FIELD

The disclosure relates to the improvement of calcium absorption and/or bone mass and the alleviation of osteoporosis using a ferrous amino acid chelate.

BACKGROUND

Bone is a metabolically active tissue composed of several types of cells, including osteoblas ts (involved in the creation and mineralization of bone tissue) and osteoclasts (involved in the breakdown of bone tissue) . When the bone synthesis rate is faster than the bone degradation rate, the volume and density of the bone would increase . However, when the bone degradation rate is faster than the bone synthesis rate, it would cause the loss of bone mineral content (bone mass), leading to osteoporosis if the condition is severe, especially in postmenopausal women who are within 5 years of menopause.

In order to increase the bone mass and alleviate osteoporosis, the conventional treatments include administering estrogenic hormone and calcium supplements (such as calcium carbonate, calcium oxide, calcium phosphate and calcium lactate gluconate) to increase the calcium intake in order to balance the loss of calcium in the bone. However, the calcium in the calcium supplements may not be absorbed effectively by the body, and the therapeutic effect thereof for osteoporosis is limited . In addition, excessive calcium supplements may increase urinary calcium level that may lead to the formation of calcium-containing kidney stones. Therefore, the applicants have endeavored to look for alternative component that can enhance calcium absorption without the aforesaid side effects, and thus can be effective for improving bone mass and/or treating osteroporosis .

The applicants' US Patent Application Publication No. 2017/0224727 A1 has disclosed a ferrous amino acid chelate, which is capable of stably passing through stomach, and which is effective in controlling body weight and enhancing lipid metabolism and lipolysis.

In addition, the ferrous amino acid chelate can also be used in the treatment of cancer and diabetes, as well as to reduce the production of lactic acid by cancer cells, as disclosed in the applicants' previous patent applications, including US Patent Application

Publication Nos. 2015/0065569 A1 and 2017/0007568 A1 and Tai anese Invention Patent Publication No.1587856. These patent applications and patent are hereby incorporated by reference in their entirety.

SUMMARY

Therefore, in a first aspect, this disclosure provides a composition for use in increasing calcium absorption, comprising a ferrous amino acid chelate which includes ferrous ions and an amino acid.

According to a second aspect, this disclosure relates to the use of the ferrous amino acid chelate for the manufacture of a composition for increasing calcium absorption .

According to a third aspect, this disclosure provides a method for increasing calcium absorption in a subj ect, which includes administering to the subject the aforesaid composition.

In addition, in a fourth aspect, this disclosure provides a composition for use in increasing bone mass, comprising the ferrous amino acid chelate.

According to a fifth aspect, this disclosure relates to the use of the ferrous amino acid chelate for the manufacture of a composition for increasing bone mass.

According to a sixth aspect, this disclosure provides a method for increasing bone mass in a subject, which includes administering to the subject the aforesaid composition.

Furthermore, in a seventh aspect, this disclosure provides a compos ition for use in treating osteoporosis, comprising the ferrous amino acid chelate.

According to an eighth aspect, this disclosure relates to the use of the ferrous amino acid chelate for the manufacture of a composition for treating osteoporosis in a subject. According to aninthaspect, this disclosure provides a method for treating osteoporosis in a subject, which includes administering to the subject the aforesaid composition .

BRIEF DESCRIPTION OF THE DRAWINGS

Other features and advantages of the disclosure will become apparent in the following detailed description of the embodiments with reference to the accompanying drawings, of which:

Figure 1 shows the determined concentration of bone- speci fi c alkaline phosphatase (BAP) in the serum of rats in each group, in which the symbols " " and "#" respectively represent p<0.05 when compared with Sham-operated group and the pathological control group; and

Figure 2 shows the determined concentration of cross-linked n- telopeptides of type 1 collagen (NTx) in the serum of rats in each group, in which the symbols " " and " # " respectively represent p< 0.05 when compared with Sham-operated group and the pathological control group.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the present disclosure belongs .

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which couldbe used in the practice of the present disclosure . Indeed, the present disclosure is in no way limited to the methods and materials described . For clarity, the following definitions are used herein.

