Process for preparation of nicotinamide riboside (NR) and cosmetic composition comprising (NR and a phosphate-binding agent

Abstract

Disclosed herein is a process for preparing nicotinamide riboside (NR) from an NR precursor and a phosphate-binding agent in a solvent. The reaction-derived mixture comprising NR may be further used without further processing in a variety of products, particularly in a cosmetic product.

Claims

1. A process for preparing nicotinamide riboside (NR), wherein said process is a chemical synthesis process performed at a temperature between 40 to 90° C. inclusive, the process comprising combining an NR precursor which is β-nicotinamide adenine dinucleotide (NAD.sup.+), with a phosphate-binding agent in a solvent, wherein said solvent comprises water, and wherein said phosphate binding agent is a salt of Ca(.sup.2+).

2. The process according to claim 1, wherein in said solvent said salt of Ca(.sup.2+) forms a salt having solubility-product constant (K.sub.sp) of lower than 10.sup.−16 with phosphate anion.

3. The process according to claim 1, wherein salt of Ca(.sup.2+) is a water-soluble salt having a water solubility at room temperature of above 3 g per 100 g of water.

4. The process according to claim 3, wherein molar ratio between said NR precursor and said salt of Ca(.sup.2+) is between 1:2 and 1:15, inclusive.

5. The process according to claim 1, further comprising combining a zirconium salt essentially concurrently with said phosphate binding agent.

6. The process according to claim 5, wherein the process as a whole is performed for no more than 6 hours.

Description

EXAMPLES

(1) Materials

(2) The nicotinamide adenine dinucleotide (NAD.sup.+), β-nicotinamide mononucleotide, adenine monophosphate and nicotinamide (vitamin PP) standards were purchased from the Sigma Aldrich. Nicotinamide riboside (NR) was purchased from ChromaDex.

(3) Methods

(4) Mass Spectrometry (MS), High Performance Liquid Chromatography (HPLC) and Nuclear Magnetic Resonance Spectroscopy (NMR) based detection methods were developed for each one of the materials.

(5) NMR spectra were recorded with 400 MHz Bruker spectrometer. Reversed phase HPLC column of RP-18 type (Chromolith performance RP18e (100-4.6 mm) UM8 086/001, Merck KGaA) using deionized HPLC-grade solvents in the following composition: 1% acetonitrile, 99% water. Flow rate was 0.1 mL/min, detection performed with UV detector at 260 nm. Mass Spectrometry (MS), 6200 series TOF/6500 series Q-TOF B.05.01 (B5125.1).

(6) HPLC-based method alone was found insufficiently accurate for unequivocal identification of the desired nicotinamide riboside (NR) due to similar retention times of the nicotinamide mononucleotide and NR under all tested separation conditions. The best separation was achieved using reversed phase HPLC column of RP-18 type (Chromolith performance RP18e (100-4.6 mm) UM8 086/001, Merck KGaA) using deionized HPLC-grade solvents in the following composition: 1% acetonitrile, 99% water. Flow rate was 0.1 mL/min, detection performed with UV detector at 260 nm. HPLC retention times under these conditions are provided in Table 1.

(7) TABLE-US-00001 TABLE 1 Material HPLC retention time (minutes) NAD.sup.+ 24.91 Adenosine 20.77 β-nicotinamide mononucleotide 14.52 nicotinamide riboside 15.18 Vitamin PP 37.27

(8) Despite nicotinamide mononucleotide and NR significant overlap, it is possible to distinguish between the two by spiking experiment (e.g. intentionally spiking the commercial NR standard into actual sample to confirm that the intensity of the observed overlapped peaks changes accordingly). The resulting HPLC chromatograms demonstrate that β-nicotinamide mononucleotide+nicotinamide riboside (NR) were separated with retention times 14.52 min and 15.18. After spiking with NR (higher intensity) the chromatogram shows major peak at 15.18 minutes. After spiking with β-nicotinamide mononucleotide (separation) two major peaks are observed at 14.52 minutes and 15.18 minutes.

(9) Proton NMR (.sup.1H NMR) and .sup.31-phosphorus NMR (.sup.31P-NMR) allow distinguishing between the starting material NAD.sup.+, the intermediate nicotinamide mononucleotide and the desired product nicotinamide riboside (NR). NAD.sup.+ and nicotinamide mononucleotide are characterized by very distinguishable .sup.31P-NMR signals (.sup.31P NMR shifts are −11.36 and −11.65, and −0.08 ppm, respectively), while the desired NR is .sup.31P-NMR-silent (see Scheme 4).

