AU2012288401B9 - Formulas comprising highly soluble elements and vitamin for the prevention and amelioration of osteoporosis - Google Patents

Description

WO 2013/014654 PCT/IB2012/053872 FORMULAS COMPRISING HIGHLY SOLUBLE ELEMENTS AND VITAMIN FOR THE PREVENTION AND AMELIORATION OF OSTEOPOROSIS 100011 This application claims the benefit of priority of Taiwanese Application No. 100126601, filed July 27, 2011, and U.S. Application No. 61/512,685 filed July 28, 2011. The entire contents and disclosures of the preceding applications are incorporated by reference into this application. 100021 Throughout this application, various references are referred to and disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. BACKGROUND OF THE INVENTION 100031 Calcium is the major element in bones with over 99% of the body's calcium existing in bone. Adequate intake of calcium from the diet is necessary for bone growth and maintenance. Osteoporosis is a disease caused by a significant loss of bone mass leading to increased susceptibility to fracture, most often occurring in women age 35 or above, but more frequently, occurring in postmenopausal women (1, 2). [00041 Dietary supplements with calcium were thought to be primary to maintaining bone health in the past 50 years (3). However, the benefit of increased overall calcium consumption on bone health has not been clearly demonstrated, and there are conflicting reports in the literature on its effectiveness. In 43 studies of calcium supplementation reviewed by Heaney published between 1988 and 1993, 16 of the 19 placebo-controlled studies in which calcium intake was controlled showed that the mineral prevented or slowed bone loss, but 16 studies showed that calcium had no effect on bone loss (4, 5). 100051 In the 12 studies that excluded women who were within 5 years of menopause, a period when estrogen deficiency overwhelms the effect of calcium supplementation (6), all showed that calcium had a significant beneficial effect. 100061 In elderly women, it was shown that there was a significant relationship between bone mineral density (BMD) and several critical nutrients: energy, protein, calcium, magnesium, zinc and vitamin C (1). It has also, however, been found that high levels of calcium intake may be linked to higher incidence of cardiovascular disease (3). 1 WO 2013/014654 PCT/IB2012/053872 100071 Since total calcium intake has not shown to be conclusive with respect to bone health, other factors have also been taken into account, such as the calcium to magnesium ratio in modern diets, and in supplement form. The ratio of Ca/Mg in the modern diet increased from 2/1 in the first 40 years of the 1900s to > 3/1 in the 1960s, to > 6/1 in the year 2000. The daily recommended intake (DRi) in the year 2000 of Ca/Mg was > 3/1 to > 4/1. This change correlates with negative consequences with respect to bone health as well as an increased risk of cardiovascular disease. 100081 It should be noted that the increase in Ca/Mg is mainly due to the increase in calcium intake, not a change in magnesium. In the early 2000s, daily calcium intake reached a new high of 2,500 mg (3). The daily requirement of calcium was recently re-evaluated (7). It was found that an average intake of 749 mg of calcium is required, an estimate lower than previously estimated. 100091 Supporting the thesis that Ca/Mg ratio, among other factors, is a more important factor in bone maintenance and health than absolute calcium consumption, is one clinical trial, wherein 43 early postmenopausal women were randomly assigned to one treatment group for administration of the following: percutaneous estradiol, oral calcium (2000 mg/day) or placebo. Bone mineral content in the forearm, the entire body and spine remained the same in the estradiol group' however, there was a decline in the calcium and placebo groups. Calcium did not show any significant effect and calcium supplementation may have a minor effect on the loss of cortical bone, but it had no effect on the trabecular bone (6). 100101 In a National Health and Nutritional Examination Survey (NHANES) conducted from 1988 to 1994, predictive models were established to evaluate parameters such as race, body composition, exercise, alcohol intake, smoking status and nutritional intake (8). The nutritional intake analysis included study of elements such as calcium, phosphorus, magnesium, iron, zinc, sodium and potassium. Among the 7,532 women in the study who were 20 years or older, elemental intake was not a predictor of osteoporosis. However, the average calcium intake was 659 mg and magnesium was 241 ig - lower than that of the RIDA of 1000 and 310 mg, respectively. 100111 Physical activity was associated with increase in vertebral bone mineral density (9). When activity was removed, vertebral bone mineral density was dependent on calcium intake. The relationship disappeared when calcium intake exceeded 800 to 1000 mg/day. A ceiling effect of calcium was also observed by Celotti and Bignamini (10). They reported that calcium 2 WO 2013/014654 PCT/IB2012/053872 supplementation is important for maintaining bone health. However, an excessive amount of calcium may be useless and could cause hypercalciuria and kidney stones. Supplementation with a small amount of magnesium was suggested. 100121 Other studies show not just the importance of the ratio of Ca/Mg consumed or administered, but the importance of optimizing zinc levels. Mutlu et al. (11) showed that magnesium and zinc levels are the lowest in postmenopausal women, lower than postmenopausal women with osteopenia, and lower than postmenopausal women with normal bone density. Calcium supplementation may reduce zinc absorption, and magnesium and zine retention. Consequently, calcium supplementation in the absence of the administration of other optimized amounts of minerals may further aggravate the severity of osteoporosis (2, 12, 13). Apart from calcium, magnesium, zinc, manganese and copper deficiencies are linked to osteoporosis (14). 100131 Angus et al. (15) showed that calcium was not a predictor of bone mineral density in pre and post-menopausal women. Magniesium and iron were, however, predictors of bone mineral density. In this study, however, the test subjects ingested less than the recommended amounts of elements, About 29% of the post-menopausal women consumed less than 500 mg of calcium per day (16), while other nutrients such as magnesium, etc. were also deficient. [00141 A study emphasizing the benefit of magnesium on postmenopausal women found that a Mg/Ca ratio of 1.2/1 was more effective at maintaining bone health than that of a ratio of 0.4/1 (17). The study used 500 mg of calcium in the form of calcium citrate and 200 mg of magnesium in the form of magnesium oxide for the 0.4/1 group and 600 mg of magnesium in the form of magnesium oxide in the 1.2/1 group. The study showed that women on the 1.2/1 diet for 6 to 12 months had an average of an I 1% increase in bone mineral density, whereas, the other group had a non significant increase of 0,7%. [00151 Although bone health is dependent on a variety of factors, there is enough evidence to show that, in the area of elemental requirements, apart from calcium, other elements such as magnesium, phosphorus, zinc, copper, etc. are also important for maintaining or improving bone health. Further, due to differences in bioavailability, it is proposed that elemental salts would be more accurately characterized in terms of absorbability, and that calcium formulas be optimized through the use of preferred salts. 3 WO 2013/014654 PCT/IB2012/053872 100161 The selection of appropriate salts for optimized formulations has not received appropriate attention because of reports showing that solubility of calcium salts is not related to the element's bioavailability. The absorption of calcium salt, soluble or insoluble, is not affected by gastric acid secretion (18). The hypothesis that calcium carbonate can be converted to a more soluble calcium salt in the stomach, namely calcium chloride, thus enhancing calcium absorption, has been tested. The results showed that calcium carbonate absorption is not influenced by gastric acid (18). The average amount absorbed in humans is 24%. 100171 The bioavailability of calcium carbonate, D-calcium lactate, L-calcium lactate and oyster shell calcium was found to be independent of the salt's solubility (19). This study used a method which was different from that of the balance study. It measured changes in the pituitary thyroid hormone (PTH), etc. instead of actual calcium absorption. However, indirect methods of measurement, such as PTI-1, do not provide truly accurate comparisons of calcium bioavailability, [00181 Using Ca 4 as a tracer, fractional absorption values of calcium carbonate and calcium citrate were found to be insignificantly different from each other at a low dose (300 mug calcium); however, calcium absorption from calcium carbonate was slightly but significantly better than calcium citrate (20). leaney (21) reported that the rates of urinary excretion for three marketed calcium products (marketed calcium carbonate, encapsulated calcium carbonate and marketed calcium citrate) were identical. 100191 Despite these observations, there are reports showing that not all calcium salts have the same bioavailability. Bioavailability of calcium ascorbate is higher than that of calcium carbonate and calcium chloride (22). [00201 Bioavailability of calcium acetate was measured using 4 Ca (23). Compared to calcium ascorbate, bioavailability of calcium acetate was significantly lower (70% vs 45% at 25 mg calcium load). A kinetic model consisting of 8 compartments was used to fit the plasma calcium vs. time data. The difference was attributed to a saturable process. It is also reasoned that the solubility of calcium acetate may be reduced in the intestine because calcium from the acetate salt may precipitate phosphate or chloride ions in the intestine. Therefore, it is not surprising that the bioavailability of calcium acetate is not different fiom that of calcium chloride and calcium phosphate. 4 WO 2013/014654 PCT/IB2012/053872 100211 Magnesium absorption from 10 organic and inorganic salts was tested in rats (24). The bioavailability of magnesium ranged from 50 to 66'%, Magnesium gluconate provided the highest value. The solubility of these salts in the small and large intestine and cecum was also measured. Solubility of these salts was quite high at the proximal section of the intestine; it dropped off very quickly as pH increased along the intestinal tract. Differences in absorption of these magnesium salts may not be important considering the variability among individuals. [00221 Zinc absorption occurs throughout the small intestine and it is dose dependent in humans (25). With respect to zinc, there was no difference in the bioavailability of zinc oxide and zinc sulfate as measured using dual isotope techniques (26); both were at approximately 24%. The bioavailability of iron was 15.9%. However, zinc sulfate tended to reduce the bioavailability of iron to 11.5% and this number is significant. Eight to 11 mg of zinc per day is the recommended intake (http ://ods.od.nih ,gov/factsheets/Zinc-THealthProfessional/). The recommended daily allowance of zinc was 6 mg (27). [00231 The following are inventions and disclosures noteworthy in the art: 100241 U.S. Patent 5,879,698 issued in 1999 for a calcium dietary supplement comprising calcium, magnesium, zinc, etc. (28). The calcium to magnesium ratio is high and the range of magnesium used was between 50 to 150 mug. The salt for calcium is calcium carbonate. The quantity of calcium and magnesium used and the type of salts employed are different from the present invention. [00251 U.S. Patent 6,716,454, awarded to Meignant and Stenger in 2004, cites a composition which consists of calcium and a vitamin D mixture. 100261 U.S. Patent 6,790,462, awarded to Hendricks in 2004, describes a dietary supplement containing calcium and phosphorus. Vitamins including vitamin D could also be included in the supplement. Hendricks emphasized the effects of phosphorus, and optionally vitamins BI, folate and Vitamin B 6 , The present application, however, does not include phosphorus. 100271 Mazer et al. were granted U.S. Patent 5,698,222 in 1997 on a calcium supplement in solid form which contains calcium glycerophosphate, vitamin D and vitamin C. The present invention does not contain calcium salt of this kind. 5 WO 2013/014654 PCT/IB2012/053872 100281 In another patent, U.S. Patent 5,075,499, issued in 1991, Walsdorf et al. described the synthesis of dicalcium citrate-lactate by mtixing stoichiornetric mixtures of citrate and lactate salts to produce the calcium salt (29). 