As used herein, the term "osteoporosis" unless otherwise specified, refers to a disease of bones in which the bones become porous like a sponge as a result of excessive loss of the bone minerals and matrix and are highly apt to be fractured, and includes both osteoporosis and osteopenia as defined by the World Health organization (WHO) . The WHO has defined acceptable bone mineral density as that which falls within one standard deviation of a normal value or corresponds to a T score greater than -1. The WHO has defined osteopenia by a bone mineral density within 1 to 2.5 standard deviations of normal or that which corresponds to a T score of from -1 to -2.5. The WHO has characterized osteoporosis as a more severe form of osteopenia, and has defined it by a bone mineral density of less than 2.5 standard deviations from normal or that which corresponds to a T score of less than -2.5.

The term "treat" or "treatment" as used herein means lessening, inducing stasis of , or postponing or reducing the progression, development, onset, or severity of the disease or condition or severity of one or more symptoms associated with a disease or disorder or condition described herein, or ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, or ameliorating or preventing the underlying metabolic causes of symptoms . Thus , the terms denote that a beneficial result has been conferred on a subj ect with a disease or symptom, or with the potential to develop such disease or symptom. A response is achieved when the subject experiences partial or total alleviation, or reduction of one or more signs or symptoms of disease, condition, or illness, such as, but not limited to, reversal or prevention of bone loss, reversal or prevention of loss of bone mass, reversal or prevention of bone fracture or risk thereof, increase or prevention of decrease in bone density, increase in boner remodeling, reduction of bone resorption, and/or bone regeneration.

The present disclosure provides a composition for use in increasing calcium absorption and/or bone mass, containing a ferrous amino acid chelate. The present disclosure also provides the composition for use in treating osteoporosis. The ferrous amino acid chelate includes ferrous ions and an amino acid.

According to this disclosure, the chelating ratio of the ferrous ions to the amino acid in the ferrous amino acid chelate ranges from 1:1 to 1:4. In certain embodiments, the chelating ratio of the ferrous ions to the amino acid in the ferrous amino acid chelate ranges from 1:1.5 and 1:2.5.

The process for preparing the ferrous amino acid chelate has been disclosed in, e.g. US 2017/0224727 A1 and includes the steps of mixing a ferrous compound with an amino acid under heating. In certain embodiments, the mixing step may be conducted at a temperature ranging from 60°C to 90°C . In certain embodiments, the mixing step may be conducted for 8 hours to 48 hours.

According to the disclosure, the weight ratio of the ferrous compound and the amino acid used in the preparation process is between 1: 1.2 and 1: 1.5. In an embodiment of this disclosure, the weight ratio of the ferrous compound and the amino acid is 1:1.3.

In certain embodiments, the ferrous compound may be ferrous sulfate, ferrous chloride, ferrous pyrophosphate, or the combinations thereof.

In certain embodiments , the amino acidmay be glycine . That is, the ferrous amino acid chelate may be ferrous glycinate chelate.

This disclosure is directed to the use of the ferrous amino acid chelate for the manufacture of a composition for increasing calcium absorption and/or bone mass.

This disclosure is also directed to the use of the ferrous amino acid chelate for the manufacture of a composition for treating osteoporosis.

In certain embodiments, the composition of this disclosure may further include a calcium supplement. Examples of the calcium supplement suitable for use in this disclosure include, but are not limited to, calcium carbonate, calcium oxide, calcium dihydrogen phosphate, dicalcium phosphate, tricalcium bi s (phosphate ) , calcium lactate gluconate, calcium

L-threonate, calcium citrate , and combinations thereof In an embodiment of this disclosure, the calcium supplement may be calcium carbonate.

In certain embodiments of this disclosure, the weight ratio of the ferrous amino acid chelate to the calcium supplement in the composition may range from about 1:3 to about 1:11.

The composition according to this disclosure may be prepared in the form of a pharmaceutical composition or a food composition.

If the composition is prepared in the form of the pharmaceutical composition, the composition may further include a pharmaceutically acceptable carrier, and made into a dosage form suitable for oral administration using technology well-known to those skilled in the art. Examples of the dosage form include, but are not limited to , solution, suspension, emulsion, powder, tablet, pill, syrup, lozenge, troche, chewing gum, capsule, slurry and the like.

Examples of the pharmaceutically acceptable carrier suitable for use in this disclosure may include, but are not limited to, solvent, emulsifier, suspending agents, decomposers, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, lubricants, absorption delaying agents, liposomes, and combinations thereof.