(10) ##STR00005##

(11) Further decomposition of NR to ribose and nicotinamide (vitamin PP) is possible as NR is unstable over prolonged exposure to acidic or basic (hydrolytic) conditions. Proton NMR (.sup.1H NMR) is indicative in tracing the decomposition process of NR (see Scheme 5).

(12) ##STR00006##

(13) .sup.1H-NMR spectrum of nicotinamide riboside reveals the following peaks: 3.88 (2H, dd, J.sub.1,2=62.25 Hz, J.sub.1,3=3.55 Hz), 4.26 (1H, t, J=4.63 Hz), 4.37-4.39 (1H,m), 4.41 (1H, t,J=4.47 Hz), 6.15 (1H, d, J=4.47 Hz), 8.17 (1H, t, J=6.81 Hz), 8.88 (1H, d, J=8.17 Hz), 9.18 (1H, d, J=8.18 Hz), 9.51 (1H, s).

(14) .sup.1H-NMR spectrum of Vitamin PP reveals the following peaks: 7.44 (1H,dd, J=8.59, 5.9 Hz), 8.09 (1H,dt, J=8.09, 1.95 Hz), 8.56 (1H,dd, J=5.10, 1.6 Hz), 8.78 (1H,d, J=2.15 Hz).

(15) However, the ultimate identification of the desired product (NR) was performed using High Resolution-Mass Spectrometry (HR-MS) technique. Calculated molecular weight (MW) for the formula C.sub.11H.sub.15N.sub.2O.sub.5.sup.+ is 255.0975. Found MW is 255.09285.

Example 1 (Comparative)

Preparation of Nicotinamide Riboside from NAD.SUP.+ by a Two-Stage Process (ZrCl.SUB.4.-Promoted NAD.SUP.+ Primary Hydrolysis Followed by Secondary Hydrolysis

(16) Stage one: In a 250-ml flask, to a mixture of NAD.sup.+ (0.3317 gr) and ZrCl.sub.4 (0.582 gr), 100 ml of deionized water was added and the reaction was heated to 80° C. for 30 minutes. Over this time, full conversion of the NAD to β-nicotinamide mononucleotide was observed by HPLC.

(17) Stage two: 1.8 gr of calcium L-ascorbate dihydrate was added to the reaction mixture and heating was continued for further 2 days at 80° C.

(18) Samples from the reaction mixture were analyzed every 4-5 hours by HPLC and NMR. Formation of the desired NR was detected albeit in low concentration (lower than 3% yield) due to extensive decomposition of NR to ribose and nicotinamide. HR-MS spectrum of the product of Stage 2 is with MW of 255.16. The yield was determined by comparing the abundance of the desired peak to the abundance of the internal standard.

Example 2

Preparation of Nicotinamide Riboside from NAD.SUP.+ by a One-Stage Process (ZrCl.SUB.4.-Promoted Primary NAD.SUP.+ Hydrolysis and Secondary Hydrolysis Carried Out Concurrently

(19) In order to reduce the extent of the NR decomposition via tertiary “over-hydrolysis”, one stage protocol was developed.

(20) In a 250-ml flask, a mixture of NAD.sup.+ (0.33 gr), ZrCl.sub.4 (0.58 gr) and 1.80 gr of calcium L-ascorbate in 100 ml of deionized water was heated to 80° C. for 2 days. Over this time period, full conversion of the NAD.sup.+ was observed by HPLC. Decomposition of the desired NR product was also observed under these conditions, but to a lower extent in comparison with the process of Example 1. HR-MS spectrum of the product is MW of 255.10. The extent of the decomposition of the desired NR was determined by comparing the abundance of the desired peak to the abundance of the internal standard.

(21) The concentrations of the products and the reactants at various time points are provided in Table 2.

(22) TABLE-US-00002 TABLE 2 Time NAD.sup.+ Relative NR Relative (hours) Concentration Concentration Nicotinamide 0 1 0 0 0.5 0.66 0.05 0.1 6 0.32 0.06 0.19 16 0.05 0.02 0.32 24 0 0 0.48

Example 3

Preparation of Nicotinamide Riboside from NAD.SUP.+ with the Aid of Phosphate Binding Agent Alone

(23) In a 250-ml flask, a mixture of NAD.sup.+ (0.34 gr) and 1.8 gr of calcium L-ascorbate in 100 ml of deionized water was heated to 80° C. for 4 days. The mixture was analyzed every 3 hours using HPLC and .sup.31P-NMR. According to HPLC analysis and .sup.31P NMR, in the absence of ZrCl.sub.4, the rate of the primary hydrolysis of NAD.sup.+ to β-nicotinamide mononucleotide was slow and comparable to the rate of the secondary hydrolysis of the β-nicotinamide mononucleotide to the NR. The concentrations of the products and the reactants at various time points are provided in Table 3.

