Summary

The current invention relates to a novel ionic liquid of creatine comprising a creatine anion called creatinate and a cation. Particularly this novel ionic liquid of creatine comprises the cation of choline called cholinium, to form the ionic liquid cholinium creatinate.

Introduction

Creatine is one of the most popular ergonomic aids, and is a key source of energy for short-term energy-demanding activities (8-10 seconds) in many sports disciplines. However, its efficacy and consequential usability are limited by physical-chemical properties such as poor solubility in water and the tendency of cyclization in a biologically inactive form — creatinine, especially at pH less than 7.

Other creatine salts with increased solubility in water exist, such as creatine hydrochloride or creatine tartrate, however their stability in beverages and bioavailability require further development.

In order to overcome the current creatine stability and solubility problems, the present invention encompasses synthetized protonated forms of creatine in the form of salts with inorganic and biologically active anions as well as neutral forms.

There are no formulations with creatine in the form of anion on the market. This ionic liquid of creatine is liquid and stable at room temperature and provides a concentrated low volume liquid dose of creatine without any solvent. It is stable at low pH and high temperatures making it ideal as an ingredient for beverages or in liquid capsules.

Statements of Invention

According to the present invention there is provided an ionic liquid of creatine comprising a creatine anion and a cation of an ergomonic aid selected from one of the following : choline, betaine and agmatine.

Most preferably the ionic liquid of creatine is choline creatinate of the following formula ( 1 ) :

or betaine ethyl ester creatinate of formula (2)

Or agmatine creatinate of formula ( 3 )

Most preferbaly the ionic liquid of creatine is choline creatinate of Formula ( 1 )

Preferably the choline creatinate has a density of between 1.18g/cm ³ and 1.17g/cm ³ .

Preferably the choline creatinate salt is water soluble at room temperature .

The ionic liquids of creatine are stabel in aqueous compositions at ph 4 or below at 4 degrees Celsius, 24

Degrees Celsius and 40 deg Celsius for over 6 months.

Preferably the is density of choline creatinate is

1.1844g/cm3 at room temperature and has a pH greater than

7.

Preferably the ionic liquid of creatine is synthesised by mixing

a) equimolar amounts of creatine monohydrate and a choline, or betaine or agmatine base in a solvent; or b) equimolar amounts of sodium creatinate and a salt of choline, or betaine or agmatine in an organic solvent; c) the mixture of a) or b) above is heated under reflux for between 15 minutes to 180 minutes at between 30 and 85 degrees Celsius;

d) the solvent is then removed.

Preferably the solvent is then removed, by known methods including evaporation especially evaporation under vacuum.

More preferably the solvent is water for the creatine monohydrate and choline base reaction.

Alternatively equimolar amounts of choline, or betaine or agmatine salt and sodium creatinate are dissolved in organic solvents and heated under reflux at between 50 and 85 degrees Celsius.

Preferably the chloride salt of choline, or betaine or agmatine is used.

Preferably the resulting undissolved sodium chloride is removed by known methods including washing.

The organic solvent is removed, preferably by evaporation especially evaporation under vacuum.

More preferably the organic solvent is ethanol, methanol or propanol or a mixture thereof.

Most preferably the organic solvent is ethanol. Preferably the ionic liquid of creatine, especially choline creatinate, is provided ready to consume free of any added solvent, for example in liquid capsules.

More preferably the ionic liquid of creatine is used to manufacture stabiliser free, ready to drink beverages, including creatine water, long shelf life Ultra High Temperature (UHT) pasteurised beverages, and beverages with a pH less than 5, especially at or below pH 4, e.g. pH 3, and pH2 for greater than 6 months at 40 deg Celsius .

Choline creatine does not form crystals of creatine mono hydrate or choline creatinate at 4 Degrees Celsius at any concentration in aqueous compositions, at ph 4 to pH 7.

Preferably the ionic liquid of creatine is added to already prepared bottled beverages using a twist and relase cap.

The most prefered ionic liquid of creatine in choline creatinate .

Choline creatinate is alkaline with a pH higher than pH

8.

At 0.1M concentration, choline creatine has a pH of 11.