100291 Krurhar and Johnson designed a diet supplement for bone health, disclosed in U.S. 7,029,703 which issued in 2006, consisting of microcrystalline calcium hydroxyapatite, protein (mostly collagen), phosphorus, fat, and other minerals. It also contains vitamin D 3 , cholecalciferol, and a preferred osteoblast stimulant, ipriflavone. In addition to these basic ingredients, the composition can further include various other minerals known to occur in bone, vitamin C, and glucosamine sulfate, all of which have been claimed to have beneficial effects on the growth and maintenance of healthy bone. [00301 Sultenfuss, in U.S. Patent 5,5 14,382, issued in 1996, described another daily vitamin and mineral supplement for women comprising vitamin A, beta-carotene, niacin, riboflavin, pantotheriic acid, pyridoxine, cyanocobalamin, biotin, para-aminobenzoic acid, inositol, choline, vitamin C, vitamin D, vitamin E, vitamin K, boron, calcium, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, selenium, zinc and bioflavonoid. For women up to 40 years of age, iron is included. For women over 40 years of age, iron is optionally included. The Ca/Mg ratio is in a range of 10-1 5/4-6. [00311 A dietary supplement consisting of an extensive list of minerals and vitamins was described in U.S. Patent 5,654,0 11 (30). The patent sets forth no quantitative description on the contribution of each component to bone health. SUMMARY OF THE INVENTION 100321 The present invention describes formulations of a dietary supplement comprising acetate salts of calcium, magnesium, zinc and vitamin D 3 . These preparations are highly soluble in water, gastric and intestinal fluids. DETAILED DESCRIPTION OF THE FIGURES [00331 Figure 1 shows mean (±S.D.) percentage-time profiles of calcium of various formiulas in artificial gastric juice (U SP). [00341 Figure 2 shows the average cumulative net amount of calcium retained (±S.EM.) in rats receiving calcium free diet over a four day period. 6 WO 2013/014654 PCT/IB2012/053872 100351 Figure 3 shows the cumulative net amount of magnesium retained (+S.E.M.) in rats receiving calcium free diet over a four day period. [00361 Figure 4 shows the cumulative net amount of zinc retained (±S.EM.) in rats receiving calcium free diet over a four day period. 100371 Figure 5 shows the plasma calcium (A), magnesium (B) and zinc (C) levels sampled from rats at the end of the treatment period while receiving calcium free diet. [00381 Figure 6 shows the average cumulative net amount of calcium retained (iS.E.M.) in rats receiving normal diet over a four day period. 100391 Figure 7 shows the average cumulative net amount of magnesium retained (±S.E.M.) in rats receiving normal diet over a four day period. 100401 Figure 8 shows the average cumulative net amount of zinc retained (±S.EM.) in rats receiving normal diet over a four day period. 100411 Figure 9 shows the plasma calcium (A), magnesium (B) and zinc (C) levels sampled from rats at the end of the treatment period while receiving normal diet. 100421 Figure 10 shows the cumulative net amount of calcium retained (±S.EM,) in rats receiving calcium free diet plus a daily consumed dose of calcium over a four day period. 100431 Figure 11 shows the cumulative net amount of magnesium retained (±S.M.) in rats receiving calcium free diet plus a daily consumed dose of calcium over a four day period. 100441 Figure 12 shows the cumulative net amount of zinc retained (iS.E.M.) in rats receiving calcium free diet plus a daily consumed dose of calcium over a four day period. 100451 Figure 13 shows the plasma calcium (A), magnesium (B) and zinc (C) levels sampled from rats at the end of the treatment period while receiving calcium free diet and a normal daily dose of calcium, 100461 Figure 14 is the body mass record of rats which received individual elemental treatments. 7 WO 2013/014654 PCT/IB2012/053872 100471 Figure 15 shows trabecular BMD of Distal Femur Averaged from 3 pQCT Slices. significantly different from OVX-control (p<0.05). 100481 Figure 16 shows trabecular BMD of Proximal Tibia Averaged from 3 pQCT Slices. *: significantly different from OVX-control (p<0.05). DETAILED DESCRIPTION OF THE INVENTION [00491 In general, soluble calcium salts have a lower percentage of calcium. For example, calcium ascorbate has only 9% of calcium. The content is several folds lower than that of the insoluble calcium carbonate (40% calcium). Among soluble calcium, calcium acetate has the highest calcium content (25% calcium), This makes calcium acetate a suitable candidate for making a solid dosage. 100501 The addition of elements and vitamins to a formula lowers the percentage of calcium. This poses a severe challenge to prepare a dosage form that has an acceptable size to consumers. [00511 The present invention describes methodologies for preparing dosage forms with acceptable sizes. [00521 The present invention also provides a method of preparing tablets comprising calcium acetate, magnesium acetate, zinc acetate and vitamin D 3 , comprising the steps of: (i) blending a calcium composition comprising calciui acetate, magnesium acetate, and zinc acetate with a composition comprising vitamin D 3 ; and (ii) blending the composition obtained from (i) with a caIcium composition comprising calcium acetate, magnesium acetate, and zinc acetate, thereby obtaining tablets comprising calcium acetate, magnesium acetate, zinc acetate and vitamin D3. In one embodiment, the calcium composition comprises at least 10 percent by weight of calcium acetate, at least 5 percent by weight of magnesium acetate, and at least 0.2 percent by weight of zinc acetate, [00531 The present invention also provides a tablet produced by the method described above. 100541 The present invention also provides a method of preparing soft gel capsules comprising calcium acetate, magnesium acetate, zinc acetate and vitamin Di, comprising the steps of: (i) dissolving vitamin D 3 in fish oil, flaxseed oil, or other oils containing either omega 3 or omega 3-6 9; (ii) mixing the composition obtained from (i) with a calcium composition comprising calcium 8 WO 2013/014654 PCT/IB2012/053872 acetate, magnesium acetate, and zinc acetate, thereby obtaining soft gel capsules comprising calcium acetate, magnesium acetate, zinc acetate. In one embodiment, the calcium composition comprises at least 10 percent by weight of calcium acetate, at least 5 percent by weight of magnesium acetate, and at least 0.2 percent by weight of zinc acetate. [00551 The invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative, and are not meant to limit the invention as described herein, which is defined by the claims which follow thereafter. [00561 Throughout this application, various references or publications are cited, Disclosures of these references or publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. It is to be noted that the transitional term "comprising", which is synonymous with "including", "containing" or "characterized by", is inclusive or open-ended and does not exclude additional, un recited elements or method steps. EXAMPLE I Formulations of Pearl Extracts [00571 A pearl extract was prepared by adapting the patented method reported by Li and Li (31). Briefly, pearls are pulverized to a size between 80 to 120 mesh. The powder is soaked in a mixture of saturated sodium chloride solution with titrated amount of acetic acid, Electrical current is applied to the mixture for several days. After dilution with water and magnetization, the mixture was filtered and precipitated. The precipitate, rich in calcium acetate, is dried and ready for consumption as a dietary supplement. A detailed list of elements present in the extract is presented on Table 1: TABLE I Content of Pearl Extract Element Quantity, ppm Calcium 233,000 Magnesium 253 Zine 3281 Potassium 1650 Manganese 1170 9 WO 2013/014654 PCT/IB2012/053872 Sodium 680 Strontiun 158 Molybdenum 55.4 Silicon 38.0 ISelenium 27.9 10058] This extract, Al, is fortified with acetate salts of magnesium to provide Ca/Mg ratios of 0.5/1 (A6), 1/1 (A4) and 2/1 (A5). The major elemental content of the pearl extract and its fortified mixtures are listed on Table 2: TABLE 2 The Content of Each Element in Each Formula (n=3) The conite nt of three elements in each Foritiua Fornmla No Ca (%) Mg (%) Zn (%) Determined Labeled Deermined Labeled Deerined Labeled content content content Al 23,3021,26 23.4 0.0253i0 0013 0.0012)*** 0.328±0.03 0.330 A4 765±0,62 7.51 7.56±0.32 7.50 0.372±0.029 0.375 AS 11.5±0.34 11.3 5.41i0.04 5.64 0.556±0.044 0.565 A6 4,58±0,09 4.50 8.290.15 8.99 0.256±0.012 0,225 Data are expressed as meaniS.D. aIn-house Data. ***p<0,001 100591 Besides Pearl, the method described in this example can also be used to extract multiple acetate salts of calcium, magnesium and zinc from natural sources such as corals, oysters, mineral mines, etc. The composition of formulas Al, A4 through A6 could also be achieved by mixing appropriate amounts of acetates salts of calcium, magnesium and zinc. 100601 Experimental Data on Elemental Solubility. The gastrointestinal tract is a complex organ. There are a number of factors which could alter the solubility of elements including calcium, magnesium and zinc; subsequently, their rate of absorption and bioavailability. Examples 2-5 highlight some of the physiological factors which have been postulated to have a significant impact on the solubility of elements. EXAMPLE 2 Solubility of Calcium In Artificial Gastric and Intestinal Juice 10 WO 2013/014654 PCT/IB2012/053872 100611 The solubility of calcium in the four formulas in an artificial gastric (pH = 1) and intestinal fluid (p- ::::7) was tested using a method developed for ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometer) (PerkinElmer Optima 4300DV). Two commercial samples, Caltrate TM and calcium acetate, were also tested in parallel for comparison, The results are shown in Table 3. [00621 Compared to Caltrate , the solubility of calcium acetate is approximately 45 times higher in the artificial gastric juice and 26,000 times higher in the artificial intestinal juice. The solubility of the pearl extract, Al, comprising mostly calcium acetate, is similar to that of calcium acetate in the artificial gastric juice and intestinal juice (p>0.05). The solubility of calcium acetate is pH dependent; it is lower in the artificial intestinal fluid when compared to the artificial gastric juice. Magnesium has a tendency to lower the solubility of calcium. When the ratio of Ca/Mg decreases, the solubility of the extract decreases, A5 > A4 > A6. Nevertheless, A6, the least soluble pearl extract formula, is ~12 times more soluble in artificial gastric juice and 8,500 times more soluble in artificial intestinal juice than that of CaltrateT . Therefore, unlike CaltrateT', solubility of acetate salts should not be an issue in gastrointestinal tract fluids because the acetate salts will still be in solution, [00631 The solubility profile of magnesium salts is very similar to that of calcium (Table 4). In general, acetate salts of magnesium are highly soluble. They are more soluble in artificial gastric juice than artificial intestinal juice. In contrast to magnesium acetate, the solubility of magnesium carbonate in CaltrateTM is low, [00641 The solubility profile of zinc salts is also similar to that of magnesium and calcium, except the magnitude of difference between salt forms under differing p-I and environmental conditions is less drastic (Table 5). [00651 This set of experiments thus leads to the conclusion that acetate salts are preferred salts in the disclosed formulations for their high solubility. TABLE 3 Saturated Solubility of Calcium in Artificial Gastric And Intestinal Fluid (n=3) saturated solubili tyofcalciumT______ Formula No. Gastric fluid (g/L) Intestinal fluid (g/L) A1 72.93±4 14 64.97i6.29 A4 33.60±118 29.90+214 11 WO 2013/014654 PCT/IB2012/053872 A5 53,97 8,34 45.50±7.24 A6 19.87+3.11 20.90+2.36 Calcium 77.73 8.13 68.43 ±2.55 Acetate Caltrate" 1.70±0.24 0.00246±0.00015 Data are expressed as Mean±S.D. TABLE 4 Saturated Solubility Of Magnesium In Artificial Gastric Fluid And Intestinal Fluid Saturated solubility of magnesium Formul-a No. --------------------------------------------------------- Gastric fluid (g/L) Intestinal fluid (g/L) Al 0.13+0.006 0.13+0.04 A2 0.11+0.01 0.10+1.0.03 A3 0.12+0.04 0.09+0.01 A4 40,78±2.46 26.57±1,81*** A5 24.97±2.95 19.03±2,73*** A6 49.30±2.61 38.67±4,33*** Calcium Acetate 0.50±0.07 0.42+0.10 Caltrate" 0.17=0.17 0.09 0.02 Data arc expressed as mcaniS.D. (n-3). ***:P <0,001 compared with solubility in the artificial gastric fluid. Table 5 Saturated Solubility Of Zinc In Artificial Gastric Fluid And Intestinal Fluid Formula No. Saturated solubility of zinc Gastric fluid (g/L) Intestinal fluid (g/L) Al 1.04±0.16 0.76±0,07* A2 3.72+0.68 2.14+0.14* A3 3.25 0.19 2.31±008** A4 2.22 0,17 1.19+0. I,*** A5 2.64±0.38 1.64±0,07* A6 1.54+0.13 1.07+0. 11 ** Calcium Acetate 0.60±0.17 0.53±0,14 Caltrate" 0.33± , 0.1 0.23+0.08 Data are expressed as mcan±S.D. (n-3). *:P<0.05, **:P<0.01, ***:P<0.001 compared with the solubility in artificial gastric fluid. EXAMPLE 3 Effects Of LIH On The Solubility Of Calcium In Different Formulations [00661 In this example, the effects of pH (ranging from I to 9) on the solubility of three elements of the four pearl formulas (Al, A4, A5 and A6), a commercial product (Caltrate