(24) TABLE-US-00003 TABLE 3 Time NAD.sup.+ Relative NR Relative (hours) Concentration Concentration Nicotinamide 0 1 0 0 0.5 0.86 0.06 0.1 6 0.62 0.11 0.16 16 0.55 0.09 0.32 24 0.38 0.08 0.48 36 0.22 0.01 0.41 48 0.2 0 0.41

(25) HR-MS spectrum of the product, as taken at the end of the reaction yielded MW of 255.09495. Calculated MW for the formula C.sub.11H.sub.15N.sub.2O.sub.5.sup.+ is 255.0975.

(26) The rate of the tertiary hydrolysis (decomposition of the NR) is slower. Under these conditions, the concentration of the NR remains essentially stable (˜10% decline) over 24 hours (steady-state). The concentration was determined by comparing the abundance of the desired peak to the abundance of the internal standard MS and .sup.1H- and .sup.31P-NMR (intensity of the signal). After this time the concentration of the NR decreased and the product was no longer present. The concentrations of the products and the reactants at various time points are provided in Table 3.

(27) Additionally, to establish the stability of the commercial NR product (Niagen® purchased from ChromaDex) under hydrolytic conditions, it was dissolved in deionized water as described herein and the mixture was analyzed every 3 hours using .sup.1H- and .sup.31P-NMR. The .sup.1H-NMR spectra obtained after 0.5 hours, 6 hours and 24 hours demonstrate two peaks ma at 6.15 ppm (1H,d, J=5.38 Hz) that represents the NR compound and 7.78 (1H,s) represents the DMF solvent (dimethylformamide) which was used as an internal standard in (1:1) ratio to help us following the decay of the NR peak at 0.5 hours, at 6 hours the NMR shifts and splits were the same but the intensity of the NR peak decreased compared to the DMF, and after 24 hours the peak in 6.15 ppm was not observed, signifying that NR has degraded. The data for various time points are provided in Table 4.

(28) TABLE-US-00004 TABLE 4 Time NR (Niagen ®) relative (hours) concentration 0.5 1 3 0.62 6 0.48 24 0.02 48 0

Example 4

Preparation of Silica Capsules Containing Reaction-Derived Mixture of NAD.SUP.+ and Phosphate Binding Agent

(29) Step 1 (Reaction-Derived Mixture):

(30) A 10-mL vial was loaded with 0.33 g of NAD.sup.+, 1.8 g of calcium L-ascorbate dihydrate. The powders were dissolved with 8.2 mL of distilled water and the mixture was heated at 80° C. for 24 hours.

(31) Step 2 (Capsules Formation):

(32) The resultant mixture from the step 1 was cooled to room temperature, and subsequently added to a 250-mL flask, containing a solution of 77.65 g of isononyl isononanoate (SABODERM ISN oil, manufactured by SABO S.p.A), 0.5 g ABIL EM90 (cetyl PEG/PPG-10/1 Dimethicone, manufactured by Evonik Industries AG Personal Care) and 11.85 g of tetraethoxysilane (TEOS). The mixture was homogenized by homogenizer at 10,000 rpm for 5 minutes, with Polytron PT-6100 homogenizer, manufactured by Kinematica, equipped with 20-mm PT-DA standard-type head/shaft (PT-DA 3020/2EC; product number 9115128).

(33) The obtained w/o emulsion was stirred at room temperature using magnetic stirrer for 24 hours to yield silica microcapsules. The microcapsules were then separated from the reaction mixture by centrifugation using 29×32×25-cm HSCEN-204 centrifuge, at 5000 rpm, washed 3 times with diethyl ether, and finally dried in a Memmert Beschickung loading model 100-800 oven (Memmert GmbH) at 54±0.1° C. for 24 h.

(34) SEM evaluation of the particles was conducted using high resolution scanning electron microscope (HR SEM) Sirion (FEI Company) using Shottky type field emission source and secondary electron (SE) detector. The images were scanned at voltage of 5 kV. TEM evaluation was performed at (S) Tecnai F20 G.sup.2 instrument (FEI company) operated at 200 kV.

(35) SE micrographs reveal capsules of size between 0.8-5.0 μm, of essentially spherical in shape, with mildly negative zeta potential of −25.4±13.0 mV. TE micrographs demonstrate capsules with a shell around, with some degree of roughness.

