KR20220050082A - Stability of vitamin D in β-hydroxy-βmethylbutyrate (HMB) - Google Patents

Abstract

The present invention preferably provides a composition comprising an acidic formulation comprising β-hydroxy-β-methylbutyrate (HMB) in free acid form, vitamin D and a stabilizing excipient, said vitamin D being stabilized against degradation .

Description

Stability of vitamin D in β-hydroxy-βmethylbutyrate (HMB)

This application claims priority to U.S. Provisional Patent Application No. 62/689,305, filed on June 25, 2018, which provisional application is incorporated herein by reference.

1. Field

The present invention relates to a composition comprising β-hydroxy-β-methylbutyrate (HMB) and vitamin D. In particular, the present invention relates to a composition containing HMB and vitamin D and a method for stabilizing vitamin D when vitamin D is combined with an acidic composition.

2. Background

Vitamin D is classically associated with calcium and phosphorus metabolism and bone strength. Until recently, adequate vitamin D levels were defined using rickets, a vitamin D deficiency disease. In recent years, vitamin D has been found to have a broader effect on metabolism in addition to calcium and bone structure. Low vitamin D status has been associated with osteoporosis, falls, type I diabetes, cancer, autoimmune diseases, hypertension, periodontal disease, multiple sclerosis, and susceptibility/poor response to infection ¹ .

Vitamin D deficiency is a worldwide problem, and the problem is only getting worse as sun exposure decreases and the use of sunscreen increases. Diet and health may also be mitigating factors for vitamin D levels. Subjects with fat malabsorption syndrome, chronic disease, taking certain medications, or obesity have a higher risk for vitamin D deficiency ² . Darker skin tone can also have an effect, as it takes more time to produce the same amount of vitamin D. Therefore, supplementation is the best way to increase vitamin D and reduce deficiency ² . Cholecalciferol (vitamin D ₃ ) can be synthesized by humans by irradiating the skin with 7-dehydrocholesterol, or extracted from lanolin.

Although naturally occurring foods contain very little vitamin D, 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 yolk. Some mushrooms provide vitamin D ₂ , but in varying amounts. Because vitamin D is found in very few foods and often only in small amounts, supplementation through fortification or dietary supplements is necessary. Beginning in 1930, food and beverage manufacturers began fortifying foods such as milk, bread, hot dogs, sodas and even beer. Vitamin D is also available as a dietary supplement.

Vitamin D ₃ is a large, hydrophobic sterol molecule with poor water solubility that is readily degraded by light and oxygen. The stability of vitamin D is excellent in the absence of water, light, acidity and at low temperatures ³ . 5,6-trans-isomers and isotachisterols can be formed upon exposure to acids and light ⁴ . Although vitamins are less susceptible to oxidation than vitamin A, oxidation of vitamin D may be the main pathway for degradation in conjugated double bond systems at positions 5,6 and 7,8 of the secosteroid structure ³ . Vitamin D is very stable in processes used for fluid milk production or in the production of nonfat formula ⁴ . No significant loss was observed when steam was injected at 95 °C ⁵ . More recently, vitamin D was found to be stable in orange juice with a pH of approximately 4 ⁶ . Bioavailability was equally available when ingested in capsule form. However, the long-term stability of vitamin D3 at a pH of 4 or less was not confirmed.

HMB

Alpha-ketoisocaproate (KIC) is the first major active metabolite of leucine. A side product of KIC metabolism is β-hydroxy-β-methylbutyrate (HMB). HMB has been found to be useful within the context of a variety of applications. Specifically, in US Pat. No. 5,360,613 (Nissen), HMB is described as being useful for reducing blood levels of total cholesterol and low-density lipoprotein cholesterol. In US Pat. No. 5,348,979 (Nissen et al.), HMB is described as being useful for promoting nitrogen retention in humans. U.S. Patent No. 5,028,440 (Nissen) discusses the utility of HMB for increasing dry tissue development in animals. Also in US Pat. No. 4,992,470 (Nissen), HMB is described as being effective in enhancing the immune response in mammals. U.S. Pat. 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 proteolysis stems from the observation that leucine has protein-sparing characteristics. The essential amino acid leucine can be used in protein synthesis or transaminated to α-keto acid (α-ketoisocaproate, KIC). In one pathway, KIC can be oxidized to HMB, which accounts for approximately 5% of leucine oxidation. HMB is superior to leucine in improving muscle mass and strength. The optimal effect of HMB can be achieved at 3.0 grams per day, or 0.038 g/kg body weight per day when given as the calcium salt of HMB, whereas the optimal effect of leucine requires more than 30.0 grams per day.