TM

) and a synthetic compound (Calcium Acetate, Ca ACE) were investigated. Solution pH was adjusted using appropriate amounts of acetic acid (AcOl), nitric acid (HNO 3 ) or arnrnoniurn hydroxide (NH1 4 01-). Saturated solutions were prepared by dissolving each preparation in a solution with a final pH value 12 WO 2013/014654 PCT/IB2012/053872 ranging from 1 to 9. The resultant mixtures were incubated in a water bath at 37C for one hour. Each sample was then filtered (with or without centrifugation) immediately, and the filtrate was diluted to an appropriate concentration for elemental analysis. The concentration of calcium, magnesium, and zinc was measured using ICP-OES. The results are shown in Tables 6-8. Statistical analysis was performed using one-way ANOVA and the P value was set at 0.05. [00671 Throughout the p1-I range tested, both Al and calcium acetate showed significantly higher calcium content in solution than the other preparations. Caltrates' had the lowest calcium content (p<O.05). Al and calcium acetate have the highest solubility at pH4 1 (Table 6) [00681 Magnesium has a negative effect on the content of calcium in solution; the rank order in TM terms of solubility is A5>A4>A6. Except for Caltrate , calcium acetate and Al, which are more soluble at p-I 1, p-i has no effect on the solubility of magnesium in solution (Table 7). [00691 Similarly, the amount of zinc in solution correlated well with the zinc content in different formulations (A5>A4>Al>A6) (Table 8). For all four acetate formulas tested, p-i values higher than 5 were associated with higher solubility of zinc than that at pH 2 and 3. [00701 Since intestinal pH values are typically higher than 6, the present formulations present advantages in terms of solubility, when compared with the solubility of calcium carbonate in Caltrate TM under such pH conditions. These results are consistent with those reported in Table 3. TABLE 6 Calcium Solubility (g/L) in Different p H Solutions (N = 3) pH CaltrateTM Ca ACE Al A4 A5 A6 1 4.60± 0.28 99.0 ± 19.2 101 ± 12.9 37.6 ± 2.5 48.7 ± 2.1 23.3 3. 2 2 4.04 0.23 613 ±0.97 64.6± 5.1 29.4 ±2.1 43.5 5.3 22.6 0.54 3 0.507 0,10 75.7 ± 4.8 71_A 22.2 38.0 ± 4.7 48.6 ± 0.98 19.5 2.5 4 0.237 0.03 85.4 ± 5.7 75.3 3.4 38.5 ± 2.9 48.3 ± 0.82 20.8± 0.98 5 0.240 0,06 75.6 5.5 65.4 11.8 39. 3 4.4 474 8.6 24.7± 2.5 6 0.317 0.10 76.0 5.9 83.8 12.7 41.2 1.3 52.3 5,0 19.3 ± 2.7 7 0.133 0.05 80.0 3.5 84.2± 16.8 34.6 33 49.5 8.1 20.6 ±3.8 8 0.160 0.03 71.2 1.6 78.3 13.0 30.0 3.8 55.3 7.9 19.8 ± 2.0 9 0.227 0.13 74.5± 6.8 84,8 8.2 35.8 3.5 50.2 1.5 19.6 ± 4.2 TABLE 7 Magnesium Solubility (g/L) in Different pH Solutions (in = 3) 13 WO 2013/014654 PCT/IB2012/053872 pH Caltrate M Ca ACE Al A4 A5 A6 1 0.197 ± 0.015 0.527 ± 0.121 0.173 ± 0.015 34.697 ±4.836 23.927 ± 1.747 41,797 ±5.622 2 0,100 ± LOGO 0360 ± 0,010 0.133 ± 0.006 29.117 ±2,204 20.020 ± 2.'14 39.957 1.050 3 0.133 ± 0.006 0.413 ± 0.035 0.13 ± 0,021 32.640 41 65 21.880 0,849 3400 5. 169 4 0.123 ± 0.012 0.490 ± 0.010 0.173 ± 0.0,15 33.560 ±2.606 21.733 ±0.248 34A153 1,5601 5 0,107 ± 0,012 0,500 ± 0040 0.3± 0.012 34.510 ±, 2817 24.367 ±3.916 45.353 ±7.2941 6 010±0.010 0.47'3 ± 0.076 0. 17-7 ± 0. 040 35.747 ±1.738 24.997 ±0.8171 :34A477 ±4.730 7 0.093 ± 0.006 0.460 ± 0.035 0.153 ± 0.015 30.197 2.818 21.677 3.127 36.983 7.234 8 0.097 ± 0.006 0.433 ± 0.040 0.157 + 0,015 31 .023 6.548 24.953 3.410 34.480 4.046 -9 0.097 ± 0.006 0.433 ± 0.045 0 1A30 + 0.0,10 33.473 ±T7169 23.607 ±1.055 34.41-0 ±-6.836 TABLE 8 Zinc Solubilitv (g/L) in Different pH Solutions (n = 3) pH CaltrateM Ca ACE Al A4 A5 A6 1 0.007 ±C0.006 0.030 5 0010 1283 ± 0.220 1,980 0.256 2.637 ± 0.143 1,440 ± 0.140 2 0,003 ±M.06 0,013 ± 0,006 0.793 ± 0,09:3 1.457 ±M003 2,220 ± 0,204 1.143 ± 0,0251 3 0.000 0.000 0.017 ± 0.006 0.843 ± 0.315 1.593 0.216 2.103 ± 0.134 0.817 ± 0.064 4 0.00 0.000 0,017 ± 0.06 1.137 ± 0.092 1.867 ±0.078 2,383 ± 0.071 0.933 ± 0,032 5 0.003 ±0.006 0.017 ± 0.006 0.993 ± 0.195 1.930 ±0.164 2.790 ± 0.305 1.250 ± 0.193 6 0.000 ±0.000 0.020 ± 0.000 1.227 ± 0.133 2,063 ±0.059 2.837 ± 0.135 0,870 ± 0.096 7 0,007 ±M.12 0,023 ± 0,006 1.23-7 ± 0,223 1.770 ± 0,132 2.49:3 ± 0,372 0.990 ± 0,1571 8 0.003 0.006 0.027 ± 0.012 1.180 ± 0.180 1.787 ± 0.306 2.903 ± 0.300 0.940 ± 0.082 9 0,07 ±0,006 0.027 ± 0,06 1.260 ± 0.087 1.970 ± 0.364 2.753 ± 0, 133 0.917 + 0.152 Experimental Data on Solubility in the Presence of Common Gastric and Intestinal Anions and Cautions 100711 The following analyses using anions which are present in abundance in gastro-intestinal tract fluids were performed on the four test formulas (Al, A4, A5 and A6), CaltrateTM and calcium acetate in order to assess the solubility and subsequently, their rate of absorption and bioavailability. The following are the standard ranges of common anions and cations in the human gastrointestinal fluids. TABLE 9 Concentration Of Ions in Human Gastric And Intestinal Fluids Concentration of ion (mM) Ions In Stomach / Gastric Fluid' In Intestine / Intestinal Fluid 3 Na* 0 - 100 (0 - 80) (155) K* 0-10(0-15) (70-150) HI+ 1 - 140 (20 - 120) (pH 7.7 - 8.2) Cl 100 - 170 (120 - 160) (30-90) 14 WO 2013/014654 PCT/IB2012/053872 Phosphate ions Up to 100** HCOJ (70 - 130) 'Values were cited from The Digestive System (ISBN 0443062455). The values in brackets were cited from The Medical Phsio logy (ISBN 07817! 9364). ** based on the solubility of sodium phosphate. EXAMPLE 4 Effects of Anions On The Solubility Of Calcium, Magnesium and Zinc In The Test Preparations [00721 The following analyses using anions which are present in abundance in gastro-intestinal tract fluids were performed on the four test formulas (A], A4, A5 and A6), Caltrate"m and calcium acetate in order to assess the solubility and subsequently, their rate of absorption and bioavailability. In this example, the effects of bicarbonate and phosphate (HCC3j and P4 q) on the solubility of calcium, magnesium, and zinc were studied at pH 7. Furthermore, the effects of chloride on the absorption of these three elements at p1H I and pI 7 were also studied. The procedures described in Example 3 for pH adjustment and solubility measurements were used. ICP-OES was used to quantify calcium, magnesium and zinc, Statistical analysis was performed using one-way ANOVA and the level of significance was set at p<:0.05. A. Chloride Effects at pH 1 [00731 Tables 10-12 are the results of chloride effects at pH 1. This condition mimics that of the acidic environment in the stomach. Chloride has the most intense effect on the solubility of calcium, magnesium and zinc in Caltrate' TM at pH 1 (Tables 10-12). At a Cl~ concentration of 200 nM, the solubility of calcium was the highest. The maximum magnesium and zine solubility was reached at Cl~ concentrations of 50 mM and 120 mM, respectively. The fluctuations of calcium, magnesium and zinc solubility are minimal in all the acetate formulations: calcium acetate, A1, A4, A5 and A6. Significant differences are often obtained at the highest C1- concentration (p<0.05). TABLE 10 The Effect of C1 Concentration On The Solubility Of Calcium (g/L) In Different Formulations At pH 1 CI Conc. CaItrate M Ca ACE Al A4 A5 A6 0 m ___ _ 4.597 ±0.276 9.950±19.224 101 .353 ±12.97_ 37.637:2.509_ _8.670±2 1202 23.37 3.162 50 mM 8.160 0 497 80.857 10.277 73.950 0.987 29.950 6.933 42.413 ±12.931 22.290 4.543 100 mM 7.333 ± 1.572 71.060 ± 1.660 85.627 ± 14.191 30.023 4.042 43.853 2.264 24.690 0.746 120 mM 8.157± 1.210 76.453 ±6.196 83.967 0.479 36.883±1.966 50.283 2.977 24.850±1.077 150 mM 5.883 ±1.416 73.353 1.037 87.340± 3.166 39.657 4.659 44.443± 5.495 24.647 0.775 180 mM 9.073 ± 0.325 80.977 ± 12.440 88.593 ± 5.579 41.710 ±2.836 50.343 1.392 26.067 1.891 200 mM 12.123± 1.178 77.257 ±12.364 97.840 ±12.364 42.313 ±6.119 63.027 3.406 29.387 4.062 15 WO 2013/014654 PCT/IB2012/053872 TABLE II The Effect of Cr~ Concentration On The Solubility Of Magnesium (g/L) In Different Formulations At pH 1 C1 Con. Caltrate Ca ACE A1__ A5 A6 0 mM 0.197 0 015 0.527 0 121 0.173 0.015 34.697 ±4.836 23.927± 1.747 41.797 5.622 50mM 0.357 ± 0471 0.440 0.075 0.133 ± 0.006 31.420 ± 6.649 20.547 ± 6.525 45.827 6.006 100mM1 0.13 ±+0.012 0.380 0.020 0.157±-t0.012 35.853 ±4.215 22.697±1.231 46.900 ±4.117 .0mMk 0.243 ± 0.163 040±006 0. 140 ±; 0.01 7 33,363 ± 2,542 23.333 ±3.312 48.827 ± 4.095 50 mM 0.220 ± 0.132 0.403 0.012 0.163 ± 0.015 36.037 ± 4.510 21.967 ± 1.260 45.653 2.449 180mM1* 0.227 ± 0.134 0. 4 20 + 0. 04 0 0. 160 -t 0. 020 38&117 ± 3.356 24.2 10 ±0. 698 46.070 ±3.290 200 mM 0.207 ± 0.074 0.427 ±0.080 0.163 ± 0.006 43.203 ± 4.646 29.41 0 ±0.11 5 81.437 ±4.319 TABLE 12 The Effect of Cl Concentration On The Solubility Of Zinc (g/L) In Different Formulations At CT Conc. Caltrate Ca ACE A: A4 A5 A6 0 mM 0.007 t 0006 0.030 0.010 1.283 0.220 1.980 0.256 2.637 0.143 1.440 ±0.140 50 mM 0.030 ±0.000 0.027 ± 0.006 0.917 0. 156 1.500 i 0.216 2.073 0.598 1.237 ± 0.110 00mM 0.130 ±0.026 0.067 0.025 1.120 0.010 1.683 0.100 2.353 0.057 1.293 0.025 120 mM 0.217 ±0.047 0.103 0.031 1.113 0.112 1.687 0.196 2.487 0.273 1.363 0.095 .50 M 0.277 ± 0.091 0.073± 0.015 1. 360± 0. 144 1.8103 ± 0. 121 2.320 ±0. 106 1.280 ± 0.046 80mMt. 0 180 ±0M60 0. 117±0. 031 1.193+0.211 1.927 00 1 2.590±0.061 1.313±0.032 200 mM 0. 190 ± 0.056 0. 123 +0. 04 0 1.4.13 ±0. 187 2.230± 0.265 3.173 ±0.248 2.083± 0. 112 B. Chloride Effects at pH- 7 [00741 At pH 7, the solubility of calcium in Caltrate TM is dramatically lower than that at pH 1 in the presence of chloride (Compare values in Tables 10 and 13). As chloride concentration increased, the solubility of calcium in Caltrate TM increased, The pH and chloride effects are not pronounced for the acetate formulations. In general, maximum calcium solubility is reached at chloride concentrations between 50 to 100 mM. 100751 In the presence of chloride, pH has less of an effect on magnesium solubility (compare values between Tables II and 14). In general, the solubility of magnesium at p-1 7 is slightly lower for all formulas and the chloride effect is not pronounced. 10100] In the presence of chloride, the solubility of zinc in Caltrate 1 " at pH 7 is less than half of that at pH 1 (compare values between 11 and 14). However, this difference is not as pronounced in the acetate formulas. There is a tendency for zinc sohibility to increase with the increase of chloride concentration. Maximum zinc solubility is reached at 120 mM chloride when CaltrateIm was evaluated, For the acetate formulas, maximum zinc solubility occurred when chloride concentration reached 200 mM. 16 WO 2013/014654 PCT/IB2012/053872 TABLE 13 The Effect of CI Concentration On The Solubility Of Calcium In Different Formulations At p. 7 C- Conc. CaitrateTM [gIL] Ca ACE [g/L] Al [g/L] A4 [g/L] A5 [g/L] A6 [g/L] ___ 0133 ± 0.051 80.017 ± 3.505 84.170 16.834 34.640 3.268 49.497 ± 8.097 20.627 3.821 50 mM 0.340 ±0.082 99373± 6 182 80u703 13.103 47.473 ±2.381 61 537± 6436 31.490 2.399 100 mM 0 557 ±0.040 87.263± 13.984 77.660± 19.779 47.867 ±7.511 66.743 ±13.191 29.053 6.684 120mM 0.370 ± 0,165 71.440 ± 5.851 61437 ± 8,616 35.400 ± 0.864 45.060 6.166 2 353 2.351 150 mM 0.567 ± 0.075 70,923 ±3,240 73,73 12.437 33.017 ± 2.455 42.980 ±2,603 2 0. 313 ±2. 00 5 180mM 0560 ± 0.165 77.823 12.314 59 '20 ± T467 3403 ± 0846 42.890 5.516 17.490 0.916 200 rnM 0.600 ± 0,.132 73.930 ± 7.785 84.707 ± 15,685 33.223 ± 2.093 46.403 ±4.643 18.627 +2.238 TABLE 14 The Effect of CI Concentration On The Solubility Of Magnesium In Different Formulations At pH 7 CF Conc. Caltrae g/L] Ca ACE [/Li A1 [g A4 [g/L] A5 g/L) A6 [g/L O0mM 0.093 0.006 0.460 0.035 0.153 0.015 30.197 2.818 21.677 ±3.127 36.983 ±7.234 50 mM 0.280 0.202 0.503 0.031 0.140 0.020 43.190 ± 2.792 29.203 ± 1.107 56.003 3.989 10r0M 0.280 0.149 0.480 0.0.7 0. 1430.006 456253 ± 66350 30.917 ± 6.111 52.953 120mMik 0. 11.0 ±0.026 0.390± 0.030 0.147 ±0.01.2 31.983 ± 3.302 19,333 ± 2,217 42431,448 150 mM 0.227 0.096 1.750 ± 2.382 0.167 0.015 29.087 ± 0.957 19.383 ± 1.482 42.643± 0.446 180 mM 0.2530.129 0.430 0.046 0.16 7 0.006 32633 ±2372 1973T 2149 36.160 133 9 200mrvM 0.283±0. 107 0AA27 0.065 0.203±0.025 32.923 ±0.802 23,067 ±2,175 47 -3159,8 TABLE 15 The Effect of CI Concentration On The Solubility Of Zinc In Different Formulations At pH 7 or Con. ua mae Ca ACE [g./Li _ A /LL_ A__ L4 I A__ ___A A6 0 mM 0.007 0.012 0023 0 006 1 237 ±0223 1.770 ±0.132 2.493 ±.372 0.990 ±0.157 50 mM 01 3±0.006 01 80+± 089 0 997 ± 0 195 2.057 ± 0.189 3.177 ±~ 0289 1.457 ±0.244 10 0 IIM 0. I14 ± 0.026 0.213 ± 0.102 0.903 ± 0.280 2.413 ± 0. 144 3.063 ± 06287 154 ± 0.380 120 mM 0.050 ±0.017 0.167 0.202 0.760 ±0.118 1.573 ±0.146 1.997 0.254 1 .10 ± 0.036 150 mM 0.087 ±0.025 0 177 0 085 0.987 0.110 2.030 0.615 2.010 .16 1.177 ± 0.072 180 mM 0.093 ±0.015 0.143 0.071 0.780 0.151 1.37 ±0.127 2090 & 167 1 077 ±0163 200 mM 0093 ±0.012 00160 0609 1.117 0.202 1.663± 0.078 1.64_ __1 1.303±0.060 C. Bicarbonate Effects at p H 7. [01011 The solubility of calcium in CaltrateTM increased with the increase of bicarbonate concentration (Table 16). However, the opposite is true for calcium acetate. The solubility was reduced at least 40%. The reduction for all the pearl extract formulas was less, approximately 20 to 25%. [01021 The solubility of magnesium in CaltrateTN' increased with bicarbonate concentration (Table 17). Bicarbonate effect was minimal for the acetate formulas. 17 WO 2013/014654 PCT/IB2012/053872 101031 The solubility of zinc in Caltrate M increased in the presence of bicarbonate (Table 18). Maximum zinc solubility was reached at 70 mM, For calcium acetate, the trend is similar to that of Caltrate Bicarbonate has very little effect on the pearl extract formulas. TABLE 16 The Effect of HCO, Concentration On The Solubility Of Calcium In Different Formulations At pH 7 HCO Co Caltrate [L] Ca ACE [g/L] Al [g/L] A41[g/L1 A5[ A6 [g/L] 0 mI 01 33 i 0,051 80.017 ±3.505 84.170± 16,834 34,640 i 3,268 9497 i 8,0 20.627 3.821 50 rnM 0.217 ±0.214 50.243 ±3.312 72.030 ±7.103 36.007 ±3.807 42.577 0.779 21.737± 1.255 70 mM 0.213± 0.098 62.090 ± 8,524 70.933 4.812 33.420± 5.263 42.130 4.734 22,343 i 0,847 100 mM 0,380 0,075 56.367 ± 9.062 83.640± 10,870 34,997 6,049 46,167 4 546 25.260 10.191 120 mM 0.440 0.167 46.023± 2.463 67.010 ±3.767 31.060 ±2.23 46.973 2.919 20.817 1.664 150 mM 0.433 0.120 70.637 3,622 65.617± 1.475 30.410±2.888 41567 4620 19,163i1.56 180 mM 0,930 1,290 46.847± 2.741 65.270± 1.781 28,680± 1,362 38,073 3,465 18.870± i1.679 TABLE 17 The Effect of HCO 3 Concentration On The Solubilitv Of Magnesium In Different Formulations At pH 7 SC CtrteT

