adjusted to 6.8±0.1 using NaOH or HCl, with dissolution and QS to 1 L with DI water, to a final pH should of 6.8±0.1. To prepare simulated gastric fluid without enzymes, 2.0 g sodium chloride was mixed with 7.0 mL concentrated HCl, then dissolved and diluted to 1 L with DI water, with the resulting pH at approximately 1.2. Prior to experimentation, these simulated fluids were filtered at 0.2 um.

[0031] After fluid preparation, 159 mg of disodium phosphocreatine were placed into each of two 250 mL volumetric flasks. The simulated gastric fluid described above was added to one of the flasks to 250 mL and the simulated intestinal fluid was added to the other of the flasks to 250 mL. 93 mg of creatine monohydrate were placed into each of two other volumetric flasks. The simulated gastric fluid described above was added to one of the flasks to 250 mL and the simulated intestinal fluid was added to the other of the flasks to 250 mL.

[0032] At test time 0 (start), a 1 mL sample was withdrawn from each volumetric flask, filtered at 0.2 um and injected and measured by high-performance liquid chromatography (UPLC) immediately. Six separate withdrawals of 20 mL of each of the samples were then placed in scintillation vials for a total of 24 vials. Three samples from each set were incubated in an orbital shaker at 100 rpm and 37 C. Three other samples from each set were retained at room temperature on an orbital shaker at 100 rpm. 1 ml samples were then drawn from all vials at 0.5, 1, 2, 4, 6, 12, 24 and 48 hours, filtered at 0.2 um and injected into the UPLC immediately. Retained filtered samples were saved refrigerated at 4 C until after the completion of the study. A creatinine internal standard of 2.5 mM, 71 mg/250 ml, 0.2 um filtered in either simulated gastric or intestinal fluid was spiked into the retained samples at 50 ul creatinine to 450 ul sample and rerun on the UPLC to ensure proper detection of creatinine formation.

[0033] Thereafter, for all samples from each formulation were monitored apical to basolateral (A-B) for 90 minutes in 15 minute increments through a Caco 2-membrane registering an average TEER of 2500 Ohm*cm2. Samples were monitored for creatine and creatinine content. No significant quantities of creatinine were detected. Creatine permeation through the Caco-2 monolayer is illustrated in FIG. 1, with data as summarized below in Table I.

TABLE-US-00001 TABLE I 15 min 30 min 45 min 60 min 75 min 90 min .diamond-solid. Disodium 194.3 220.1 245.8 254.3 280.3 299.7 phosphocreatine .square-solid. Disodium 206.8 243.4 289.4 295.7 317.5 343.9 phosphocreatine w/cyclodextrin .box-tangle-solidup. Creatine 124.8 136.7 153.3 193.6 194.6 220.3 hydrochloride .circle-solid. Creatine 117.2 136.0 157.1 174.6 188.7 208.5 hydrochloride w/cyclodextrin

[0034] Creatine permeation was measured in micrograms per square centimeter (μ/cm.sup.2) for each formulation at 30, 45, 60, 75 and 90 minutes after test start. As is readily apparent, the disodium phosphocreatine formulations had substantially greater creatine permeation at all times throughout the test, as compared to creatine permeation from the creatine HCl formulations. This result was unexpected. Indeed, at test end, creatine permeation of the disodium phosphocreatine formulation in which cyclodextrin was not premixed, was measured at 299.7 μ/cm.sup.2, which is 136% of the creatine permeation of the creatine HCl formation measured at 220.3 μ/cm.sup.2.

[0035] The formulations of creatine HCl without the presence of cyclodextrin showed little difference in creatine permeation throughout the 90 minute test period as compared to the formulations of creatine HCl premixed with cyclodextrin. In contrast, the formulations of disodium phosphocreatine exhibited consistently increased creatine permeation, which ranged from 6.4% at 15 minutes after test start (206.8 μ/cm.sup.2 as compared to 194.3 μ/cm.sup.2) to 15% at 90 minutes after test start (343.9 μ/cm.sup.2 as compared to 299.7 μ/cm.sup.2) when premixed with the cyclodextrin, as compared to disodium phosphocreatine permeation when no cyclodextrin was present. This result of the presence of the cyclodextrin was completely unexpected.

[0036] Further studies were then undertaken to confirm above results comparing creatine dissolution of coated and uncoated disodium phosphocreatine granules in simulated gastric and intestinal buffers (without enzymes). Once again, the CaCo2 cellular diffusion model substantially similar to that described by A. K. Dash, et al., “Evaluation of Creatine Transport Using Caco-2 Monolayers as an In Vitro Model for Intestinal Absorption” was used. More particularly, two formulations were studied, one coated with Eudraguard® control applied in a fluidized bed coater.