Once produced or ingested, HMB appears to have two fates. The first fate is simply excretion in urine. After HMB is supplied, the urine concentration increases, resulting in approximately 20-50% loss of HMB into the urine. Another fate is related to the activation of HMB to HMB-CoA. Upon conversion to HMB-CoA, further metabolism can occur with dehydration of HMB-CoA to MC-CoA or direct conversion of HMB-CoA to HMG-CoA, which provides a substrate for intracellular cholesterol synthesis. Several studies have shown that HMB can be integrated into the cholesterol synthesis pathway and become a source of new cell membranes used to regenerate damaged cell membranes. Human studies have shown that muscle damage after strenuous exercise, as measured by elevated plasma CPK (creatine phosphokinase), is reduced with HMB supplementation within the first 48 hours. The protective effect of HMB lasts up to 3 weeks with continued daily use. Numerous studies have shown that the effective dose of HMB is 3.0 grams per day as CaHMB (calcium HMB) (about 38 mg kg body weight ^-1 ·day ^-1 ). HMB has been tested for safety and has been shown to be free from side effects in healthy young adults or the elderly. HMB in combination with L-arginine and L-glutamine has also been shown to be safe when supplemented in patients with AIDS and cancer.

A delivery form of HMB, HMB free acid (“HMBFA”) has been developed. This form of delivery has been shown to be absorbed more rapidly and has greater tissue clearance than CaHMB. Such delivery forms are described in US Patent Publication Ser. No. 20120053240, which is incorporated herein by reference in its entirety.

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

Therefore, it is desirable to combine HMB with vitamin D to maximize the effects of HMB on muscle mass, strength and muscle function. However, it has been found that vitamin D, particularly vitamin D3, is not stable when combined with acidic compositions comprising the free acid form of HMB (HMBFA). The vitamin D level of the composition comprising only HMBFA and vitamin D decreases significantly over time. Since HMBFA may be a more effective form of HMB delivery in terms of effects on muscles, it is desirable to combine HMBFA with vitamin D to maximize the efficacy of HMB. Therefore, there is a need for the development of stable formulations containing HMBFA and vitamin D. To this end, it has been found that vitamin D is stabilized against degradation in acidic formulations, including formulations containing HMB comprising at least one stabilizing excipient.

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

Another object of the present invention is to provide a composition containing HMBFA and vitamin D, wherein 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 reference to the following specification, drawings and claims.

The present invention seeks to overcome the difficulties encountered hitherto. To this end, a composition comprising HMBFA and vitamin D is provided.

1 shows the stability of vitamin D of formula 7.
Figure 2 shows the stability of vitamin D of formula 10.
Figure 3 shows the stability of vitamin D of formula 11.

Formulations for improving the stability of vitamin D when it is combined with HMB are disclosed herein. It has been found that the inclusion of HMB and additional ingredients acting as stabilizing excipients in the formulation of 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, carnosolic acid and/or sorbic acid. Inclusion of at least one of these additives in a formulation containing HMB and vitamin D results in minimal loss of vitamin D in the formulation over time. Addition of additives to formulations containing vitamin D in combination with an acidic component (eg, HMBFA) stabilizes vitamin D against degradation over time.

Vitamin D is intended to include vitamins D ₂ , D ₃ and D ₄ and related analogs.