M

[/L] Ca ACE [ 1 / Al [g/L A4 [g/L A5 [J/L A6 [g/L] 5 aI 009 000C 0.460±0.035 0.153 0.015 30.197±2.818 21.677±3127 36.983±7.234 50 nHV 0.1090 ±0.035 0.297 ±0.055 D. 19()±D05 6 34.600 ±4.638 20.4217± 1.272 48.140 ± 1.653 70 mM 0,093± 0,012 0.347 ± 0,031 0.160 ± 0.000 32.057 4.407 22.000 ± 0.141 42.767 ± 0,40 100mM 0.223±0.111 0.101 0 167 ±0.065 41.580± 12.984 26.393±4720 43.883± 8.28 120M 0.JI O220 ± 0 069 0. 303 ±0. 015 0 A83 ± 0. 551 30.960 ± 2.164 22.87 7 ±1.082 46. 990 ± 5.278 150 mM 0.227 0.072 0.410 0.061 0.150 ± 0.017 28.950 ±2.262 18.850 2.169 42.877± 7.608 180 mM 0.240± 0.095 | 0.293 0.049 0.163 ±0.015 30.787± 1.021 19.607± 1,529 36.957± 0.839 TABLE 18 The Effect of HC0 3 Concentration On The Solubility Of Zinc In Different Formulations At pH 7 H CO3 Conc. atrate /L Ca ACE /L A4 [gL] A5g/L] A6 [gL 0mMI 0.007 ± .02 1 0.023 ± 0.006 3 0.23 1.770 ± .132 2.493 ±0.372 0.990 ± 0.1S57 50 mM 0.057 0.015 0.050 ±0.010 0.953 ±0 .01 1.663 ±0.205 2. 0.142 1.260.0.017 70 mM 0.070 0.020 0.100 ± 0.061 0,990 i0 082 1.560 ± 0.236 2.237 ± 0099 1.147± 0±O,1 100 mM 0,070 0,017 0.157 ±0.055 1.190 o.101 2 067 0 654 2.660 0.442 1.193 ± 0.023 120mM 0.093 0.025 0.210 ±0.096 0.907 ±0 042 1.513 0.127 2.290 0.115 1.317 ±0.182 150 mM 0.087 ± 0.021 0.137 ± 0.083 0.863 ± 0.081 1.427 0.059 1.887 ± 0. 144 1.237 ±0.235 180 mM 0,070 ± 0,017 0.160 ±0.078 0.933± 0.072 | 1.517 0.119 1.997 ±0.157 1.023± 0.042 D. Effects of Phosphates at p1H 7 [01041 Phosphates have insignificant effects on the solubility of calcium in Caltrate " (Table 19). As phosphate concentrations increased the solubility of calcium decreased in all acetate formulations. Maximum reduction (up to 40%) of the solubility of calcium was observed in formulas containing higher percentage of magnesium (A4, A5 and A6). Considering the range of phosphate concentration tested, 10,000-fold, the change of calcium solubility is not significant. 18 WO 2013/014654 PCT/IB2012/053872 101051 Magnesium solubility decreased as phosphate concentration increased (Table 20). The reduction (80%) is most significant for the magnesium in Caltrate

T

. For th other formulas, th maximum reduction was approximately 50%. Again, the effect of phosphates was not that significant considering the range of concentration tested, [01061 Among the three elements, phosphates have the most intense effect on the solubility of zinc (Table 21). All formulas were affected to the same extent and the maximurn reduction was approximately 70%. Considering the range of phosphate concentration tested, again, the effects of phosphates were not that significant. TABLE 19 The Effect of PO4 Concentration On The Solubility Of Calcium In Different Formulations At pH 7 Conc. C L L] CaACE [1L) A1 [ A4 [g/L] A5 [g/L] A6 [gL) 0.01 mM 0.587± 0.200 77.517 6.084 84.270 9.511 34.950 6.725 47.823 ±3.080 22.287 ±2.539 1 riM 0.510± 0.252 68.220± 19,638 56.450 9.879 39.923± 10.060 42,363 i 3,572 23.530 ±0.159 10 mM 0.430 ± 0.046 78.417 ± 7.046 64 697 9 058 25.703 7.033 41.287 ± 3.584 21 687 ± 156 10 0mM 0.4 53 ± D. 158 64.770 ± 4 58. 607 ± 9415 25.090 ±3.181 34.650 ± 6.972 15.437 ± 2.428 TABLE 20 The Effect of PO Concentration On The Solubility Of Magnesium In Different .Formulations At pH 7 T~ on . C ltae a C [0~i Al A4 1_ __ A5 g 66 ___ A L 0.01-m 0.280 ±0.070 0.493 ± 0.025 0.203 ± 0.006 380 17 ± 2532 24.733 ±0.886 -12. 000 ± 5647 1 mMk 0.317 0.087 0 .450,± 0.095 0.217±0.031. 35.647 ± 10.790 83.583±+ 1.676 48.967± 1.486 10 mM 0.240 0.050 0.477 0.035 0.173 ± 0.012 20.837 5.545 18.163 1.368 37,140i 2,681 10 mM 0.073 0.006 0.350 0.017 0.127 ±0.012 21 490 1 830 16.720± 4.514 31.163 4.838 TABLE 21 The Effect of PO.g Concentration On The Solubility Of Zinc In Different Form ulations-At ilH 7 Po Conc. Catrate [g/L] Ca ACE [g/L] Al [g/L] A4 [g/L] AS (g/L] A6 (g/L] 0.01 mMik 0,1117 i 0,042 0.190 ±0.070 1.r193 ±0.097 1.950 0.040 2.7150 0. 135 1.470 0.154 1 mM 0.100 i0.044 10.197 ±0.110 0.780 0.151 1.800 0.394 1.993 0,093 1380 0,079 10 mM 0.070± 0010 0 180 ±0089 0.767 ±0.137 0.937 0.253 1.740 0. 173 1.023 0.060 100 mM 0.053 ± 0.023 0.527 ± 0.119 0.623 ± 0.087 1.013 0.345 0.510 0.131 EXAMPLE 5 Effects of Cations On The Solubilitv Of Calcium, Magnesium and Zinc In The Test Preparations [01071 The following analyses using cations which are present in abundance in gastro-intestinal tract fluids were performed on the four test formulas (A], A4, A5 and A6), Caltrate"M and calcium acetate in order to assess the solubility and subsequently, their rate of absorption and bioavailability. 19 WO 2013/014654 PCT/IB2012/053872 A. Effects of Na* at pH 1 [01081 The effects of Na- concentration on the solubility of the three elements in the four formulations (Al, A4, A5, and A6), Caitrate T M m and CaACE were investigated at gastric p-I (pH::1) and intestinal pH (pH= 7 ), respectively. Tables 22 and 23 show the results tested at pH 1. No significant effects of Na concentration on calcium and magnesium solibility of all formulations were observed. Solubility of zinc in Caltrate TM and calcium acetate, which contained trace amounts of Zn, increased significantly with an increase in sodium concentrations; however, no significant differences were obtained for all the acetate formulations (Table 24). TABLE 22 Effect Of Concentration Of Na -On The Solubility Of Calcium Of Each Formula At pH I Na+ Solubility of calcium (g/L) Conc.(mM) Caltratem CaACE Al A4 A5 A6 0 4.597 0.276 98.950 ±19.224 101.353 ±12.947 37.637 2.509 48.670 ± 2.102 23.337 ± 3.162 5 5.447 2.061 84.800± 13.912 72.233± 1.501 36.467 5.173 46.100 ±0.721 22.000 ± 1.323 10 4.340 0.035 66,967 17.377 80,000 1,852 40.033 4.623 49.833 ± 2.503 27 900 ± 3 736 50 4.64 0 070 90.167 9.343 83.467 3.313 36.633 1.877 49.033± 452 25.467 ±0.231 80 5.530 0.946 87.167 3.630 83.067 6.813 37.033 1.069 55.733 ± 5.372 3f0.600 ± 1 709 100 5.360 0.742 79.233 15.964 84.900 11.609 39.100 5.696 48.733 ±3.968 25.0 6 7± 0 1 53 Data are expressed as meaniS.D. No statistical differences in all Na concentrations tested for all formulations tested. Table 23 Effect Of Concentration Of Na* On The Solubilitv Of Magnesium Of Each Formula At pH I Na+ Solubility of magnesium (g/L) Conc.(mM) CaltrateM CaACE Al A4 A5 A6 0 0 197 ± 015 0.527 ± 0.121 0 173 ± 0015 34.697 ± 4.836 23.927 ± 1.747 41.797 ± 5.622 5 0.223 ± 0.006 0.700 0.183 0.283 ± 0.040 36.400 ± 4.854 24.467 ± 1.361 39.933 ± 1.343 10 1.037± 1.109 0483 ±0.115 0.317 0.050 38.967 5.745 23.900 ±1.800 49.100 3.305 50 0.807 ± 0.889 0.620 ± 0.115 0.237 ± 0.031 35.733 ± 1.909 22.667 ± 2.055 45.500 ± 2.211 80 1.087 + 1.264 0.580± 0.061 0.960 ± 1.031 35.033 t 3.625 27.767 + 3 700 50.900 ± 7.375 20 WO 2013/014654 PCT/IB2012/053872 100 0.577 ±0.525 0.497 ±0.025 0.223 ±0.032 36.000 ±5.629 21.267 2.120 16.233± 1.401 Data are expressed as meaniS.D. No statistical differences in all Na-, concentrations tested for all formulations tested. TABLE 24 Effect Of Concentration Of Na t On The Solubility Of Zinc Of Each Formula At pH 1 Na+ Solubility of zinc (g/L) Conc.(rnM) CaltrateM CaACE Al A4 A5 A6 0 0.007 0.006 0.030 0.010 1.283 ±0.220 1.980 ±0.256 2.637 ±0.143 1.440 0.140 5 0.087 0.006 0123 0.006 0.660 ±0.128 1.393 ±0.316 2.180± 0.413 1.183 ±0.121 10 0.173 0.015 0.317 0.106 0.883 ± 0080 1.767 ± 0.280 2.080 0.160 1.760 + 0617 50 0.240 0.053 0.400 0.139 1.023 ± 0.075 1.727 ± 0.060 2.250 0.114 1.410 ± 0.125 80 0.210 0.026 0,397 0163 0.907± 0.211 1.730± 0.479 2.613± 0.270 1.747 ±0.015 100 0.223 0.031 0.363 0.095 0.947 ± 0188 1.490 ± 0.105 2.207 ± 0.506 1493 ± 0630 Data are expressed as mean±S.D B. Effects of Na* at pH 7 [01091 Tables 25-27 show the effects of sodium ion at p-I 7. Na has no significant effects on calcium, magnesium and zinc solubility in general. It is interesting to note that all three elements in CaltrateTM could be not detected in the presence of Na' at pH 7. TABLE 25 Effect Of Concentration Of Na* On The Solubility Of Calcium Of Each Formula At pH 7 Na+ Solubility of calcium (g/L) Ccnc.(nM) CaltrateFm CaACE Al A4 A5 A6 0 0.133 0,051 98,950 ±19.224 101.353 ±12.947 37.637 ±2.509 48.670 ± 2.102 23.337 3.162 10 - 83.300 26.469 67 433 4.460 37.433 ±4.822 43.800 4.703 39.367 16.110 50 - 69.000± 1.015 99.333 21.548 35.533 0.814 48.367 ±4.359 23.833 2.219 100 71.467±10.891 71.433 1.193 36.867 ±3.139 46.267 ±1.380 24.567 4.104 140 - 83.067 6.596 68.900± 7.400 32.300± 1.153 47 200± 6 023 25.633 3.754 170 - 72.33 15.467 71.433 ±0.551 37.567 ±10.473 43.133 ±4.876 25.867 3.175 Data are expressed as mean±S.D, No statistical differences in all Na concentrations tested for all formulations tested, 21 WO 2013/014654 PCT/IB2012/053872 TABLE 26 Effect Of Concentration Of Na* On The Solubility Of Magnesium Of Each Formula At pH 7 Na+ Solubility of magnesium (g/L) Conc.(nM) CaltratemTM CaACE Al A4 A5 A6 0 0.093 0.006 0.527 ± 0.121 0173 0,015 34.697 ± 4.836 23,927 1,747 41.797 5.622 10 - 0.740 0.165 0.10 01010 35.300 3.579 19 500 1 769 75.167 34.360 50 -0.27 ±0.081 0.193± 0.015 35.933 5.139 23.000± 4.327 52.167 4.852 100 -- 0.510 ± 0.066 0.157 ± 0.006 33.267 3.889 20.667 ± 0.493 45.867 3.329 140 --- 0.497 ± 0.099 0.157 0.021 28.867 2.255 20.567 2.610 51.000 6.963 170 - 530 ± 0.036 0,167 ± 0,021 45,633 ±11.097 21,600 2,476 53.500 ± 3.650 Data are expressed as mean±S.D. TABLE 27 Effect Of Concentration Of Na* On The Solubility Of Zinc Of Each Formula At pH 7 solubility of zinc (g/L) Na+ Conc.(mM) Caltratem CaACE Al A4 AS A6 0 00070012 0.030 0.010 1.283 0.220 1.980 ±0.256 2637 ±0.143 1.440 ±0.140 10 - 0.213 0.102 0.600 0.040 1.453 ±0.185 1.543 ±0.215 2.337 1.351 50 0.280 0.118 0.963 0.280 1,700 ± 0,779 2.317± 0.798 1,687± 0.466 100 -- 0.293± 0.129 0.707 i 0.107 1,243 0,211 1.790 ± 0.087 1,667 ±0 275 140 -- 0.320 0.165 0.690 0.137 1.113 0.144 1.770 0.056 1.643 0 402 170 - 0.223 0.102 0.730 0.079 2.230 0.397 1.933 ±0.838 1.577 ±0.529 Data are expressed as mean±S.ED. C. Effects of K at pHT 1 [01101 There is a tendency for the solubility of calcium to increase with an increase in potassium ion concentration (Table 28). However, most of the differences are not statistically different (p<0.05). In A5, the calcium solubility increased by more than 50%; this difference is significant (p<0.