Examples of these beverages includes 60ml to 100ml shots or 200 to 500ml creatine water with or without collagen peptides, whey protein peptides or Leucine; ready to consume gels or in capsules that include other ingredients like amino acids especially glutamine, Leucine, isoleucine and valine; carnitine, glycerol, carbohydrates, proteins, peptides, sugars, poly unsaturated fatty acids and lipids.

Description

The invention will now be described by way of example only with reference to the accompanying figures and examples .

A Brief description of the Figures 1 to 8 :

Figure 1: Chemical formula of choline creatinate

Figure 2: The cyclization of the zwitterionic form of creatine in creatinine

Figure 3: Synthesis of choline creatinate

Figure 4: Display of the final compound

Figure 5 1H NMR spectrum of choline-creatinate

Figure 6: 13C NMR spectrum of choline-creatinate

Figure 7: Thermal stability of cholinum-creatine

Figure 8: The bioavailability of Creatine and choline creatinate over the course of 24 hours

The invention provides the choline creatinate compound as well as the process of its synthesis and the ability to be used as an active ingredient in the formulations of sports supplements, the chemical structure of (Figure 1 ) . The poor solubility of creatine in water is due to strong electrostatic attraction between its zwitter ionic structures and the formation of hydrogen bonds between them. By translating creatine molecules into the anionic form, these ions are mutually repulsive, and therefore better solvated by the water molecule, resulting in increased solubility and consequent bioavailability.

The cyclization of the zwitter ionic form of creatine to creatinine takes place in two phases, via a cyclic transition state, as shown in Figure 2. By translating creatine molecules into the aniioniic form, the electrophilicity of the carbonyl C atom is significantly reduced, which significantly reduces the possibility of creating a cyclic transient conditions, as transformation into creatinine (step 2) requires the presence of the H ⁺ ion and therefore the reaction in the base environment is no longer performed.

Example 1

The synthesis of choline creatinate can be accomplished in several ways (Figure 3) particularly l)by adding an equimolar amount of creatine and choline hydroxide and mixing in an aqueous solvent and refluxing at between 50 and 95 degrees Celsius for 15 to 180 minutes. It is possible to remove excess solvent by known means like evaporation under vacuum. The choline creatinate compound is rendered in a liquid aggregate state .

(2) The reaction between choline chloride and sodium- creatinate is carried out in organic solvents and refluxed at between 50 and 85 degrees Celsius for 15 to 180 minutes. The sodium chloride by product is not dissolved in the organic solvent, which allows it to be removed by washing, while the organic solvent is removed by evaporation under vacuum.

An aqueous solution of choline chloride can be passed through an anion exchange resin to form choline chloride.

The resulting pure compound as shown in FIG. 4 is an ionic liquid whose high osmotic pressure prevents the development of microorganisms and allows for easier storage and practically unlimited shelf life, for density, viscosity and thermal expression of choline creatinate, as can be seen density is stable at temperatures between 291.15K ( 1.1844g/cm ³ ) and 313.15 ( 1.17135g/cm ³ ) , with an average thermal expansion over this same temperature range of 5.54E-04, i.e. between 291.15K and 313.15K.

Table 1

This high pH of choline creainate (above pH 8) prevents the conversion into creatinine, and the hydrophilic properties of good solubility in water, thereby obviating the need for stabilisers and gums like xanthan gum, guar gum, carboxy methyl cellulosea and carageenan that are routinely used to suspend creatine monohydrate in ready to drink beverages (see patent applications US2017156359 and US2014066512 ) .

Example 2

Synthesis of the choline creatinate compound was carried out according to the following procedure. A 1.2 mole of creatine monohydrate was dissolved in water and an equimolar amount of choline hydroxide was added. The mixture was transferred to a round bottom flask, and heated at reflux for 2 hours at 80 deg Celsius. Upon completion of the reaction, the excess solvent was evaporated and a choline creatinate was obtained in the form of a light brown colour.

Example 3

Synthesis of the choline creatinate compound was carried out according to the following procedure. A 1.2 mole of a sodium creatine salt was dissolved in ethanol and an equimolar amount of choline chloride was added. The mixture was transferred to a round bottom flask, and heated at reflux for 2 hours at 60 deg Celsius. Upon completion of the reaction, sodium choride is precipitated out of solution, and excess solvent was evaporated. The choline creatinate was obtained in the form of a light brown colour.