Stabilizing excipients encompassed by the present invention are EDTA, or antioxidants including BHT, BHA, ascorbyl palmitate, propyl gallate, vitamin E, ascorbic acid, cysteine, methionine, monothioglycerol, sodium thiosulfate or sulfite. includes the

It has been found that vitamin D degrades significantly over time when vitamin D is included in the acidic formulation. Since it is desirable to combine vitamin D and HMBFA in an acidic formulation at low pH, there is a need for a stable formulation of HMB and vitamin D. It has been unexpectedly discovered that 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, HMB used in clinical studies and marketed as an ergogenic aid was in the form of a calcium salt. Recent developments have allowed HMB to be prepared in free acid form for human and animal use, preferably as a nutritional supplement or to be incorporated into food. The free acid form of HMB (HMBFA) has been developed and has been shown to be absorbed more rapidly than CaHMB, resulting in faster and higher peak serum HMB levels and improved serum clearance of tissues. The pH of HMBFA is less than 3.

Thus, HMB free acid may be a more effective way to administer HMB than the calcium salt form, especially when administered immediately prior to intense exercise. However, one of ordinary skill in the art will recognize that the present invention encompasses any form of HMB.

HMB in any form may be incorporated into delivery and/or dosage forms in such a way as to produce a typical dosage range of from about 0.5 gram HMB to about 30 gram HMB.

Any suitable dose of HMB may be used within the context of the present invention. Methods for calculating an appropriate dose are well known in the art.

When the composition is administered orally in edible form, the composition is preferably in the form of a dietary supplement, food or pharmaceutical medium, more preferably in the form of a dietary supplement or food. Any suitable dietary supplement or food product comprising the composition may be used within the context of the present invention. Those skilled in the art will understand that the composition may include amino acids, vitamins, proteins, peptides, carbohydrates, fats, sugars, minerals and/or trace elements, regardless of form (eg, dietary supplement, food or pharmaceutical medium). .

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

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

Although any suitable pharmaceutical medium comprising the composition can be used within the context of the present invention, preferably, the composition is combined with a suitable pharmaceutical carrier such as dextrose or sucrose.

Moreover, the composition of the pharmaceutical medium may be administered intravenously in any suitable manner. For administration via intravenous infusion, the composition is preferably in an aqueous, non-toxic form. Intravenous administration is particularly suitable for inpatients receiving intravenous (IV) therapy. For example, the composition may be dissolved in an IV solution (eg, saline or glucose solution) administered to a patient. The composition may also be added to a nutritional IV solution which may include amino acids, glucose, peptides, proteins and/or lipids. The amount of the composition administered intravenously may be similar to the level used for oral administration. Intravenous infusion may be more controlled and more precise than oral administration.

Methods for calculating the frequency at which a composition is administered are well known in the art, and include any suitable period of time (eg, a single dose may be administered over a period of five minutes or a period of one hour, or alternatively multiple Any suitable frequency of administration over an extended period of time (e.g., a 6 g dose once a day or a 3 g dose twice a day) can be used in the context of the present invention. . The composition may be administered over an extended period of time, such as weeks, months, or years.

Any suitable dose of HMB and vitamin D may be used within the context of the present invention. Methods for calculating an appropriate dose are well known in the art.

The term administering or administering includes providing the composition to a mammal and consuming the composition and combinations thereof.

Experiment example

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

The purpose of the experiment below was to test the stability of vitamin D ₃ in different formulations of HMBFA. A loss of less than 10% vitamin D ₃ was considered acceptable.

Methods: Eleven different HMBFA/vitamin D ₃ formulations were tested. These formulas were tested for the stability of vitamin D ₃ over time. The formula was:

1. HMBFA + Vitamin D3 (100 HP, BASF, Denmark)

2. Vitamin D3 in HMB FA + Choline Chloride (Acros Organics) (100 HP, BASF, Denmark)

3. Vitamin D ₃ in HMB FA + Betaine (Sigma-Aldrich) (100 HP, BASF, Denmark)

4. Buffered HMBFA + Vitamin D ₃ (100 HP, BASF, Denmark)

5. Buffered HMBFA + BASF Protected Vitamin D3 (100GFP/HP, BASF, Denmark)

6. HMBFA + NovaSol D®, protective vitamin D ₃ (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 ® Vitamin D ₃ (AquaNova, Germany) stabilized with 60 ppm disodium EDTA (Versene™ NA Chelating Agent, Dow Chemical, Midland, MI)