0 5 ), [01111 Magnesium solubility profiles for the acetate formulas show a similar trend (Table 29) to that of calcium. There is a three-fold increase in the magnesium solubility in Caltrate'M, (p<0.0). However, the magnitude of increase in inconsequential when compared to that of A4, A5 and A6. 22 WO 2013/014654 PCT/IB2012/053872 10112] An increase in potassium is associated with an increase in zinc solubility for Caltrate and CaACE (Table 30). Potassium has insignificant effect on the solubility of zinc in the four formulas (p>0.05). Again, the magnitude of increase in zinc solubility is inconsequential when compared to A4, A5 and A6 TABLE 28 Effect Of Concentration Of K* On The Solubility Of Calcium Of Each Formula At pH 1 K+ Solubility of calcium (g/L) Conc.(mM) CaltrateTM CaACE Al A4 A5 A6 0 4.597 ± 0.276 98 950 19,224 101.353 ±12 947 37 637 ± 2.509 48.670 ± 2.102 23,337 3.162 2 4.300± 0.403 78,933 1,320 71.833± 9.338 34.033± 1.739 35.833 ±5.314 24.067 1.474 5 3.607 0.540 71.033 13.079 73.733 3.412 36 967± 1.159 47 500± 5.272 23,500 1,778 10 6.497 3.381 158333 40.624 83.733 14.093 40.467 ±7.823 66567 21.033 30.867 ±10.262 15 6.877 0.956 161.667 46.918 92167 14.793 41.867 7.019 63.333 7651 26.667 0473 20 3.567 0.501 100.800 ± 3.811 103.333 15.822 42.633 ± 4.674 103.567 ± 64 463 29,300 3,751 Data are expressed. as meaniS .D. TABLE 29 Effect Of Concentration Of K* On The Solubility Of Magnesium Of Each Formula At pH 1 K + Solubility of magnesium (g/L) Conc.(mM) GaitrateTM CaACE Al A4 A5 AG 0 0.197 0.015 0. 52 7 0.121 0.173 ± 0.015 34.697 ± 4.836 23927 ±1 747 41.797 ± 5.622 2 0.223 ±0.087 0.693 0.283 0.203 ±0.029 34.933± 1.716 21.633± 4.300 49.700± 1.249 5 0.490 ± 0.419 0.453 0.112 0.170 ± 0.030 32.667 ± 2.542 23.433 ± 3.408 43.000 ± 2.406 10 0.703 0 846 0.820 0.193 0.270 0 130 38.733 5.552 30.067 ±8.429 55.400 ±18.187 15 0.730 ± 0.912 0.687 ± 0.215 0.327 ± 0.185 41.467 8. 617 31.067 ± 4.050 54.800 3,897 20 0.660 0 764 0.650 0.020 0.883± 1 140 52.067 2.859 55.733 ±34.208 54.233 14.632 ata are expressed as meaniS.D. TABLE 30 Effect Of Concentration Of K On The Solubility Of Zine Of Each Formula At p l I K+ Solubility of zinc (g/L) Conc.(rnM) CaltrateTM CaACE Al A4 A5 AG 0 0.007 ±0.006 0. 0 3f0± 0.010 1.283 ±0.220 1.980 ±0.256 2.637 ±0.143 1.440 ±0.140 23 WO 2013/014654 PCT/IB2012/053872 2 0.053 ± 0.015 0.077 ± 0.006 0.607 ± 0.108 1,377± 0,221 1.937± 0.591 1.360 0.122 5 0.173± 0.035 0.240 ±0.075 0.790± 0.147 1,297± 0169 2.593 ±0.821 1.143 0.278 10 0.203 ± 0.058 0.357 ± 0.111 1.127± 0.142 1,630 ± 0185 2.373± 0.658 1.627 0.225 15 0.193±0.023 1.307 1.199 1 060 0.600 1 953 0 590 2.963 ± 309 1.630 0.161 20 0 167 ±0 15 0293 ±0 093 1.100 0.140 2.500 0.236 5.450 ±3.159 2.540 1.424 Data are expressed as mean+S.D. C. K+ Effects at p'1 7 [01131 There was a tendency for the solubility of calcium to increase with an increase in potassium concentration, however, the difference is riot significant, p>0,05 (Table 31). No calcium could be detected in preparations using Caltrate'. 101141 Similar observations to that of calcium were obtained for the solubility of magnesium and zinc (p>0.05) in all formulas containing acetate salts (Tables 32-33). No measurable nagnesium and zinc was reported for preparations using Caltratet TABLE 31 Effect Of Concentration Of K* On The Solubility Of Calcium Of Each Formula At pH 7 K+ The solubility of calcium (g/L) Conc.(mM) CaltrateM Ca ACE Al A4 A5 A6 0 0.133 0.051 98.950 ±19.224 101.353±12.947 37.637±2.509 48.670±2.102 23.337±3.,162 10 - 14000 i 14.731 66.800± 1.539 32.100 ±0.361 64.033 8.892 17.100 ±0.173 50 - 174.467± 79. 146 68.533 3.259 33.933 ± 2.515 64.867 17.244 19.033 ± 3.630 100 - 156.333 ± 64 361 68.600 5,356 30 500 3.672 82.000 ± 35,508 20 667 ± 2 363 140 130.033± 32.461 60.400 25.999 56.767 32,771 68.400 7.100 42.000± 18,340 170 - 134.567 ± 55 048 126.133 72.997 68.433 29905 64.800 26 352 30.900 ± 14 912 Data are expressed as mean±S.D. No statistical differences in all K' concentrations tested for all formulations tested. TABLE 32 Effect Of Concentration Of K On The Solubilitv Of Magnesium Of Each Formula At pH1 7 K+ The solubility of magnesium (giL) Conc.(mM) CaltrateTM CaACE Al A4 A5 A6 24 WO 2013/014654 PCT/IB2012/053872 0 0.093 ±0.006 0.527±0.121 0.173±0.015 34.697±4.836 23.927 1.747 41.797 ±5.622 10 --- 0.767 0.189 0.140 0.010 32.033 2.829 30.967 ± 2.136 46.800 ± 3.158 50 --- 1.027 0.587 0.347 0.316 33.533 2.084 31,867 8151 48 200 1 253 100 --- 0.807 0.278 0.183 0.047 34.067 3.465 39.233 16.350 54.000 2.955 140 -- 0.87±0.303 0.160 0.035 57.833 34279 32.833 5.541 90.467 42.518 170 --- 0.760 0.310 0.230 0.062 64.200 26 513 31.333 12.507 61.900 30.685 Data are expressed as mnean+AS.D. No statistical differences in all K' concentrations tested for all formulations tested. TABLE 33 Effect Of Concentration Of K On The Solubility Of Zine Of Each Formula At pH 7 K+ The solubility of zinc (g/L) Conc.(mM) Caltratem CaACE Al A4 AS A6 0 0007 ± 012 0030 0010 1 283 0220 1.980 0.256 2.637 ±0.143 1.440 ±0.140 10 --- 0,293 0110 0 727 0 064 1.173± 0.163 3.243± 0.725 1.090 0.070 50 --- 0,627 0,437 1140±0036 1.447± 0.135 3.127 ± 0.720 1.247 ±0.045 100 --- 0,257 0110 1197 0 068 1.587 ± 0.106 3.417 ± 1.252 1.460 0.122 140 - 0 38 10 1 86 0.827± 0.506 2.583 ± 0.755 2.747 ± 1.432 2.607 ± 1.301 170 --- 0.287 0 .142 1.223±0.541 2.437 ±0.618 2.873 ±0.771 1.720 ±0.624 Data are expressed as mean+S.D. EXA MPLE 6 In Vivo Evaluation of Calcium, Magnesium And Zinc Balance 10115] The objectives of the balance studies were to evaluate the effects of dietary conditions and formulations on calcium, magnesium and zinc balance. A. Dietary Conditions 101161 Two diets, one with normal calcium and the other is calcium free, were used for the studies The nutrient composition of the diets is listed on Table 34: TABLE 34 Composition Of Normal And Calcium Free Diet Normal Calciumn Free Protein, % 2-1.0 19.0 Fat, % 4,5 (ether extract) 10.0 25 WO 2013/014654 PCT/IB2012/053872 6.0 (acid Cholesterol, ppm 101 48 Fiber, % 5. 3 5. 4 Carbohydrates, % 21.5 (starch) 60.6 0.2 (Glucose) 0,2 (Fructose) 3.4 (Sucrose) 0 (Lactose) Pot asiu, % 10 0 62 Sodiuifm, % 0.40 0.27 Chlorine, % 0,70 0,27 Calcium, % 0.95 0.0 [Magnesium % 0.2 2.07 Zinc, 0.011 0.0031 1ro opm 290 60 Mangane se, ppm 110 65 C opper pp 17 3. Vitamin K, ppm 3.2 10.4 Riboflavin, ppm 12 20.0 Py'idoxine, ppm 8,0 16.5 B. Materials and Methods [01171 Male Sprague-Dawley rats (about 6-7 weeks), with an initial weight between 220g to 250g were randomly divided into different treatment groups. All the rats were housed in individual metabolic cages in a temperature-controlled room, Each rat received free access to the normal diet (Table 34) before the experiment. Both normal and calcium free diets (Table 34) were used in this set of studies, De-ionized water was provided ad libitum. All the rats were weighed before treatment. C. Treatments [01181 Two sets of studies were performed: a normal diet and calcium free diet. In each study, there were seven treatment groups. Thirty five animals were randomly assigned to one of the treatment groups in which one of the following were administered: Caltrate

