JP7520731B2 - Stability of Vitamin D in β-hydroxy-β-methylbutyrate (HMB) - Google Patents

Description

This application claims priority to U.S. Provisional Patent Application No. 62/689,305, filed June 25, 2018, which is incorporated herein by reference.
1. Field
The present invention relates to compositions comprising β-hydroxy-β-methylbutyrate (HMB) and vitamin D. In particular, the present invention relates to compositions containing HMB and vitamin D, and methods for stabilizing vitamin D when combined with an acidic composition.

2. Background
Vitamin D has classically been associated with calcium and phosphorus metabolism, and bone strength. Until recently, rickets, a vitamin D deficiency disease, was used to define adequate vitamin D levels. In recent years, vitamin D has been found to have broader effects on metabolism other than calcium and bone structure. Low vitamin D status has been associated with the following: osteoporosis, falls, type I diabetes, cancer, autoimmune diseases, hypertension, periodontal disease, multiple sclerosis, susceptibility/poor response to ^infection1 .

Vitamin D deficiency is a worldwide problem, and as exposure to sunlight decreases and sunscreen use increases, the problem will only worsen. Diet and health conditions can also be mitigating factors for vitamin D levels. Subjects with fat malabsorption syndrome, chronic illness, on certain medications, or ^obesity are at higher risk for vitamin D deficiency.2 Skin tone may also have an effect, with darker skin tones taking more time to produce the same amount of vitamin D. Thus, the optimal method to increase vitamin D and decrease deficiency is ^{supplementation.2} Cholecalciferol (vitamin _D3 ) can be synthesized by humans through irradiation of 7-dehydrocholesterol in the skin or extracted from lanolin.

Very few naturally occurring foods contain vitamin D, but fatty fish meat and fish liver oil are the best sources. Small amounts of vitamin D are found in beef liver, dairy products, and each egg yolk. Some mushrooms provide vitamin _D2 , but in varying amounts. Vitamin D is found in only a few foods, and often in small amounts, so supplementation through food fortification or dietary supplements is necessary. Since 1930, food and beverage manufacturers have begun fortifying foods such as milk, bread, hot dogs, soda, and even beer. Vitamin D is also available in dietary supplements.

Vitamin _D3 is a large and hydrophobic sterol molecule, slightly water soluble, and easily degraded by light and oxygen. The stability of vitamin D3 is excellent in the absence of water, light, acidity, and at low temperatures. 5,6-trans isomers and isotaxosterol can be ^formed when exposed to acid and light. 5,6-trans isomers and isotaxosterol can be formed. ⁶ Although the vitamin is less susceptible to oxidation than vitamin A, oxidation of vitamin D can be the main route of degradation at the conjugated double bond systems at the 5,6 and 7,8 positions of the secosteroid structure. ⁷ Vitamin D is very stable in processes used for liquid milk production or in skim milk powder production. ⁸ No significant losses were observed with steam injected at 95°C ^{. 9} More recently, vitamin D was found to be stable in orange juice, which has a pH of approximately 4. 10 Bioavailability is equally available when consumed in capsule form. However, the long-term stability of vitamin D3 at pH 4 and lower has not been determined.

HMB
Alpha-ketoisocaproic acid (KIC) is the first major and active metabolic product of leucine. A minor product of KIC metabolism is β-hydroxy-β-methylbutyrate (HMB). HMB has been found to be useful in a variety of application contexts. In particular, in U.S. Pat. No. 5,360,613 (Nissen), HMB is described as useful for reducing blood levels of total cholesterol and low density lipoprotein cholesterol. In U.S. Pat. No. 5,348,979 (Nissen et al.), HMB is described as useful for promoting nitrogen retention in humans. U.S. Pat. No. 5,028,440 (Nissen) discusses the usefulness of HMB for increasing lean tissue development in animals. Also, in U.S. Pat. No. 4,992,470 (Nissen), HMB is described as effective in enhancing immune responses in mammals. U.S. Patent No. 6,031,000 (Nissen et al.) describes the use of HMB and at least one amino acid to treat disease-related wasting.

The use of HMB to inhibit protein breakdown stems from the observation that leucine has protein-sparing properties. Leucine, an essential amino acid, can either be used in protein synthesis or transaminated to an α-keto acid (α-ketoisocaproic acid, KIC). In one pathway, KIC can be oxidized to HMB, which accounts for approximately 5% of leucine oxidation. HMB is superior to leucine in promoting muscle mass and strength. Optimal effects of HMB can be achieved with 3.0 grams per day, or 0.038 g/kg of body weight per day, when given as the calcium salt of HMB, while optimal effects of leucine require more than 30.0 grams per day.