9. Buffered HMBFA + Vitamin D ₃ (Sigma-Aldrich) stabilized with 60 ppm disodium EDTA (Versene™ NA Chelating Agent, Dow Chemical, Midland, MI)

10. Buffered HMBFA + NovaSol D® Vitamin D ₃ (AquaNova, Germany) + L-Carnitine stabilized with BHT (Butylated Hydroxytoluene, Sigma-Aldrich)

11. Buffered HMBFA+ NovaSol D ® Vitamin D3 (AquaNova, Germany) + L-Carnitine stabilized with NovaSol Rosemary®, NovaSol DS/4® and BHT (butylated hydroxytoluene, Sigma-Aldrich)

Vitamin D3 was determined by HPLC by Heartland assay.

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

Table 1

1, 2 and 3 illustrate the stability of vitamin D3 in formulas 7, 10 and 11, respectively. In conclusion, these studies demonstrate that vitamin D ₃ is stable when formulated with buffered HMBFA and disodium EDTA or when BHT displaces disodium EDTA. Protective vitamin D ₃ such as NovaSol D is also needed.

Preparation of buffered HMB (HMBFA) in free acid form

Briefly, 400 g HMBFA (TSI lot 12111036) was weighed into a 1 liter beaker, and then 37.8 g potassium carbonate (K ₂ CO ₃ ) was added to the HMBFA. A stir bar was added and the mixture stirred for 60 hours. The resulting pH was approximately 4.5. Alternatively, after mixing water (35 g) and potassium carbonate (37 g), HMBFA (400 g) can be added to accelerate the reaction.

Vitamin D3 in Formula 7 is derived from AquaNova and is described as NovaSOL D. These vitamin D3 products are protected with polysorbate 80. The product is soluble in water and oil and produces a clear solution. To calculate the amount of ingredient required, the following was used: to use 250 IU vitamin D3 per capsule (calculated 1 μg = 40 IU), 2.5 mg per capsule at 6.25 μg based on this 0.25% NovaSOL D product. It means NovaSOL D is required.

The EDTA used in formulas 7 and 8 was Dow's disodium EDTA (Versene™ NA Chelating Agent, Dow Chemical, Midland, MI). In formulas 10 and 11, disodium EDTA was replaced with the preservative BHT. In addition, Formula 11 also contained the antioxidant NovaSol Rosemary Extract high in carnosolic acid and the preservative NovaSol DS/4 (sorbic acid).

The above description and drawings contain exemplary embodiments of the present invention. The embodiments and methods described herein can be varied based on the abilities, experience, and preferences of one of ordinary skill in the art. However, listing the steps of the method in a particular order does not place any limitation on the order of the steps of the method. The foregoing description and drawings merely describe and illustrate the present invention, which is not limited thereto, but only by the claims. Those skilled in the art, having read the disclosure of the present invention in advance, will be able to make changes and modifications herein without departing from the scope of the present invention.

references

Claims (10)

A stable formulation comprising β-hydroxy-β-methylbutyrate (HMB) in its free acid form, vitamin D and at least one stabilizing excipient. The formulation of claim 1 , wherein the stabilizing excipient is 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 in a delivery form selected from the list consisting of capsules, softgels, liquids and food products. A method of stabilizing vitamin D in an acidic formulation comprising the step of including at least one stabilizing excipient in the formulation comprising vitamin D and β-hydroxy-β-methylbutyrate (HMB). 6. The method of claim 5, wherein the HMB is in the free acid form. 6. The method of claim 5, wherein the stabilizing excipient is selected from the list consisting of EDTA, butylated hydroxytoluene (BHT), carnitine, rosemary extract, carnosolic acid and sorbic acid. A method of minimizing the degradation of vitamin D in an acidic formulation comprising β-hydroxy-β-methylbutyrate (HMB) and vitamin D, comprising the step of including at least one stabilizing excipient in said formulation. 9. The method of claim 8, wherein the HMB is in the free acid form. 9. The method of claim 8, wherein the stabilizing excipient is selected from the list consisting of EDTA, butylated hydroxytoluene (BHT), carnitine, rosemary extract, carnosolic acid and sorbic acid.

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