T

M Calcium Acetate (Ca ACE), Al, A4, A5, A4 plus vitamin D3 and A5 plus vitamin D3 (n = 5 per group). Rats participating in the normal diet study received normal diet ad libitum throughout. Rats participating in the group of calcium free diet received the calcium free food ad lihitun starting five days before and throughout treatment. In both study groups, animals received one dose a day for five days. Amounts of calcium, magnesium and zinc in individual formulation and in each diet were determined using ICP-OES. Values of dosage and dietary intake were measured for the calculation of elemental balance. For rats that were fed the normal diet, average daily elemental intake of calcium, magnesium and zinc was 625, 155 and 10 mg/kg/day, respectively. Daily elemental dosages, similar to that of human's, are 53,14 mg/kg for calcium, 0.38 to 55 mg/kg/day 26 WO 2013/014654 PCT/IB2012/053872 for magnesium and 0,017 to 2.5 mg/kg/day for zinc. Vitamin D 3 , 1.06 pg/kg/day (42.512 IU/kg/day; I IU=0.025 pg), was added to each dosage preparation prior to administration. The vehicle for preparing each dose was de-ionized water. The concentration of calcium in all dosage preparations was 15,94 mg/mL One mL of each preparation was administered by gavage. Body weight, elemental dosage and diet consumption were recorded daily. D. Sample Collection, Handling and Analysis [01191 Animals were housed individually in a metabolic cage five days before the study. Food consumption was evaluated daily. Urine and feces were collected daily for four days and the content of calcium, magnesium and zinc was determined, On Day 5. each animal received its treatment, These treatments were administered once a day for four days. After the last treatment, each animal was anesthetized shortly before peak elemental blood concentration was achieved. Blood was collected using a heparinized syringe via cardiac puncture. Immediately after blood collection, the animal was then sacrificed with an overdose of isoflourane. Each blood sample was centrifuged at 1900 rpm at room temperature; plasma was harvested and stored at -20 'C until analysis. Urine was measured daily; it was diluted with de-ionized water, filtered and an aliquot was stored at -20 0 C until analysis. Daily fecal output was collected and lyophilized, Each sample was weighed and digested using a mixture of three volume of nitric acid and one volume of perchloric acid, For every gram of dried feces, 10 mL of acid mixture was added, Each sample was digested for three days. The volume of the digested sample was measured and an aliquot of the digest was stored at -20 'C until analysis, The content of calcium, magnesium and zinc in plasma, feces and urine were determined using ICP-OES. [01201 Daily calcium balance was calculated using equation 1: [01211 Ca Balance = total Ca intake (dose and dietary intake) - Ca excreted in urine- Ca excreted in feces (1) [01221 While, percentage of Ca balance was determined using equation 2: 101231 % Ca balance:= Ca balance / (total Ca intake) x 100% (2) [01241 Cumulated calcium balance and % cumulated net calcium balance were calculated using equations (1) and (2), except, the sum of daily intake and excretion was used for calculation, The balance for magnesium and zinc was also calculated using the concept of equations (1) and (2). 27 WO 2013/014654 PCT/IB2012/053872 Cumulated elemental balance and % cumulated net elemental balance were calculated in a similar fashion as described above. [01251 In general, urinary excretion accounted for less than 5% of fecal excretion. Therefore, fecal excretion practically determines the quantity of elemental balance. E. Statistical Analysis 101261 All results were analyzed using two-way ANOVA. P<0.05 was considered to be significantly different. The data are presented as mean ± S.D. and mean ± S.E.M. in tables and figures, respectively. F. Results: Calcium free diet 101271 Table 35 shows the body weight of rats during the study. Stools from study animals were soft and this observation could be related to low elemental intake. Insufficient elements from the diet and dosage may have also caused the lack of weight gain for this set of animals. There is a statistical difference (p<0.05) among the starting body weights of the study animals (Table 35). There is also a slight in decline in body weight during the treatment period; is not the difference significantly different. TABLE 35 Body Weight Of Rats In Each Treatment Group With Calcium Free Diet (n=5) Treatment Body weight of rats (g) group Day 1 Day 2 Day 3 Day 4 Day 5 Caltratem 184.6± 77 178.6 ±99 1772 ±8.8 179.2 13.7 1754 14.2 Ca ACE 202.4 ±9.3 194 - 9.3 196.8 10.9 193.4 11.9 188.0 12.2' Al 190.4 ±11.9 185,6± 14.0 187.6 10.9 186.6i 11.3% 182.8 ±15,4 A4 188.4 12I 9*' 184.2 13.24 184.0 ± 12 7 182.4 13.5"' 183.2 ±'14.0' A5 187.8 ±8.8" 184.01 60" 184.2 ±5.6"' 185.4 6.0 **" 1824 9.2'" A4 + Vit D 207.6 11.9 200.0 5.2$' 198.6 ± 4.5' a 204.2 ±4.4"4 199.8 6.4 A5 + Vit D 204.8 14.4'_* 195.6 8.3' 196.4 ± 7.7'" 201.0 5.0'* 196.8 8. 2 $:<.05, compared with Cal trate ; P< .05, cmpa red with Ca ACE; +P<. compare ed wit Al; & : PrO . 05, compa red with A4; % P<O 01, cormared with A5; P<10.00 compared with AO + Vit D ; @:P<05, compared with AS - Vit D. [01281 The addition of magnesium and zinc to a formula promotes the retention of calcium. AL, a composition with niniscule amounts of magnesium and zinc, has a lower calcium retention (17%, 28 WO 2013/014654 PCT/IB2012/053872 Table 36); whereas the retention of calcium is significantly higher when the ratio of Ca/Mg was increased to 2/1 (A5), the calcium retention is 49% (Table 36). A higher proportion of magnesium, such as that present in A4, does not produce more changes in calcium retention (49%, Table 36). With respect to the minimum amount of magnesium required to provide the highest calcium retention, it appears a 2/1 Ca/Mg ratio is optimal. [01291 The addition of vitamin D3 increases calcium retention significantly (Figure 2 and Table 36). Calcium retention increased to 62% when vitamin D 3 was added to A5 (Table 36). This value is more than five times higher than that of the Caltrate' T and ('aACE groups. TABLE 36 Cumulative Net Percentage Of Calcium In Rats Treated With Elemental Supplements While Receiving Calcium Free Diet (n=5 per group) Treatment Cumulative net percentage of calcium (%) group Day 1 Day 2 Day 3 Day 4 CaltraterM 23.8 159 22.3 ± 16,8 -2.27 ± 40.0 0,734 35.7 Ca ACE -30.6 51.3 -9.88 ±26.5 4.88 24.0 11.1 20.0 Al 37.5 18.7 20.9 ± 15.5 20.8 15.0 17.2 12, 1 A4 40.9 ± 19.1 486 13. 7 49.1 ± 10.2 49 1 7.7" AS 36.4 24. 1 46.8 19.5 48.7± 18.4* 48.6 19.1 " A4 + Vit D 46.6 22.3 50.3 10.9 47.9 ± 14.8* 50.8 11.2* A5 + Vit D 43.7 ± 19,2 52.7 11,8 59.2 ± 7.6 '+ 62.0 ±5.2" <PO .n5, compared with Caltratem * : <0 05, ared with Ca CE +: P<0 ,0 5, c compared with Al; # :P < 0 . 0 5, compared with A4 + Vit. D. 101301 Magnesium appears to be required in order to maintain magnesium balance (i.e. to avoid magnesium depletion) (Table 37). Formulas (Caltrate m , CaACE and Al) that have miniscule amounts of magnesium caused a net loss of magnesium (Figure 3 and Table 37), 101311 The addition of vitamin D 3 has no significant effect on the retention of magnesium. The cumulative net percentage of magnesium did not change significantly after vitamin D was added to A4 and A5 (Figure 3 and Table 37). TABLE 37 Cumulative Net Percentage Of Magnesium In Rats Treated With Elemental Supplements 29 WO 2013/014654 PCT/IB2012/053872 While Receiving Calcium Free Diet (n=5 per group) Cumulative net percentage of magnesium (%) Day 1 Day 2 Day 3 Day 4 Gatrate -191.9 ±139.1 -1256±51.0 -1118 39.1 -116.5 37.7 Ca ACE -197,2 105.2 -150.4± 88 9 -115.3 62,7 -93.6± 37.2 A1 -7.3 ± 22.4* -67.9 33.3* -55.2 13.4 -64.4 24.6 A4 66.5 ± 8.7'* 68.1 6.4** 65.8 5.9** 60.9 4.7'* A5 23,7 46 3$ 37.6 37. 1'- 41.1 34.2'-* 39,6 ± 33.3 A4 + Vit D 46.3 2 49.3 1 49.3i 15.3** 48.9 ± 15.5' A5 + Vit D 16.0 20.0" 23 9± 21.57* 28.9± 17,9'* 27,223.. P- 0 .05, corpar ed wiLth Cal-tratem; P< 0 opared withaACE; :P<0, cormipa red with Al. [01321 The retention of zinc is highly variable; it is particularly true with formulas such as Caltrate, calcium acetate and Al that contain minute amounts of zinc (Table 38). The results also show that zinc balance became negative when the amount of zinc is low. [01331 The addition of zinc to formulas such as A4 and A5 did not significantly improve zinc balance (Table 38). The addition of magnesium to the formulas may have caused zinc balance to stay negative (Figure 4). [01341 However, the addition of vitamin D 3 to A4 and A5 made zinc balance positive (Figure 4 and Table 38). The importance of vitamin D 3 on zinc is clearly demonstrated in this set of studies. [01351 Figure 5 shows plasma elemental profiles after each treatment. There were no significant differences observed after elemental treatments. TABLE 38 Cumulative Net Percentage Of Zinc In Rats Treated With Elemental Supplements While Receiving Calcium Free Diet (n::::5 per group) Treatment Cumulative net percentage of zinc group Day 1 Day 2 Day 3 Day4 Caltrate Tm ~ -50. 6 50.0 -38.7 ± 23 8 -36.9 ± 264 -39.5 i 23 7 Ca ACE -107.1 85. 5 -77.7 59.0 -65.7 ± 66.7 -50.5 46.4 30 WO 2013/014654 PCT/IB2012/053872 A1 10.1 1 8.7 -0.348 22.2y 4.22 i 7.3y -2.79 ±64 A4 -61.0 38.8' -55.3 29.3" -58,3 ± 24.5 -33,8 23.9 A5 -8.05 45.3" 9.737 ± 39.5"' 9.96 ± 40.3'* 876 401** A4 + Vit D 27.2 40.7"' 43.7 ± 18.8 512 ±15. 1' 54.2 ±11. 2" A5 + Vit D 22,8 17.91 35.8 ± 17.8' 42.9 ± 1291 44.6 ± 10 14 : <0 0 5, compared with Caitrate ; * : P<0 .05, compared with Ca ACE; &:P<0.05, compared with A-4 G. Results: Normal diet [01361 Rats that received normal diet gained weight (Table 39). Elemental treatments have no significant effect on weight gain (p>0.05). TABLE 39 Body Weight Of Rats Receiving Normal Calcium Diet (n=5) Body weight of rats (g) Treatment group Day 1 Day 2 Day 3 Day 4 Day 5 CaltrateM 228.8 ± 4 6 232.8 2.6 233.8 ± 3.5 243.6 8.9 243.8 5.1 Ca ACE 242.0 ± 7.4 237.0 12.5 239.2± 139 238.6 13.9 244.0 12.8 Al 230.0± 415 233.8 8.0 238.2 ± 6.1 244.6 72 244.6 3.5 A4 234.8 7 7 238.6 5.1 238.2 ± 5.9 239,0 5.1 245,8 4.9 A5 239.6 10.3 243.0 13.9 245. ±13.6 245.4 13.4 248.6 14.4 Data are expressed as mean±iS.D. [01371 The pattern of calcium retention appears to be similar to that obtained from rats that received calcium free diet (compare Tables 36 and 40); suggesting calchim balance is dependent upon elemental treatments, despite the fact that the amount of calcium administered was approximately 10% of the animal's daily dietary intake (-130 to 140 ng of calcium per day). This observation strongly suggests that dietary calcium, present in the least absorbable carbonate form, was enhanced by elemental treatments. The treatment with Caltrate

T

"M has minimal effect, It is not surprising because CaltrateTM contains only calcium carbonate. The treatment with A5 has the most pronounced effect (Figure 6 and Table 40). TABLE 40 Cumulative Net Percentage Of Calcium In Rats Treated With Elemental Supplements 31 WO 2013/014654 PCT/IB2012/053872 While Receiving Normal Diet (n=5 per group) Treatment Cumulative net percentage of calcium (%) group Day 1 Day 2 Day 3 Day 4 Caltratem -6.9 ± 246 173 ± 7.5 21 3 ± 10.0 175 ± 10.2 Ca3 ACE 14.4± 24 0 26,9 ± 9.0 30.3 ± 4.9 31.9 ± 3.0 A1 31.4 33

.

5 ' 49.2t 3.8' 39,2 27.3 31,3 21.9 A4 19.3 12.6 23,7 ±9.4 26.2± 9.6 22.7± 7.3 A5 52.1 ±21.7 49.0 i 9.8 48.9 ± 20.4 45.3 ± 22.7 S :P<0.05, compared with Calt-rate T M ; *PO. 05, compared with Ca ACE; &:P<,.05, compared with A4 101381 Average dietary intake of magnesium by the study animals was approximately 35 mg. Magnesium balance for all study groups was positive (Figure 7 and Table 41). This observation is consistent with the observation obtained from animals receiving the calcium free diet, in that magnesium intake is required to maintain a positive balance (Tables 37 and 41). Interestingly, the day to day trend showed that animals treated with acetate formulas (CaACE, A1, A4 and A5 vs. Caltrate M 4 ) had consistently higher percentages of magnesium balance. TABLE 41 Cumulative Net Percentage Of Magnesium i Rats Treated With Elemental Supplements While Receiving Normal Diet (n=5 per group) Net accumulative percentage of magnesium (%) Treatment group ---------------------------------------------

------------------------------

Day 1 Day 2 Day 3 Day 4 -2.82 ± 1 ./'23. 3 P, 1 _ GaltraUeM -28 127. 3 ± 10.0 24.6± 6.9 16.7± 1.2 Ga ACE1 29.9 38 34.3 ± 25 37.7 27 Al 11.7± 1. 44.1 30.7 38,9± 22.9 31.5 17.0 A4 28. 2 ± 9.1' 34.0 7.8 36.8 7.2 35.0 ±4.4 AS 489± 25.3 489 ± 20.9 506 20.1 486 ± 21.0 $: P<0 . 5, compared with Caltrate M; P:<0 , 05, compared with A5 [01391 There were no statistical differences among elemental treatments in terms of zinc balance (Figure 8 and Table 42). The quantity of zinc administered via elemental formulas was no more than 30% of the daily dietary intake. It was noted that the addition of a high quantity of 32 WO 2013/014654 PCT/IB2012/053872 magnesium tended to lower zinc balance, a trend observed with A4 treatment (Figure 8 and Table 42). This observation is similar to that observed in the calcium free diet study (Table 38). 101401 Contrary to the calcium free diet study (Table 38), zinc balance was positive in this study (Table 42). This was achieved without vitamin D 3 (Figures 4 and 8, Tables 38 and 42). This apparent discrepancy may be due to the quantity of total zinc intake and/or the rate at which zinc was consumed. Elemental consumption, along with other nutrients, occurred throughout the feeding period which may last tip to 12 hours; whereas elemental treatments were given as a bolus. Concentration and ratio of nutrients presented to the intestinal wall may have a huge difference between bolus administration and dietary consumption. These differences could account for the difference in zinc balance. 101411 The results from the calcium free and normal diet studies clearly suggest that adequate dietary intake of elements is key to elemental balance. Elemental and vitamin D 3 supplementation are necessary if the diet in deficient in these nutrients. 101421 Figure 9 shows plasma concentration of calcium, magnesium and zinc after individual elemental treatments. There were no statistical differences in the concentration of these elements in plasma after elemental treatments (P>0.05). TABLE 42 Cumulative Net Percentage Of Zinc In Rats Treated With Elemental Supplements While Receiving Normal Diet n=5e group) Cumulative net percentage of zinc (%) Day1 Day2 Day 3 Day4 Caltratem 0.67 34.7% 29.5± 7.5 33.8 10.2 32.0i 7,8 Ca ACE 27.5 16.0A 0.9 7.3 45.6 5.9 48.4 ±4.4 Al 26.6 11. 2* 50.8 26.8 46.3 20.4 38.7 18.8 A4 17.7 10.3% 24.9 6.3' 27.6 7 2 27.6 5 0 A5 54.7 21.9 52.6 21.7 538 21.0 51,2 23.0 %:P<0,05, compared with A5 H. Results: Calcium Free Diet with Daily Consumed Doses of Calcium [01431 The objective of this study was to evaluate elemental balance when the daily intake of calcium, magnesium and zinc was replaced with elemental treatments. Animals, received de 33 WO 2013/014654 PCT/IB2012/053872 ionized water ad libitum (DI Water group), were fed normal calcium diet. Animals, substituting their daily calcium intake by Al or A5, were fed calcium free diet. It is apparent that the gavage procedure did not have an effect on the body weight of the animals (Table 43). Elemental treatments, however, induced a significant reduction in body weight. TABLE 43 Body Weight Of Rats Receiving Calcium Free Diet And Daily Consumed Doses Of Calcium (::::4) Treatment Body weight of rats (g) group Day 1 Day2 Day3 Day4 Day5 DI Water 200.8 ± 2.50 207.0 ± 3.9 209.0 ± 8 7 209.5 ± 9.9 215.5 11.7 Al 198.0± 9.1 183.5± 7.7 178.3± 81 180,8 10.2 186.0 8.0 A5 194.0 8,2 182.3± 7.1 179.8± 7 2 179.0± 7.7 181.5 6.8 Note: There is no statitical si gni fi cnt di erence between Al and A5. There is statistical difference between Al and Dl (p < 0.001), and between A5 and DI (p<0.01). [01441 Contrary to the results obtained from the normal and calcium free diet studies, magnesium has a minor effect in enhancing calcium retention (Figure 10 and Table 44). The administration of a soluble form of calcium, calcium acetate, significantly enhanced calcium balance (Figure 10 and Table 44). Table 44 Cumulative Net Percentage Of Calcium In Rats Treated With A Daily Consumed Dose Of Calcium While Receiving Calcium Free Diet (n=4 per group) Treatment Net accumulative percentage of Ca (%) group Day 1 Day 2 Day 3 Day 4 DI Water 2.87 5.4 3 89 7.6 5.72 ± 43 5.41 ± 2 A1 46.3 14.7 37.7 ±8.9 37.4 ± 1.3 42.7 ± 3.1 A5 54.9± 12. 7 567± 10 .3" 501 ±7.5 47.4 ±8.0* :P< 005, when compared with DI; @:p<C.05m when compared to Al [01451 Consistent with the calcium free diet study described above, magnesium was required to maintain a positive magnesium balance (Figure 11 and Table 45). 34 WO 2013/014654 PCT/IB2012/053872 Table 45 Cumulative Net Percentage Of Magnesium In Rats Treated With A Daily Consumed Dose Of Calcium While Receiving Calcium Free Diet (n=4 per group) Treatment Net accumulative percentage of Mg (%) group Day 1 Day 2 Day 3 Day 4 DI Water -32.2 ± 12.4 -17.0 ± 10A -7.9 10.0 -2.59 10.4 Al -75.9 ±500 -276± 27.7 -6.54 19.4 36 ± 18.0 A5 18.3 ± 12.7 14.4 ± 8.6' 7.0 5.2 4.4i 84 *w:P<0.05, when compared with Dl; @:P<0.05m when compared to Al [01461 Despite a higher amount of zinc administered with A5, zinc balance was significantly lower than that of the DI Water group, providing further support that high calcium and magnesium concentration in the intestine could have diminished zinc absorption. (Figure 12 and Table 46). The amounts of zinc administered between the DIl Water and Al groups were similar. However, similar to that of A5, zinc balance was significantly lower than that of DI Water (Figure 12 and Table 46); suggesting high solution concentration of calcium in the intestine may interfere with zinc absorption. 101471 This set of results suggest that elemental dietary intake of elements does not produce the same effects when compared to that of an equivalent bolus dose. 101481 Taking all the study results into consideration, A5 produces the most consistent calcium balance under different experimental/dietary conditions (compare results on Tables 36, 40 and 44). The addition of vitamin D 3 enhances calcium retention of A5 when the subject is deficient in dietary elements (Table 36). 101491 Figure 13 shows plasma concentrations of calcium, magnesium and zinc after each elemental treatment. No statistical differences were found in these profiles (P>0,05). TABLE 46 Cumulative Net Percentage Of Zinc In Rats Treated With A Daily Consumed Dose Of Calcium While Receiving Calcium Free Diet (n=4 per group) Treatment Net accumulative percentage of Zn (%) group Day 1 Day 2 Day 3 Day 4 35 WO 2013/014654 PCT/IB2012/053872 D! Water -26.5 ±37.7 -10.8 22.9 -4.45 ± 17.3 -1 80 ± 12 2 Al -42.9 ± 25.9 -67.3 16.3* -69.5 ± 7.3* -58.5 6.2* A5 23.7 ± 16 2* -9.09± 19