[0037] EUDRAGUARD® control, a product of Evonik Nutrition & Care GmbH is, is a functional coating designed for enteric release, sustained-release formulations and gastro retention. The product is specifically designed for dietary supplement applications that require reliable and reproducible controlled-release profiles. EUDRAGUARD® control meets the regulatory requirements for use in dietary supplements in the European Union (E 1206) and the United States (GRAS).

[0038] EUDRAGUARD® protect, also a product of Evonik Nutrition & Care GmbHis, a polymer designed for immediate release formulations. It offers reliable taste and odor masking and protects ingredients from light, moisture and oxygen, which could impact ingredient effectiveness. The coatings give a smooth and even finish. EUDRAGUARD® protect ReadyMix is a ready-to-use powder that contains all the ingredients needed to form effective protective coatings for nutraceuticals. The spray suspension is formed by adding water to the ReadyMix while stirring. EUDRAGUARD® protect ReadyMix, a one-component system, is a ready-to-use mixture of the polymer plus excipients that can be augmented. It can be applied to solid oral dosage forms such as tablets, capsules and multiparticules. And because it is based on EUDRAGUARD® protect, the mix meets the regulatory requirements for use in dietary supplements in the European Union (E 1205) and the United States (GRAS).

[0039] For all samples 10 Mm creatine was present in the initial solution. Samples from each formulation were monitored apical to basolateral (A-B) for 90 minutes in 15 minute increments through a Caco 2-membrane registering an average TEER of 2500 Ohm*cm.sup.2. Samples were monitored for creatine and creatinine content. No significant quantities of creatinine were detected for the duration of the study.

[0040] Creatine permeation through the Caco-2 monolayer is illustrated in FIGS. 2, 3 and 4, below with data as summarized below, respectively, in Tables II, III and IV which follow:

TABLE-US-00002 TABLE II 15 30 60 90 120 min min min min min Disodium phosphocreatine  47.4  80.4 156.7 216.1 302.7 coated Disodium phosphocreatine 111.8 307.9 444.6 466.4 471.4 w/cyclodextrin uncoated

TABLE-US-00003 TABLE III 30 min 60 min 120 min 180 min 240 min 300 min 360 min Disodium phospho- 161.81 172.1 266.1 328.3 358.0 373.0 384.6 creatine coated Disodium phosphor- 84.4 321.1 431.3 449.4 455.1 457.1 462.2 creatine uncoated

[0041] These data follow with the loss of matter observed from the samples (samples retained). It was observed that in simulated gastric buffer there was only one sample/time point that showed significant creatinine formation. The amount at that time point calculated to a total of 15 mg/500 ml creatinine formation in 2 hours. There was no formation of creatinine observed in the coated gastric samples. Measurements were made, with the following results:

TABLE-US-00004 TABLE IV 15 min 60 min 120 min 180 min 240 min 300 min 360 min Disodium 0.86 1.42 1.83 2.24 3.70 3.08 3.39 phosphocreatine coated Disodium 0.87 1.71 2.33 2.89 3.42 3.89 4.32 phosphocreatine uncoated

[0042] Dissolution studies were also undertaken in which granules were prepared containing 85.73% (w/w) disodium phosphocreatine, 4.5% (w/w) Eudraguard® protect, and 4.75% (w/w) PEG 3350. In other embodiments other polyethylene glycols may be used. Uncoated granules contained 5% (w/w) lactose, coated granules contained 5% (w/w) Eudraguard® control. Wet granulation of particles was performed using a 10% solution of PEG 3350 in ethanol slowly added while powders were mixed using a high sheer mixer. Wet granules were sieved through 30 and 60 mesh sieves. Granules were then dried and granules between 100 and 140 mesh by sieve were collected to produce granules between 100 and 150 μm. For purposes of experimentation, blue dye was added to the Eudraguard® control <0.1% w/w, and particles were coated using the following parameters:

TABLE-US-00005 Parameters Values Atomization Rate 1.5 Barr Airflow rate 52 m.sup.3/hr Inlet Temperature 80° C. Drying Chamber Temperature 50° C. Filter Cleaner knob 2 Spray Rate Dial reading 5 (1 ml/min)