The composition according to this disclosure may be in the form of a food additive (an exemplary example of the food composition) , which can be added into an edible material to prepare a food product for human or animal consumption. Examples of the food product according to this disclosure may include, but are not limited to, fluid milk products, e.g., milk and concentrated milk; fermented milk, e.g., yogurt, sour milk and frozen yogurt; milk powder; ice cream; cream cheeses; dry cheeses; soybean milk; fermented soybean milk; vegetable- fruit juices; fruit juices; sports drinks; confectionery; jelly; candies; health foods; animal feeds; and dietary supplements.

This disclosure also provides a method for improving calcium absorption and/or bone mass in a subject, which includes administering to the subject the aforementioned composition.

In addition, this disclosure provides a method for treating osteoporosis in a subject, which includes administering to the subject the aforementioned composition.

As used herein, the term "subject" refers to any animal of interest, such as humans , monkeys , cows, sheeps, horses, pigs, goats, dogs, cats, mice and rats. In certain embodiments, the subject may be a human. In an exemplary embodiment of this disclosure, the subject may be a perimenopausal , menopausal or postmenopausal woman.

As used herein, the term "menopausal" refers to the time from the beginning of the failure in ovarian function to the cessation of menstruation.

As used herein, the term "perimenopausal" refers to the menopause transition years in which the ovarian response to pituitary gonadotropins

(follicle-stimulating hormone [FSH] and luteinizing hormone [ LH] ) decreases, initially resulting in shorter follicular phases (thus, shorter menstrual cycles), fewer ovulations, decreased progesterone production, and more irregularity in cycles.

The term "postmenopausal" is defined to include not only women who have not experienced any menstrual flow normally, prematurely or artificially for a minimum of 12 months, as a result of decreased ovarian function, but also women who have been hysterectomized or for some other reason have suppressed estrogen production, such as those who have undergone long-term administration of corticosteroids, have gonadal dysgenesis, have undergone radiation therapy, etc.

The dosage and the frequency of administration of the composition according to this disclosure may vary depending on the following factors: the severity of the disease to be treated and the weight, age, physical condition and response of the subject to be treated. For instance, the daily dosage of the composition according to this disclosure may be 12 to 36 mg per kg of the body weight, and may be administered in a single dose or in several doses.

This disclosure will be further described by way of the fol lowing examples . However, it should be understood that the following examples are solely intended for the purpose of illustration and should not be construed as limiting the disclosure in practice.

Example

General Experimental Materials:

1. Preparation of ferrous amino acid chelate:

The ferrous amino acid chelate was prepared based on the procedure as disclosed in Preparation Example 1 of US 2017/0224727 A1. Specifically, ferrous sulfate was mixed with glycine (above 98% purity) in a weight ratio of 1:1.3, followed by heating from 60°C to 90 ^° C for 8 hours to 48 hours, so as to obtain the ferrous amino acid chelate for further experiments as described below. The chelating ratio of ferrous irons to the amino acid in the obtained ferrous amino acid chelate was between 1 : 1 and 1:4.

2. Experimental animals:

Female Sprague Dawley (SD) rats (4 weeks of age) purchased from BioLASCO Taiwan Co., Ltd., were kept in an animal room under the following laboratory conditions : a temperature of 22±3°C, a relative humidity ranging from 40% to 60%, and a 12-hour 1ight/ 12-hour dark cycle. Diet and water were provided ad libitum for all of the experimental animals.

General Experimental Procedure:

Statistical Analysis:

In the following examples, the experimental data are expressed as mean + standard deviation (SD) . All the data were analyzed using analysis of variance (ANOVA) followed by Duncan's multiple range test, so as to evaluate the differences between the groups. Statistical significance is indicated by p<0.05. Example 1

The female SD rats were randomly divided into a sham-operated group, a pathological control group, a positive control group and an experimental group (n=4/group) . After being raised in the animal room for one week, the rats in the pathological control group, the positive control group and the experimental group were subjected to ovariectomy, which was conducted according to the method described in D. D. Thompson et al . (1995) , Bone, 17 (4) : 125S-133S. The rats in the sham-operated group were subj ectedtoa similar surgical procedure, except that the ovaries of the rats in the sham-operated group were not removed. One week after the surgery, the rats (6 weeks of age) in each group were administered by oral gavage with the test material (s) at a given dosage as shown in Table 1 once a day, 7 times a week, during a 16-week period, in which the ferrous amino acid chelate was prepared as described in the section, entitled "1. Preparation of ferrous amino acid chelate" of the "General Experimental Materials".