.

3 -45.1± 11.8' -63 2 16.2* *:P<cc5, when compared with DI; < P0 .05, when compared to Al EXAMPLE 7 101501 The objectives of this study were to evaluate the effects of salt, mineral composition and vitamins on the rate of bone loss in an ovariectomized rat model. [01511 One hundred 4.5-month-old female Sprague-Dawley rats were used and housed at the Laboratory Animal Services Center at the Chinese University of Hong Kong with 12-h light-night cycle. Free cage movement was allowed with access to the normal calcium pellets and tap water. Daily consumption of calcium was approximately 140 mg, similar to that recorded in animals who participated in the balance studies, Ovariectomy (OVX), the removal of ovaries from the female rats, was performed on all rats at 6-month of age with the exception of the sham control, 101521 Three weeks after OVX, all the rats recovered from the trauma of the surgery. The rats were randomly divided into different treatment groups or control groups and each group contained six rats. Four calcium formulas (Al, A4, A5 and A6) and Caltrate' M were investigated in the present study. The CaltrateTM group served as an elemental treatment control, All formulas were dissolved in distilled water, while Caltrate' was in suspension in distilled water. The solution or suspension was given to the rats daily for 8 weeks by gavages. The dose of all formulas was calculated based on a calcium dose of 53.14 mg/kg/day. Dose of vitamin D3 and vitamin K2 was 12.75 1U/kg/day (equivalent to 800 IU/70 kg man/day) and 1.71 pg/kg/day (equivalent to 120 pg! 70 kg man/day), respectively. All the treated rats were weighed daily and the mass data were recorded. The rats in two control groups (sham control and normal control) were given the equivalent volume of distilled water in parallel. For the groups with the treatment of bisphosphonate, alendronate (14 Ag/kg/2-week) was injected subcutaneously on the back of the rats once every two weeks. 101531 At the end of 8 weeks, the rats were anesthetized using isoflourane. Blood sample was then taken via heart puncture. The rats were then euthanized under anesthesia by neck dislocation, and right hip, right femur and right tibia of each rat were collected for analysis. Plasma was 36 WO 2013/014654 PCT/IB2012/053872 collected from blood samples centrifuged at 1500 g for 15 in. Plasma concentrations of calcium, magnesium, and zinc were measured using ICP-OES. 101541 Results show that plasma calcium levels were not statistically different from that of the sham control (p>0.05) and the values are all within normal levels (90-110 mg/L). All plasma concentrations of Mg were within the normal range (18-36 mg/L). No significant difference in magnesium plasma concentrations was observed except normal control (without surgery) has a mean value higher than that of A4+Vit D+Vit K (p<0.05). Similarly, plasma concentrations of Zn in all rats reached the rat normal concentration at about 1.26 mg/L. Zn plasma concentrations of rats in the normal control was significantly higher than that of sham control rats and also the rats treated with A5+vitarnin D and A4+vitamrine D+vitarnin K (p<0.0S), 101551 Body weight changes for different treatment groups are shown in Figure 14. As expected, weight gains in the OVX rats were significantly greater than the nonnal rats (p<0.05). [01561 The effects of test substances on bone mineral density (BMD) are shown on Figures 15 and 16. Trabecular BMD of Distal Femur BMD values of groups AL, A5+Vit D, Bis-IA1+Vit D, Bis+A4-+Vit D, Bis+A5'+Vit 1) and Bis- Caltrate -Vit D are significantly higher than that of the OVX control (Figure 15), suggesting these treatments significantly slow down the rate of loss of bone mass. The addition of vitamin K did not have any significant effect on reducing the rate of bone loss. Similar observations were obtained for the average values of trabecular BMD of Proximal Tibia, except the value of CaltrateTM was high enough to become statistically different (p<0.05, Figure 16). Again, vitamin K did not have any significant contribution. The treatment with A5-Vit D provided consistently higher BMD at distal femur and proximal tibia, suggesting this formula may have an advantage over the other elemental formulas. Although, the addition of bisphosphonate provides consistently better results, the difference, when compared to A5-Vit D and other elemental formula, such as Al, was not significant (Figures 15 and 16). [01571 The BMD results of A1 are similar to that of A5 - vit D. This is not surprising because Al animals were fed normal calcium diet which contains a significant amount of magnesium. 101581 The OVX rat model used in this study did not permit evaluation of maximum bending force and failure energy after each treatment because the values obtained from the OVX control and that of the Sham were insignificantly different from each other (P>0.05). 37 WO 2013/014654 PCT/IB2012/053872 EXAMPLE 8 Fortification of Juices with A5 101591 Fruit juices contain a number of acids such as malic acid, citric acid, etc. which may alter the solubility and hence the recovery of the three key elements in the formulae, hence changing the absorbability of these elements when administered in juice format. 101601 The objectives of this study were to evaluate the effects of temperature and storage on the recovery of calcium, magnesium and zinc in A5 after mixing with filtered and unfiltered orange, grape and carrot juice. [01611 A 2.6 g or 500 mg amount of A5 was weighed accurately and mixed with 330 ml of water or either filtered or unfiltered grape, orange or carrot juice. The specimens were prepared at either 4 or 21 "C. The elemental content was measured using ICP-OES. [01621 Small quantities of calcium, magnesium and zinc were found in orange, grape and carrot juice (Tables 47, 50 and 53). Temperature arid filtration had no effects on the recovery of calcium, magnesium and zinc of A5 when 2.6 g of A5 was used for the study (Tables 48, 51 and 54). TABLE 47 Content of the three key elements in fresh orange juice Content. (mna/L) S ame l e -------------------------------------------------------------- Ca Mg Zn Fresh o-rane7.7 ±0.87 115 ±0.9 0.47 ± 0.03 Data are expressed as Mea.n ± S. D . (n=3) TABLE 48 Elemental recovery of the 3 key elements of A5 2.6 g)in orange juice at 40C and 21"C Solubitty (g/L) sa mIl e Ca Mg , Zn 21 OC 4 C 21 OC 4C 2 1 . .997-1 0.528 0.5 0.047 0.o4 nitere i 0 .012 +o, 70.004 0.000 0.78 094 421 0.52 0.045 0.045 1tered 0.008 i.08 0.007 i .. 001 k, 0~ C -9 09 002 01 0.003 Data are expressed as mean S. D. (n=3) 38 WO 2013/014654 PCT/IB2012/053872 TABLE 49 Elemental recovery from 500 mgof A5 in 330 ml orange juice stored at 4 C Solubility (g/L) Ca Mg Zn Filtered Unfiltered Filtered- Unfiltered Filtee.d Uflt ere Fresh 0.273 i 0.24 0.170 i 0.167 i 0.005 0.0089 0.005 0.009 0.001 0.003 0.0012 0.0003 - -ne 0.272 i 0.172 i .171 0.104 11 -.- i 0.0139 Week 0.005 0.017-' 0.001 0.010 0.001 0.0088 Data are expressed as Mean i S.D. (n=3) ***P<0.001 comparing wih fresh group TABLE 50 Content of the three key elements in fresh grapefruit juice Same Content (mg/L) Ca Mg Zn Fresh graperuit 48.2 ± 0.79 104 ± 1.6 0.536 ± 0.008 juice DLta are expressed as mean ± S.D. (n=3) TABLE 51 Comparison of elemental recovery of A5 (2.6 g) in grapefruit juice at 4"C and 21C Solubility (g/L) Sample Ca Mg Zn 40C 210C 40C 210C 40C 210C 0.958 0.968 0.55 0.518 0.046 0.046 Unfiltered 0.010 0.016 0.005 0.010 0.001 0.001 0.981 0.975 0.516 0.520 0.045 0.048 Filtered i i i i 0.018 0.004 0.027 0.005 0.002 0.002 Data are expressed as mean i S.D. (n=3) TABLE 52 Elemental recovery from A5 (2.6 g) in distilled water at 4 and 21 "C Solubility (p/L) Temperature --------------------------------------------------- Ca Mg Zn '1 Oc 0.875 t 0.407 i 0.024 i 0.018 0.000 0.002 21 00 0.897 ± 0.404 + 0.028 i 0.016 0.009 0.001 Data are expressed as Mean r S.D. (n=3) 39 WO 2013/014654 PCT/IB2012/053872 101631 Similarly, temperature has no effect on the recovery of A5 elements in distilled water (Table 52). 101641 Storage at 4 'C for a week did not change the recovery of calcium, magnesium and zinc when 2.6 g of A5 was dissolved in 330 ml of filtered and unfiltered orange and grape juice (Tables 48 and 51). However, when 500 mg of A5 was used instead, the recovery of calcium and magnesium was significantly lowered from the unfiltered orange juice (Table 49). The lower recovery of calcium from unfiltered orange juice suggests that the pulp in orange juice may bind Ca and Mg in A5. Carrot juice did not have this problem (Table 54). TABLE 53 Content of the three elements in fresh carrot juice Content (mg/L) CaMg Zn i 37.499 ± 75.279 0. 7045 ± Fresh carrot uice I 1 0 4.-.30 6. 1-R3 0 .0 9 3 Data arKe expreIssed as Mean i S. D. (nl=3) TABLE 54 Elemental recovery from A5 in 330 ml carrot juice stored at 4 0 C Solubility (/ L) Ca Mq Zn 2 500 mg 26 500 mg 26 500 m g L- r m. grams Fresh 0. ' t .196 . t >.5 0.0235 i 0 0.0 1 0.009 0 01 0.001 ?0 C0 0.0 one Week 0 0.188 C .48 i -1 i 0.0491 001 0 .033 0.006 0.016 0 .00 0.004 ** q.0001 Datlia arce expresse as Mean i S.D. ( * comparing w g group 101651 This set of studies suggests that A5 can be used to fortify a number of juices and water. The 2.6 g of A5 provides a daily requirement of the three key elements for the prevention of osteoporosis: 300 mg of calciun, 150 trig of magnesium and 5.6 ig of zinc. 500 trig of A5 is intended to provide a serving of these elements in the functional food format. 40 WO 2013/014654 PCT/IB2012/053872 EXAMPLE 9 Tablet Formulation Compositions [01661 The relatively low calcium content in A5 has posed a challenge in creating a solid form with a size that is acceptable to end-users, The following formulation was created in tablet form (Table 55): TABLE 55 Ingredients Wt./Tablet % Comp. Wt. for 2000 (mg) tablets batch (g) Calcium 550 93.43 110 Acetate blend Dry Vitamin D 3 3.25 0.55 6.5 100 GFP/HP* Kollidon VA 64 32.5 5.52 65 MagnesiUm 2.93 0 .50 5.86 STearat e Total 588.68 100.00 1177.36 * Equivalent to 250 IU of Vitamin D 3 101671 The calcium acetate blend in the above table comprises 14% calcium acetate, 7% muagnesiun acetate and 0.7% zinc acetate, Magnesium stearate was used as a lubricant. 101681 The Dry Vitamin D3 100 GFP/I-P composition (as mentioned in the certificate of analysis provided by BASF) is as follows: Ingredients (CAS No.) Concentration (w/w) Sucrose- -1) 30 .0 . 0% Starch (q05-25-8) 20.0 - 30.0% Gum AraiJc (9000-01-5) 15.0 - 2 % Gl cerides (73398-61-5) 5.0 -. 0% Water (7732-18-5) 1.0 -- 5.0 Tricalcium phosphate (7758-87- < 1.0% /4) Vitamin D (67-97-0) >=0 .25% DL - alpha - Tocopherol <0.2% (10 191 -4 1-0) 41 WO 2013/014654 PCT/IB2012/053872 101691 Assay value: 100,000 IUJ Vitamin D3/g (=2500 microgram cholecalciferol/g). The target weight of Vitamin D 3 per tablet is 2.5 mg. 30% extra Vitamin D 3 has been added per tablet as overage. The manufacturer assay value is 100000 IU/g i.e. 100 lU/mg. Since 2.5 mg (3.25 mg with 30% overage) has been used each tablet has ~250 IU of Vitamin D 3 . [01701 The tablets were created according to the following steps: [01711 Step 1: Calcium Acetate blend provided was sieved through 40 mesh screen and 100/120 mesh screen. The fraction that passed through the 40 mesh screen and was retained on 100/120 mesh screen was used for formulation. The fraction of calcium acetate above 40 mesh and below 100 mesh was not used for formulation. This fraction was chosen to keep the particle size similar to other ingredients - Vitamin D 3 and Kollidon Va 64. 