Example 4

The structure was confirmed by 1H and 13C NMR and IR techniques (Figure 5 and 6). The product obtained is hygroscopic and has a distinctive odour. Thermal stability of the product was done using the thermogravimetric method (Figure 7), using simultaneous TG / DSC technique on the SDT Q600 (TA Instruments, USA). The mass of the sample was about 2.94 mg, heating was done in nitrogen stream (flow: 100 cm3 · min -1) from room temperature to 400 ° C and heating rate of 10 ° C · min-1. The [Hoi] [Cre] compound is stable up to about 115 oC, which makes it easy to store under the usual temperature conditions. Continuous drop in mass to Tdecomp is due to water evaporation. Example 5

Toxicological studies of the obtained choline creatinate were performed on the MRC-5 cell line of fibroblastic lungs, where the ionic liquid showed lower toxicity than vitamin C as a reference value. IC 50 is over 2000 micrograms / ml, which is about 5 times higher than the results obtained with vitamin C on the same cell line. This indicates that choline creatinate is non-toxic and safe for human use. Functional studies of the obtained choline creatinate showed higher bioavailability (greater Tmax and shorter tl / 2 ) of choline creatinate compared to creatine monohydrate over 24 hours in oral route in an equimolar amount of a case study in an active athlete (Figure 8 ) .

Example 6

8ml choline creatinate (100%) and 1ml of liquid flavouring were put into 10ml sachets or twist and release caps and sealed after nitrogen flushing.

Example 7

Ready to drink beverages having a final 100ml volume (pH 3.8) were formulated without stabilisers as follows percentage weight for weight ( % ) :

8% choline creatinate (100%)

2.2% carnitine tartrate (Carnipure®)

2% natural flavour (Strawberry Kiwi Qpharma®)

0.7% citric acid

Water q.s. to 100% All ingredients were mixed together in water and heated to 30 deg celcius, pH was monitored and maintained at less than pH 4 by citric acid.

Example 9

10% choline creatinate (100%)

6% collagen peptides (Peptan®)

2% natural flavour (Strawberry Kiwi Qpharma®)

0.7% citric acid

Water q.s. to 100%

All ingredients were mixed together in water and heated to 30 deg celcius, pH was monitored and maintained at less than pH 4 by citric acid.

Example 10

Ingredient % by weight of the composition

Whey Protein hydrosylate 5%

Choline Creatinate (100%) 3%

Stabiliser (Carrageenan) 0.100%

Skimmed Milk (liquid) 90%

Flavourings 0.3%

The stabiliser was dissolved in water by heating to 35 deg Celsius, the skimmed milk and choline creatinate were then added, followed by the whey protein and flavourings. The beverage composition was UHT sterilised.

Futhermore, as creatine is recognised for its medical use in heart, brain, muscle and other major organ health, the immediate solubility of creatine cholinate in water makes creatine cholinate ideal for use in medical applications like kidney dialysis, organ preservation solutions and intra venous drip solutions. Creatine cholinate can be added to these solutions directly prior to use or used in their manufacture.

Example 11

An organ preservation solution was formulated and chol ine creatinate was added at 6 %

Choline creatinate (100%) 60ml/L

Lactobionic Acid (as Lactone): 35.83 g/L

Potassium Phosphate monobasic: 3.4 g/L

Magnesium Sulfate heptahydrate : 1.23 g/L

Raffinose pentahydrate : 17.83 g/L

Adenosine: 1.34 g/L

Total Glutathione: 0.922 g/

Potassium Hydroxide: 5.61 g/L

Sodium Hydroxide/Hydrochloric Acid: Adjust to pH 7.4 Water for Injection: q.s.

Example 12

8mls Choline creatinate was added to a Myers Cocktail solution

8 ml of choline creatinate (100%)

5 mL of magnesium chloride hexahydrate (20%)

3 mL of calcium gluconate (10%)

1 mL of hydroxocobalamin (1,000 m/mL)

1 mL of pyridoxine hydrochloride (100 mg/mL)

1 mL of dexpanthenol (250 mg/mL)

1 mL of B-complex 100 containing 100 mg of thiamine HC1, 2 mg of riboflavin, 2 mg of pyridoxine HC1, and 2 mg of pantheno

100 mg of niacinamide, 2% benxyl alcohol

5 mL of vitamin C (500 mg/mL)

20 mL of sterile water.