Table 1

After administering the test material ( s ) for 16 weeks, the rats were fasted for 12 hours and then sacrificed. Subsequently, the peritoneal venous blood of each rat was collected, followed by centrifugation of the collected blood at 3000 rpm for 10 mins to obtain the serum sample for analysis of Item A listed below. In addition, the right femur of each rat was dissected, and then the connective tissue and muscle tissue were removed . Theobtainedbones ample wasusedintheanalysis of Item B listed below. A. Determination of the concentration of bone-specific alkaline phosphatase (BAP) in serum sample

The concentration of BAP, serving as an indicator of bone formation, in the serum sample of the rats in each group obtained above, were determined using Rat bone- speci fi c alkaline phosphatase (BAP) ELISA kit (Sunlong Biotech Co., Ltd) according to the protocol provided by the manufacturer . The experimental data were analyzed according to the method as described in the section, entitled "Statistical analysis", of the General Experimental Procedures.

Results :

Figure 1 shows the determined concentration of BAP in the serum of rats in each group. It can been seen from Figure 1 that, the concentration of serum BAP in the pathological control group is lower than that in the sham-operated group, indicating that the bone formation and mineralization in rats were inhibited after surgical removal of the ovaries. In addition, the concentration of serum BAP in the experimental group is higher than that in the sham-operated group, or even higher than that in the positive control group. These results indicate that the ferrous amino acid chelate of this disclosure may enhance the utilization of calcium carbonate (i.e., the calcium supplement) , thereby improving bone formation. The results also indicate that the ferrous amino acid chelate of this disclosure may be effective in treating osteoporosis.

B. Determination of morphological parameters of trabecular bones

The bone samples from all rats were imaged using a SkyScan-1076 Micro-Computed Tomography ( //—CT ) System (Skyscan, Belgium) and reconstructed using standard 3-D filtered backproj ection techniques, so as to determine the severity of osteoporosis.

For trabecular bone analysis, the pCT scanner was operated at the following conditions : an aluminum filter of 0.5 mm, a maximum scan resolution of 9 pm, x-ray voltage of 59 kV, x-ray current of 167 mA, a rotation step of 0.8 ^° and an exposure time of 540 m-seconds. The scanned area is within a region that starts approximately 3 mm away from the growth plate of the distal femur and extends approximately 3 mm to the proximal direction. The following morphological parameters were measured: trabecular number (Tb. N, 1/mm) , trabecular separation (Tb. SP, mm) and trabecular bone pattern factor (Tb. Pf, 1/mm) . The lower Tb . N value or the higher Tb . SP value means that the degree of osteoporosis is more severe. If the Tb . Pf value is positive, it could be interpreted as having osteopenia or occurrence of osteoporosis .

The experimental data were analyzed according to the method as described in the section, entitled "Statistical analysis", of the General Experimental Procedures .

Results :

Table 2 shows three morphological parameters of the bone sample in each group. Table 2

: p<0.05 when compared with the sham-operated group * : p<0.05 when compared with the pathological control group

It can be seen from Table 2 that, as compared to the sham-operated group, the rats in the pathological control group have a lower Tb . N value, a higher Tb . SP value and a positive Tb . Pf value, indicating that osteoporosis would be induced by ovariectomy. In addition, the positive control group shows a slight change in all the three parameters when compared to the pathological control group. The experimental group exhibits a significant improvement in all the three parameters as compared to the pathological control group and even to the positive control group, indicating that the combination of the ferrous amino acid chelate and calcium carbonate can be useful in alleviating osteoporosis .

Therefore, the applicants contemplate that the ferrous amino acid chelate of this disclosure is capable of assisting in calcium absorption, so as to treat osteoporosis .

Example 2

The female SD rats were randomly divided into sham-operated group, pathological control group, positive control group and experimental group (n=5/group) . The rats in each group were subjected to the same surgical procedure as described in Example 1, and then administered with the test material (s) similar to that as shown in Table 1, except that the dosage of the ferrous amino acid chelate administered to the rats of the experimental group was 12 mg/kg/day, and that the administration was carried out for 32 weeks.