Once produced or ingested, HMB appears to have two fates. The first fate is simple excretion in the urine. After HMB is ingested, urinary concentrations increase, resulting in approximately a 20-50% loss of HMB to the urine. The other fate involves activation of HMB to HMB-CoA. Once converted to HMB-CoA, either dehydration of HMB-CoA to MC-CoA or direct conversion of HMB-CoA to HMG-CoA, which provides a substrate for intracellular cholesterol synthesis, can occur. Several studies have shown that HMB can be incorporated into the cholesterol synthesis pathway and become a source of new cell membranes to be used in the regeneration of damaged cell membranes. Human studies have shown that muscle damage after intense exercise, as measured by plasma CPK (creatine phosphokinase) elevation, is reduced with HMB supplementation within the first 48 hours. The protective effects of HMB persist for up to three weeks with continuous daily use. Many studies have shown that an effective dose of HMB is 3.0 grams per day (~38 mg kg body weight ^-1 day ^-1 ) as CaHMB (calcium HMB). HMB has been tested for safety with no side effects observed in healthy young or older adults. The combination of HMB with L-arginine and L-glutamine has also been shown to be safe when supplemented in AIDS and cancer patients.

A delivery form of HMB, HMB free acid ("HMBFA"), has been developed that has been shown to be absorbed more rapidly and have higher tissue clearance than CaHMB. This delivery form is described in U.S. Patent Publication No. 20120053240, which is incorporated herein in its entirety.

HMB has been shown to be effective in increasing muscle mass, strength, and improving muscle function. These effects are most significant when blood levels of vitamin D are sufficient, which has been further defined as at least about 25 ng/ml or higher.

Therefore, it is desirable to combine HMB with Vitamin D to maximize the effects of HMB on muscle mass, strength and function. However, it has been found that Vitamin D, and especially Vitamin D3, is not stable when combined with an acidic composition containing HMB in its free acid form (HMBFA). Vitamin D levels in compositions containing only HMBFA and Vitamin D are significantly degraded over time. Because HMBFA may be a more effective delivery form of HMB with respect to its effects on muscle, it is desirable to combine HMBFA with Vitamin D to maximize the effectiveness of HMB. Thus, there is a need to develop a stable formulation containing HMBFA and Vitamin D. To this end, it has been found that Vitamin D is stabilized in acidic formulations, including those containing HMB, by including at least one stabilizing excipient.

SUMMARY OF THEINVENTION One object of the present invention is to provide a composition containing HMBFA and vitamin D that is stable.

Another object of the present invention is to provide a composition containing HMBFA and Vitamin D, in which the Vitamin D does not decrease significantly over time.
These and other objects of the present invention will become apparent to those skilled in the art upon review of the following specification, drawings, and claims.

The present invention is intended to overcome the difficulties previously encountered. To this end, a composition is provided that includes HMBFA and vitamin D.

FIG. 1 shows the stability of Vitamin D in Formulation 7. FIG. 2 shows the stability of Vitamin D in Formulation 10. FIG. 3 shows the stability of Vitamin D in Formulation 11.

Disclosed herein are formulations for improving the stability of vitamin D when combined with HMB. It has been discovered that the inclusion of additional components that act as stabilizing excipients in formulations of HMB and vitamin D improves the stability of vitamin D. Representative examples of stabilizing excipients include, but are not limited to, EDTA, butylated hydroxytoluene (BHT), carnitine, rosemary extract, calnosolic acid, and/or sorbic acid. The inclusion of at least one of these additives in a formulation containing HMB and vitamin D minimizes the loss of vitamin D in the formulation over time. The addition of an additive to a formulation containing vitamin D in combination with an acidic component (e.g., HMBFA) stabilizes vitamin D against degradation over time.

Vitamin D is intended to include vitamins _D2 , _D3 , and _D4 and related analogues.
Stabilizing excipients included in the present invention include antioxidants, including EDTA, BHT, BHA, ascorbyl palmitate, propyl gallate, vitamin E, ascorbic acid, cysteine, methionine, monothioglycerol, sodium thiosulfate, or sulfites.

Vitamin D has been found to degrade significantly over time when included in acidic formulations. Because it is desirable to combine Vitamin D with HMBFAs in low pH acidic formulations, a need exists for a stable formulation of HMB and Vitamin D. The inclusion of stabilizing excipients, such as EDTA, BHT, carnitine, rosemary extract, carnosolic acid, and/or sorbic acid, results in a formulation in which Vitamin D is stabilized against degradation over time.

In most instances, the HMB utilized in clinical studies and commercially available as an ergogenic aid is in the calcium salt form. Recent advances have allowed HMB to be produced in the free acid form for human and animal use, and preferably for use as a dietary supplement or incorporated into food. Free acid forms of HMB (HMBFAs) have been developed that have been shown to be absorbed more rapidly than CaHMB, resulting in more rapid and higher peak serum HMB levels, and improved serum clearance into tissues. The pH of HMBFAs is <3.