Example 13

Choline creatinate was added to a 1 litre dialysis solution :

Chole creatinate (100%) 8mls

Glucose, 5.5g

Sodium Chloride, 5.38 g

Sodium Lactate 4.48 g

Magnesium Chloride Hexahydrate, 50.8 g

Calcium Chloride Dihydrate, 257 mg,

Water For Injections, BP

QS

Example 14

Human study, tissue uptake and performance enhancement

Rationale: While creatine monohydrate (CRM) is a well known 'gold standard' among different creatine formulations, its large-scale use remains somewhat limited due to poor solubility in water and limited performance in specific medical conditions. Choline creatinate is a novel 100% water-soluble ionic liquid creatine formulation.

Methods: Three apparently healthy men (age 24.3 ± 1.8 years; body mass index 23.7 ± 1.2 kg/m ² ) voluntarily participated in this double blind, randomized, crossover pilot trial. All participants were allocated to receive either 3 grams per day of Choline creatinate or CRM for 7 days, with 4-week washout period between interventions.

Choline creatinate intervention resulted in a more potent rise in muscle creatine levels comparing to CRM at 7-day follow-up, as evaluated with 1.5 T MR spectroscopy (4.8 ± 2.9% vs. 3.0 ± 3.7 %; P < 0.05). Choline creatinate was similar to CRM to increase both upper and lower body strength, as evaluated by maximal number of repetitions until volitional fatigue for bench press and leg press exercise. No subjective side effects were reported during each intervention period.

Choline creatinate appears to be more superior alternative to CRM in terms of tissue uptake while a new experimental treatment is safe and not unacceptably less efficacious than CRM for exercise performance improvement in a small cohort of young healthy men.

From blood analysis, it is clear the choline creatinate separates into creatine mono hydrate and choline in the stomach and digestive system before entering the blood stream, as HPLC analysis of blood samples after ingestion, only show creatine mono hydrate not choline creatinate (data not shown). This is supported by the increased muscle creatine concentrations after ingestion of choline creatinate (see above).

Discussion

The liquid form of the product allows easier transportation during the technological process using different pumps as well as easier mixing with other ingredients in the commercial preparation of multicomponent forms of dietary (sports) supplements. An additional advantage of the fluid formulation of creatine, which would form a completely formulated form of supplements compared to conventional solid formulations, is faster and easier preparation and application by users in the sport, without the need for its prior dissolution in water.

The immediate solubility of creatine cholinate makes it ideal for twist and realse caps whereby 5 to 20mls of creatine cholinate are contained in a cap. This cap can be sold separately or sold on a bottled beverage. This cap is ruptured by twisting of the cap by a user, the contents are released into the beverage in the bottle and after a mild shake, the user can drink the beverage with fresh creatine cholinate in it.

Liquid creatine-based creatine with choline as cation and creatine as an anion prevents limited solubility and creatinine cyclization, characterized by having higher bioavailability .

The ionic liquids of creatine described herein provide a new presentation of creatine. These ionic liquids are safe, can be added to beverages without changing their formulaitons to prevent creatine to creatinine conversion, and can be simply dropped into current products without significant mixing. Alternatively they can be put into capsules or small 5ml sachets for small volume doses. Shelf life at room temperature beyond 18 months is also provided without the need for preservatives .

These ionic liquids are clean label, vegan friendly ergonomic aids with an extra ergonomic aid attached, e.g. choline is a source of methyl groups needed for many steps in metabolism, betaine is a methylating agent and agmatine is deemed to assist with nervous system functions .

Surprisingly although these ionic liquids of creatine are stable for long periods in low pH beverages, it is clear from the choline creatinate studies that the creatinate anion separates from the choline cation to create creatine mono hydrate and choline in the stomach and small intestine. Creatine mono hydrate appears in the blood and creatine mono hydrate levels are raised in muscle tissue of humans after ingestion of choline creatinate .