After administering the test material (s) for 32 weeks, the rats were fasted for 12 hours and then sacrificed. Subsequently, the peritoneal venous blood of each rat was collected, followed by subjecting the blood to centrifugation at 3000 rpm for 10 mins to obtain the serum sample for analysis of Item A listed below. In addition, the right femur of each rat was dissected, followed by removal of the connective tissue and muscle tissue. The obtained bone sample was used in the analysis of Item B listed below. A. Determination of the concentration of cross-linked n- telopeptides of type 1 collagen (NTx) in serum sample

The concentration of NTx (a degradation product of type I collagen by osteoclast) in the serum sample of the rats in each group obtained above were determined usingRat cross-1 inked n-telopeptides of type 1 collagen ELISA kit (Sunlong Biotech Co., Ltd) according to the protocol provided by the manufacturer . The experimental data were analyzed according to the method as described in the section, entitled "Statistical analysis", of the General Experimental Procedures.

Results :

Figure 2 shows the determined concentration of NTx in the serum of rats in each group. As shown in Figure 2, the concentration of serum NTx in the pathological control group is higher than that in the sham-operated group, indicating that the ovariectomy would cause osteoporosis. In addition, the experimental group has lower concentration of serum NTx when compared to the sham-operated group, and even to the positive control group. These results indicate that the ferrous amino acid chelate of this disclosure may enhance the effect of calcium supplement in inhibiting the breakdown of bone tissues, thereby being effective in alleviating osteoporosis . B. Determination of morphological parameters of trabecular bone and femur length

The bone sample of each rat was subjected to the same morphological parameters determination as described in Item B of Example 1. In addition, the femur length of each rat was measured with a measuring scale. The experimental data were analyzed according to the method as described in the section, entitled "Statistical analysis", of the General Experimental Procedures. Results :

Table 3 shows three morphological parameters of the bone sample in each group.

Table 3

: p<0.05 when compared with the sham-operated group * : p<0.05 when compared with the pathological control group

It can be seen from Table 3 that, as compared to the sham-operated group, the rats in the pathological control group have a lower Tb . N value, a higher Tb . SP value and a positive Tb . Pf value, indicating that osteoporosis has been successfully induced by ovariectomy. In addition, the positive control group shows a slight change in all the three parameters when compared to the pathological control group. The experimental group exhibits a significant improvement in all the three parameters as compared to the pathological control group and even to the positive control group, which indicates that the combination of the ferrous amino acid chelate and calcium carbonate can be effective in mitigating osteoporosis.

Table 4 shows the femur length measured in the rats of each group.

Table 4

As shown in Table 4, the rats in the pathological control group have shorter femur compared to the rats in the sham-operated group, while the rats in the experimental group have the longest femur among all the groups, indicating that the combination of the ferrous amino acid chelate and calcium carbonate can increase the growth rate of the femur.

Based on the above results the applicants surprisingly found that the ferrous amino acid chelate of this disclosure in combination with the calcium supplement increases the serum BAP concentration, decreases the serum NTx concentration, and exhibit a significant improvement in the three morphological parameters of trabecular bone, which indicate that the ferrous amino acid chelate can effectively increase calcium absorption and bone mass, and may be useful in the treatment of osteoporosis.

All patents and literature references cited in the present specification as well as the references described therein, are hereby incorporated by reference in their entirety. In case of conflict, the present description, including definitions, will prevail.

In the description above, for the purposes of explanation, numerous specific details have been set forth in order to provide a thorough understanding of the embodiments. It will be apparent, however, to one skilled in the art, that one or more other embodiments may be practiced without some of these specific details.

It should also be appreciated that reference throughout this specification to "one embodiment," "an embodiment," an embodiment with an indication of an ordinal number and so forth means that a particular feature, structure, or characteristic may be included in the practice of the disclosure. It should be further appreciated that in the description, various features are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of various inventive aspects, and that one or more features or specific details from one embodiment may be practiced together with one or more features or specific details from another embodiment, where appropriate, in the practice of the disclosure.

While the disclosure has been described in connection with what are considered the exemplary embodiments, it is understood that this disclosure is not limited to the disclosed embodiments but is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to encompass all such modifications and equivalent arrangements.