Example 5

Preparation of Silica Capsules Containing Reaction-Derived Mixture of NAD.SUP.+ and Phosphate Binding Agent, with Smaller Particle Size

(36) Step 1:

(37) The materials were used as in Example 4, and the preparation was repeated according to step 1 thereof.

(38) Step 2 (Nanocapsules Formation):

(39) The resultant mixture from the step 1 was cooled to room temperature, and subsequently added to a 250-mL flask, containing a solution of 77.65 g of isononyl isononanoate (SABODERM ISN oil, manufactured by SABO S.p.A), 0.5 g ABIL EM90 (cetyl PEG/PPG-10/1 Dimethicone, manufactured by Evonik Industries AG Personal Care) and 11.85 g of tetraethoxysilane (TEOS). The mixture was homogenized by homogenizer at 10,000 rpm for 5 minutes, with Polytron PT-6100 homogenizer, manufactured by Kinematica, equipped with 20-mm PT-DA standard-type head/shaft (PT-DA 3020/2EC; product number 9115128), and further sonicated for 30 minutes using Sonics Vibralcell VCX 130 Ultrasonic Cell Disruptor with an output of 130 W and 20 kHz.

(40) The obtained w/o emulsion was stirred at room temperature using magnetic stirrer for 24 h to yield silica capsules. The capsules were then separated from the reaction mixture by centrifugation using 29×32×25-cm HSCEN-204 centrifuge, at 5000 rpm, washed 3 times with diethyl ether, and finally dried in a Memmert Beschickung loading model 100-800 oven (Memmert GmbH) at 54±0.1° C. for 24 h.

(41) SEM evaluation of the particles was conducted using high resolution scanning electron microscope (HR SEM) Sirion (FEI Company) using Shottky type field emission source and secondary electron (SE) detector. The images were scanned at voltage of 5 kV. Particles' morphology, SEM, TEM and zeta potential were similar to the particles of the Example 4, with particle size 200-500 nm.

Example 6

Preparation of β-Nicotinamide Mononucleotide

(42) A 10-mL vial was loaded with 0.33 g of NAD.sup.+, 0.29 g of zirconium dioxide (ZrO.sub.2). The powders were dissolved/dispersed with 9.5 mL of distilled water, and the mixture was heated at 80° C. for 48 h. ZrO.sub.2 was removed by centrifugation (10,000 rpm for 30 min) using 29×32×25-cm HSCEN-204 centrifuge.

(43) The .sup.31P-NMR demonstrated a small peak at −11.15 ppm, attributed to NAD.sup.+, a peak at −0.03 ppm attributed to β-nicotinamide mononucleotide, a peak at 0.09 ppm attributed to adenosine, and two peaks at 0.027 and 0.3 ppm, attributed to the two P atoms of adenosine diphosphate (ADP).

Example 7

Direct Incorporation of an Encapsulated NR Mixture into a Cosmetic Preparation (Cream)

(44) Reaction-derived mixture was obtained as silica capsules dispersion according to the Steps 1 and 2 of the Example 4 above. The capsules were separated and were applied as described below.

(45) Separately, water, propanediol, potassium cetyl phosphate, allantoin, and glycerin were combined together at 75° C. in the main vessel of 10-L reactor, in relative amounts of 62.6:3.0:0.5:0.2:4.0, respectively. In the side vessel, the components cetearyl alcohol/cetearyl glucoside mixture (provided as Montanov 68 product), isononyl isononanoate, cyclomethicone, cetearyl ethylhexanoate, cetyl alcohol, triethanolamine, and butyrospermum parkii (shea butter) were mixed at 75° C., in relative amounts of 5.0:5.0:6.0:4.0:2.5:0.1:1.0, respectively. The components of the side reactor were introduced into the main reactor under homogenization, and further mixed for 20 minutes. The temperature was then cooled to below 50° C., and a mixture of phenoxyethanol/ethylhexylglycerin (provided as Euxil PE-9010 product), and fragrance (provided as Fragrance Rich Beauty product), in relative amounts of 1.0:0.1 parts were added, and mixed and homogenized for further 10 minutes. Finally, the temperature was lowered to below 40° C., and the encapsulated reaction-derived mixture in oil was, consisting of silica/calcium-l-ascorbate/NAD.sup.+/isononyl isononanoate/mixture of cetyl PEG/PPG-10/1 dimethicone, as described in greater detail in the example 4 above. The mixture in the vessel was slowly mixed for additional 5 minutes, and the resultant product was transferred for packaging.