[0043] Coated granules were sieved and granules in the size range 150-250 μm (between 60 and 100 mesh) were collected for dissolution studies. Dissolution of coated and uncoated granules sized 150-250 μm was performed by weighing approximately 470 mg of granules into weighed tea bags and heat sealed. The dissolution protocol was as follows: [0044] 1) Apparatus: USP type II Basket Apparatus [0045] 2) Temperature: 37°±0.5° C. [0046] 3) Volume of the dissolution media: 500 mL [0047] 4) Dissolution Media: [0048] USP Simulated Gastric Buffer pH 1.2 [0049] USP Simulated Intestinal Fluid pH 6.8 [0050] 5) Sampling Points: [0051] Gastric exposure: 15, 30, 60, 90, 120 min [0052] Intestinal exposure: 15, 30, 60, 120, 180, 240, 300, 360 min [0053] 6) Sample Volume: one mL [0054] 7) Samples withdrawn were replaced by addition of equal volume of fresh dissolution medium, removed sample amounts were accounted for in the calculations. [0055] 8) Samples were filtered and analyzed by HPLC to determine the concentration of Creatine and Creatinine

[0056] In one experiment, coated and uncoated granules were placed in sealed teabags. The Eudraguard® control <0.1% w/w prior to coating, resulted in teabags with blue contents, as compared to teabags with uncoated white granules. The top row above shows 3 teabags containing blue coated granules on the left and 3 teabags with uncoated white granules on the right. The lower row illustrates the teabags after placement in the gastric buffers. The 3 teabags on the left contain intact granules, as evidenced by blue coating. The 3 teabags on the right are mostly empty, the granules having dissolved in the gastric buffer.

[0057] Preferred ranges of the compositions of the present invention contain 75% to 95% (w/w) disodium phosphocreatine, 0 to 10% cyclodextrins as a mixture selected from the group consisting of α, β and γ-cyclodextrins, 0 to 10% (w/w) Eudraguard® protect, and 0 to 10% (w/w) PEG 3350. Coated granules preferably contain 1% to 10% (w/w) Eudraguard® control. Preferable use of these compositions are as oral supplementation for mammals. They are preferably dosed in powder, tablet, and beverage forms.

[0058] Preferred methods of manufacturing a creatine supplement of the present invention include co-granulating one or more creatine compounds, cyclodextrin, and a sodium source to form a granular composition having an average particle size of from 150 to 850 microns; and then coating the granular composition with methacrylate copolymers to produce a coated creatine supplement. The one or more creatine compounds can include phosphocreatine and the sodium source can include trisodium citrate. Also, the one or more creatine compounds may include disodium phosphocreatine and the disodium moiety of the phosphocreatine may constitute all or part of the sodium source. Also, the creatine compound may be selected from the group consisting of creatine monohydrate, creatine HCl and phosphocreatine, and sodium of the sodium source may be present in a molar ratio of sodium to creatine of from 1:1 to 3:1. In the present invention, the coating step may comprise spray drying the granular compound utilizing a fluidized bed technique. The resulting coated creatine supplement resulting from the methodologies described herein is included with the scope in the present invention.

[0059] An oral creatine supplement of the present invention most preferably comprises disodium phosphocreatine and cyclodextrin selected from the group consisting of alpha, beta and gamma cyclodextrins. This oral supplement is preferably in the form of a powder, an encapsulated product, a tablet or beverage. Preferably, the creatine supplement is coated, most preferably with a methacrylate copolymer.

[0060] In other embodiments, an oral creatine supplement of the present invention comprises disodium phosphocreatine, polyethylene glycol and cyclodextrin selected from the group consisting of alpha, beta and gamma cyclodextrins. Such supplements are preferably coated with a coating comprising a methacrylate copolymer, most preferably, Eudraguard® protect as the methacrylate copolymer coating. Preferably, the creatine supplement comprises 85% disodium phosphocreatine, 5% polyethylene glycol, 5% cyclodextrins and is coated with a 5% by weight of the formulation coating comprising methacrylate copolymer. Other embodiments of the oral creatine supplement formulation composition comprise 75% to 95% (w/w) disodium phosphocreatine, 0 to 10% (w/w) cyclodextrins as a mixture selected from the group consisting of α, β and γ-cyclodextrins, 0 to 10% (w/w) Eudraguard® protect methacrylate copolymer, and 0 to 10% (w/w) PEG 3350. Also preferred are embodiments of the oral creatine supplement formulation composition which comprise 75% to 95% (w/w) disodium phosphocreatine, 2% to 10% (w/w) cyclodextrins selected from the group consisting of α, β and γ-cyclodextrins, and 2% to 10% (w/w) Eudraguard®.

[0061] In other embodiments of the oral creatine supplement of the present invention, the formulations include coated granules preferably containing 1% to 10% (w/w) utilizing Eudraguard® control as the methacrylate copolymer.

[0062] Each of the oral creatine supplements of the present invention are preferably used as a supplement for mammals, most preferably dosed as a powder, tablet, capsule or as beverage forms. They can be used to treat a muscle wasting disorder such as cachexia, sarcopenia or the like. These products can also be used to enhance body muscle mass for sports performance applications, sports injury recovery or for maintenance of normal muscle tone during the aging process of a mammal.