HMB free acid may therefore be a more efficient method of administering HMB than the calcium salt form, especially when administered immediately prior to vigorous exercise. However, one of ordinary skill in the art will recognize that the present invention encompasses any form of HMB.

Any form of HMB may be incorporated into the delivery and/or administration form in a manner that results in a typical dosage range of about 0.5 grams HMB to about 30 grams HMB.
Any suitable dose of HMB may be used within the context of the present invention. Methods for calculating appropriate doses are well known in the art.

When the composition is administered orally in an edible form, the composition is preferably in the form of a dietary supplement, food or pharmaceutical vehicle, more preferably in the form of a dietary supplement or food. Any suitable dietary supplement or food containing the composition may be utilized within the context of the present invention. Those of ordinary skill in the art will appreciate that the composition, regardless of its form (e.g., dietary supplement, food or pharmaceutical vehicle), may contain amino acids, vitamins, proteins, peptides, carbohydrates, fats, sugars, minerals and/or trace elements.

To prepare the composition as a dietary supplement or food product, the compositions will typically be combined or mixed in a manner such that the composition is substantially uniformly distributed in the dietary supplement or food product. Alternatively, the composition may be dissolved in a liquid, for example, water.

The dietary supplement composition may be a powder, gel, liquid, or may be tableted or encapsulated, for example, in a softgel formulation. The following examples examine the stability of vitamin D when combined with HMB in a softgel or capsule delivery form. The invention is not limited to any particular delivery form, and may include combinations of HMB and vitamin D in liquid formulations, gels, and foods.

While any suitable pharmaceutical vehicle containing the composition may be utilized within the context of the present invention, preferably the composition is combined with a suitable pharmaceutical carrier, such as dextrose or sucrose.

Additionally, the pharmaceutical vehicle composition may be administered intravenously in any suitable manner. For administration via intravenous infusion, the composition is preferably in a water-soluble, non-toxic form. Intravenous administration is particularly suited to hospitalized patients undergoing intravenous (IV) therapy. For example, the composition may be dissolved in an IV solution (e.g., saline or glucose solution) that is administered to the patient. The composition may also be added to nutritional IV solutions, which may include amino acids, glucose, peptides, proteins and/or lipids. The amount of the composition to be administered intravenously may be similar to the levels used for oral administration. Oral infusion may be more controlled and precise than oral administration.

Methods for calculating the frequency of administration of the composition are well known in the art, and any suitable administration frequency may be used within the context of the present invention (e.g., a 6 g dose once daily or a 3 g dose twice daily), and for any suitable period of time (e.g., a single dose administered over a 5 minute period, or over a 1 hour period, or multiple doses administered over an extended period of time). The composition may also be administered over an extended period of time, e.g., weeks, months, or years.

Any suitable doses of HMB and Vitamin D may be used within the context of the present invention. Methods for calculating appropriate doses are well known in the art.
The term administering or administration includes the step of providing the composition to a mammal, which will consume the composition and combinations thereof.

The following examples will illustrate the invention in further detail. It will be readily understood that the compositions of the invention as generally described and exemplified in the examples herein may be synthesized in a variety of formulations and dosage forms. Thus, the following more detailed description of presently preferred embodiments of the methods, formulations and compositions of the invention are not intended to limit the scope of the invention as claimed, but are merely representative of presently preferred embodiments of the invention.

The purpose of the following experiment was to test the stability of Vitamin _D3 in HMBFA in different formulations. Less than 10% loss of Vitamin _D3 was considered acceptable.
Methods: Eleven different HMBFA/Vitamin _D3 formulations were tested. These formulations were tested for stability of Vitamin _D3 over time. The formulations were as follows:
1. HMBFA + Vitamin D3 (100HP, BASF, Denmark)
2. Vitamin D3 in HMB FA (100HP, BASF, Denmark) with Choline Chloride (Acros Organics)
3. Vitamin _D3 (100HP, BASF, Denmark) in HMB FA with betaine (Sigma-Aldrich)
4. Buffered HMBFA + Vitamin _D3 (100HP, BASF, Denmark)
5. Buffered HMBFA + BASF Protected Vitamin _D3 (100 GFP/HP, BASF, Denmark)
6. HMBFA + NovaSol D® Protected Vitamin _D3 (AquaNova®, Germany)
7. Buffered HMB free acid + NovaSol D® protected vitamin _D3 (AquaNova, Germany) stabilized with 60 ppm disodium EDTA (Versene ^™ NA chelating agent, Dow Chemical, Midland, MI).
8. HMBFA + NovaSol D® Protected Vitamin _D3 (AquaNova, Germany) stabilized with 60 ppm disodium EDTA (Versene ^™ NA chelating agent, Dow Chemical, Midland, MI).
9. Buffered HMBFA plus Vitamin _D3 (Sigma-Aldrich) stabilized with 60 ppm disodium EDTA (Versene ^™ NA chelating agent, Dow Chemical, Midland, MI).
10. Buffered HMBFA + NovaSol D® Vitamin _D3 (AquaNova, Germany) + L-carnitine stabilized with BHT (butylated hydroxytoluene, Sigma-Aldrich) 11. NovaSol Rosemary®, NovaSol DS/4®, and Buffered HMBFA + NovaSol D® Vitamin D3 (AquaNova, Germany) + L-carnitine stabilized with BHT (butylated hydroxytoluene, Sigma-Aldrich) Vitamin D3 was measured by HPLC with the Heartland assay.

Results: Of the 11 formulations tested, formulation 7 containing NovaSol D protected Vitamin D3 stabilized with 60 ppm disodium EDTA (Versene ^™ NA) plus buffered HMBFA, formulation 10 containing NovaSol D protected Vitamin D3 stabilized with BHT plus HMBFA, and formulation 11 containing NovaSol Rosemary, NovaSol DS/4, and buffered HMBFA + NovaSol D Vitamin D3 stabilized with BHT were stable. The percent change in Vitamin D3 is presented in the table below.

Table 1

Figures 1, 2, and 3 illustrate the stability of Vitamin D3 in formulations 7, 10, and 11, respectively. In conclusion, these studies demonstrate that Vitamin _D3 is stable when formulated with buffered HMBFA and disodium EDTA, or when BHT is replaced with disodium EDTA. Protective Vitamin _D3 , such as NovaSol D, is also required.

Preparation of Buffered HMB Free Acid ( _HMBFA ) Briefly, 400 g HMBFA (TSI Lot 12111036) was weighed into a 1 liter beaker and then 37.8 g potassium carbonate ( _K2CO3 ) was added to the HMBFA. A stir bar was added and the mixture was stirred for 60 hours. The resulting pH was approximately 4.5. Alternatively, the reaction may be accelerated by mixing water (35 g) with potassium carbonate (37 g) and then adding HMBFA (400 g).

The Vitamin D3 in Formulation 7 is from AquaNova and is listed as NovaSOL D. This Vitamin D3 product is protected with polysorbate 80. The product is water and fat soluble and produces a clear solution. To calculate the amount of components needed, the following was used: 250 IU Vitamin D3 (calculated 1 μg=40 IU) per capsule, or 6.25 μg based on this 0.25% NovaSOL D product, so 2.5 mg of NovaSOL D is needed per capsule.

The EDTA used in Formulations 7 and 8 was Dow's disodium EDTA (Versene ^™ NA chelating agent, Dow Chemical, Midland, Mich.). In Formulations 10 and 11, disodium EDTA was replaced with the preservative BHT. Additionally, Formulation 11 also contained the antioxidant NovaSol rosemary extract, rich in carnosolic acid, and the preservative NovaSol DS/4 (sorbic acid).

The foregoing description and figures include exemplary embodiments of the present invention. The foregoing embodiments and methods described herein may be varied based on the ability, experience, and preferences of those of ordinary skill in the art. The mere recitation of method steps in a particular order does not constitute any limitation on the order of method steps. The foregoing description and figures merely explain and illustrate the present invention, and the present invention is not limited thereto unless the claims are so limited. Those of ordinary skill in the art, having been presented with this disclosure, will be able to make modifications and variations therein without departing from the scope of the present invention.

References

Claims (5)

A stable formulation comprising beta-hydroxy-beta-methylbutyrate (HMB) in its free acid form, vitamin D and at least one stabilizing excipient, the stabilizing excipient being selected from the list consisting of EDTA, butylated hydroxytoluene (BHT), carnitine, rosemary extract, carnosolic acid, and sorbic acid. The formulation of claim 1, wherein the stabilizing excipient is selected from the list consisting of EDTA and butylated hydroxytoluene (BHT). The formulation of claim 1, wherein the formulation is contained within a delivery form selected from the list consisting of a capsule, a softgel, a liquid, and a food product. 1. A method for stabilizing vitamin D in an acidic composition comprising the step of including at least one stabilizing excipient in a formulation containing vitamin D and the free acid form of β-hydroxy-β-methylbutyrate (HMB), wherein the stabilizing excipient is selected from the list consisting of EDTA, butylated hydroxytoluene (BHT), carnitine, rosemary extract, carnosolic acid, and sorbic acid. 1. A method of minimizing degradation of vitamin D in an acidic formulation containing beta-hydroxy-beta-methylbutyrate (HMB) in its free acid form and vitamin D, comprising the step of including at least one stabilizing excipient in said formulation, wherein the stabilizing excipient is selected from the list consisting of EDTA, butylated hydroxytoluene (BHT), carnitine, rosemary extract, carnosolic acid, and sorbic acid.