101721 Step 2: Blending 01: 6.5 g of dispensed Dry Vitamin D3 100 GFP/HPI and 65 g OF Kollidon VA 64 were blended for 5 minutes at a speed of 25 rpm using a small tumble blender to produce Blend 01, 101731 Step 3: Blending 02: 250 g of dispensed Calcium Acetate blend (Blend 01 * 3.49) prepared in Step I was mixed with Blend 01 prepared in Step 2 for 5 minutes to produce Blend 02 (using tumble blender at 25-30 rpm), 101741 Step 4: Blending 03: 250 g of dispensed Calcium Acetate blend prepared in Step I was mixed with Blend 02 prepared in Step 3 for 5 minutes to produce Blend 03 (using double cone blender at 25-30 rpm). [01751 Step 5: Blending 04: 600 g of dispensed Calcium Acetate blend prepared in Step I was mixed with Blend 03 prepared in Step 4 for 9 minutes to produce Blend 04 (using double cone blender at 25-30 rpm) [01761 Step 6: Blending 05: 5.86 g of dispensed Magnesium Stearate was mixed with Blend 04 prepared in Step 5, for 2 minutes. [01771 Step 7: The final blend prepared above was dispensed using a Rotary table press with target tablet weight of 588.7 g. 42 WO 2013/014654 PCT/IB2012/053872 101781 The following formulation was also created in tablet form: Ingredients Wt./Tablet % Comp. Wt. for 2000 (mg) tablets batch (g) Calcium 1102 93.43 2200 Actate blend Dry Vitamin D 3 6.5 0.55 13 100 GFP/HP* Kollidon VA 64 65 5.52 130 Magnes ium 5 .8 6 0 . 50 11. 72 Stea rate Total 1177.36 100.00 2354.72 * Equivalent to 500 TU of Vitamin D 3 [01791 The calcium acetate blend in the above table comprises 14% calcium acetate, 7% magnesium acetate and 0,7% zinc acetate. Magnesium stearate was used as a lubricant. 101801 The Dry Vitamin D 3 100 GFP/HP composition (as mentioned in the certificate of analysis provided by BASF) is as presented above. [01811 Assay value: 100,000 IU Vitamin D 3 /g (=2500 microgram cholecalciferol/g). The target weight of Vitamin D 3 per table is 5 mg. 30% extra Vitamin D.3 has been added per tablet to account for loss due to degradation. The manufacturer assay value is 100000 IU/g i.e. 100 IU/mg. Since 5 ig (6.5 mg with 30% overage) has been used each tablet has ~500 IU of Vitamin D 3 . 101821 The tablets were created according to the following steps: [01831 Step 1: Calcium Acetate blend provided was sieved through 40 mesh screen and 100/120 mesh screen. The fraction that passed through the 40 mesh screen and was retained on 100/120 mesh screen was used for formulation. The fraction of calcium acetate above 40 mesh and below 100 mesh was not used for formulation. This fraction was chosen to keep the particle size similar to other ingredients - Vitamin D 3 and Kollidon Va 64, [01841 Step 2: Blending 01: 13 g of dispensed Dry Vitamin D_ 100 GFP/HP1 and 130 g OF Kollidon VA 64 were blended for 5 minutes at a speed of 25 rpm using a small tumble blender to produce Blend 01. 43 WO 2013/014654 PCT/IB2012/053872 101851 Step 3: Blending 02: 500 g of dispensed Calcium Acetate blend (Blend 01 * 3.49) prepared in Step I was mixed with Blend 01 prepared in Step 2 for 5 minutes to produce Blend 02 (using double cone blender at 25-30 rpm). 101861 Step 4: Blending 03: 500 g of dispensed Calcium Acetate blend prepared in Step 1 was mixed with Blend 02 prepared in Step 3 for 5 minutes to produce Blend 03 (using double cone blender at 25-30 rpm). 101871 Step 5: Blending 04: 1200 g of dispensed Calcium Acetate blend prepared in Step 1 was mixed with Blend 03 prepared in Step 4 for 9 minutes to produce Blend 04 (using double cone blender at 25-30 rpm). 101881 Step 6: Blending 05: 11.72 g of dispensed Magnesium Stearate was mixed with Blend 04 prepared in Step 5, for 2 minutes. [01891 Step 7: The final blend prepared above was dispensed using a Rotary table press with target tablet weight of 1.17 g. [01901 The size of these two formulations has proven to be acceptable to a test population. EXAMPLE 10 Gel Cap Formula Consisting of Fish Oil 101911 A gel cap formula for the Calcium Acetate blend was created to enhance end user acceptance, increased solubility of vitamin D, and increased efficacy on bone mineral density. [01921 Vitamin D 3 is an oil soluble vitamin. It can be dissolved using lipophilic substances. [01931 Fish oil containing omega 3-6-9 fatty acids is known to have beneficial effects on bone health (32). This oil also has the advantage of dissolving vitamin D 3 , obviating the granulation process of Calcium Acetate blend as described in Example 9. 101941 Fish oil has been found to have the ability to increase the bulk density of Calcium Acetate blend by displacing air from the powder. 44 WO 2013/014654 PCT/IB2012/053872 101951 Examples of oil to Calcium Acetate blend ratios include, but are not limited to, about 1:1, 1.5:1 and 2:1. 101961 Examples of oil to calcium ratios include, but are not limited to, about 1:0.14, 1,5:1 and 2:1. [01971 Examples of oil to magnesium ratios include, but are not limited to, about 1:0,07, 1.5:0.07 and 2: 0,07. 101981 Examples of oil to zinc ratios include, but are not limited to, about 1:0.007, 1.5:0.007 and 2:0.007. 101991 The dosage of vitamin D 3 ranges from 30 to 300 IU, [02001 Soft gel capsules can be manufactured using conventional methods (33) [02011 Gel capsules made with this blend in dose sizes amounting to two to four capsules a day will be acceptable. The size of a gel capsules will be equivalent to or smaller than that described in Example 9. EXAMPLE 11 Optimization of Elemental Formula [02021 The objective of this example is to design an elemental formula which would provide an optimal mix of vitamin D 3 and acetate salts of calcium, magnesium and zinc for supporting bone health. [02031 It is a general belief that the bioavailability of calcium is independent of the solubility of calcium salts (Heaney, 1999). Low levels of magnesium and zinc are associated osteoporosis (Mutlu et al,, (11). Vitamin D 3 enhances calcium absorption (Christakos et, al., 2011) and therefore, is an important component of an ideal elemental formula, 102041 Results presented in this invention clearly show that the bioavailability calcium is dependent on the solubility of a calcium salt in the gastrointestinal fluids, An optimal ratio of calcium to magnesium is required to enhance calcium absorption. Vitamin D3 is responsible for increasing calcium absorption and preventing zinc depletion. 45 WO 2013/014654 PCT/IB2012/053872 102051 A formula containing calcium, magnesium, zinc and vitamin D 3 may not work because the form of the elements and the amount of vitamin D 3 , are not necessarily formulated in the right ratios in terms of absorbable fractions. The lack of clinical effect of a blend of calcium, magnesium, zinc and vitamin Di is a good example (Braam et. al, 2003). The confusion in the literature relating to calcium absorption and the equivocal clinical trial results on bone mineral density by calcium supplementation has created problems for experts skilled in the art in designing an optimal formula of a calcium blend. [02061 Using the acetate salts of calcium, magnesium and zinc with the appropriate addition of vitamin D 3 , an optimum calcium supplement is designed. The ratio of calcium to magnesium is generally 2:1, the ratio of magnesium to zinc is 10:1 and the daily dosage of vitamin D3 ranges from 500 to 1000 IU. [02071 The bioavailability of calcium described in this invention is appropriately 2 to 3 times higher than that of Caltrate. The dosage of calcium should be half to one third of that of CaltrateTM, [02081 The recommended intake of calcium from all sources is 1000 mg. The average intake of calcium from dietary sources is 400 mg. It is recommended that 600 nmg of calcium should be provided as a supplement: usually this implies that the source of calcium is from calcium carbonate. The recommended dose of calcium from this invention is 200 to 300 mg. This will provide 100 to 150 mg of magnesium and 5 to 7.5 mg of zinc. In addition to dietary intake, the supplementation of magnesium and zinc will also provide an adequate daily requirement of the elements. References 1. Ilich JZ, Brownbill RA, & Tamborini L (2003) Bone and nutrition in elderly women: protein, energy, and calcium as main determinants of bone mineral density. European journal of clinical nutrition 57(4):5 54-565. 2. Iiich JZ & Kerstetter JE (2000) Nutrition in bone health revisited: a story beyond calcium. Journal of the Amnerican College vfNutrition 19(6):715-7 37. 3. Seelig MS, Altura 13M, & Altura 13T (2004) Benefits and risks of sex hormone replacement in postmenopausal women. Journal of the American College ofNutrition 23(5):482S-496S. 4. Heaney RP (1993) Thinking straight about calcium. The New England journal of medicine 328(7):5 03-505. 5. Heaney RP (1993) Nutritional factors in osteoporosis. Annual review ofnutrition 13:287 46 WO 2013/014654 PCT/IB2012/053872 316. 6. Riis B, Thomsen K, & Christiansen C (1987) Does calcium supplementation prevent postmenopausal bone loss? A double-blind, controlled clinical study. The New England Journal of medicine 316(4):1 73-177. 7. Hunt CD & Johnson LK (2007) Calcium requirements: new estimations for men and women by cross-sectional statistical analyses of calcium balance data from metabolic studies. Am J Cin Nuir 86(4):1054-1063. 8. Bass M, Ford MA. Brown B, Mauromoustakos A, & Keathley RS (2006) Variables for the prediction of femoral bone mineral status in American women. Southern medicaljournal 99(2): 115-122. 9. Kanders B, Dempster DW, & Lindsay R (1988) Interaction of calcium nutrition and physical activity on bone mass in young women. JBone Miner Res 3(2): 145-149. 10. Celotti F & Bignamini A (1999) Dietary calcium and mineral/vitamin supplementation: a controversial problem. The Journal of international medical research 27(1):1-14. 11. Mutlu M, Argun M, Kilic E, Saraymen R, & Yazar S (2007) Magnesium, zinc and copper status in osteoporotic, osteopenic and nonnal post-menopausal women, The Journal of international medical research 35(5):692-695. 12. Abrams SA & Atkinson SA (2003) Calcium, magnesium, phosphorus and vitamin 1) fortification of complementary foods. JNutr 133(9):2994S-2999S. 13. Lowe NM, Lowe NM, Fraser WD, & Jackson MJ (2002) Is there a potential therapeutic value of copper and zinc for osteoporosis? The Proceedings of the Nutrition Societv 61(2):181-185, 14. Saltman PD & Strause LG (1993) The role of trace minerals in osteoporosis. Journal of the American Cioliege of Nutrition 12(4):384-389. 15. Angus RM, Sambrook PN, Pocock NA, & Eisman JA (1988) Dietary intake and bone mineral density. Bone and inneral 4(3):265-277. 16. Angus RM, Pocock NA, & Eisman JA (1988) Nutritional intake of pre- and postmenopausal Australian women with special reference to calcium, European journal ofclinical nutrition 42(7):617-625. 17. Abraham GE & Grewal H (1990) A total dietary program emphasizing magnesium instead of calcium, Effect on the mineral density of calcaneous bone in postimeniopausal women on hormonal therapy. The Journal ofreproductive medicine 35(5):503-507. 18. Bo-Linn GW, et al. (1984) An evaluation of the importance of gastric acid secretion in the absorption of dietary calcium. The .Journal of clinical investigation 73(3):640-647, 19, Tsugawa N, et aL (1995) Bioavailability of calcium from calcium carbonate, DL-calcium 47 WO 2013/014654 PCT/IB2012/053872 lactate, L-calcium lactate and powdered oyster shell calcium in vitamin D-deficient or replete rats. Biological & pharmaceutical bulletin 18(5):677-682. 20. Heaney RIP, Dowell MS, & Barger-Lux MJ (1999) Absorption of calcium as the carbonate and citrate salts, with some observations on method, Osteoporos Int 9(1):19-23. 21. Heaney RP, Dowell MS, Bierman J, Hale CA, & Bendich A (2001) Absorbability and cost effectiveness in calcium supplementation. Journal of the American College ofNutrition 20(3):239-246. 22. Tsugawa N, et al. (1999) Intestinal absorption of calcium from calcium ascorbate in rats. Journal of bone and mineral metabolism 17(1):30-36. 23. Cai J, Zhang Q. Wastney ME, & Weaver CM (2004) Calcium bioavailability and kinetics of calcium ascorbate and calciurn acetate in rats. Experimental biology and medJicine (Mdvwood, N.J 229(1):40-45. 24. Coudray C, et al. (2005) Study of magnesium bioavailability froi ten organic and inorganic Mg salts in Mg-depleted rats using a stable isotope approach. Magnes Res 18(4):215-223. 25. Lee HH, Prasad AS, Brewer GJ, & Owyang C (1989) Zinc absorption in human small intestine. The American journal ofphysiolog' 256(1 Pt 1):G87-9 1, 26, Abrams SA, Griffin IJ, & Herman S (2002) Using stable isotopes to assess the bioavailability of minerals in food fortification programs. Food and nutrition bulletin 23(3 Suppl):158-165. 27. Smith JC, Jr., Morris ER, & Ellis R (1983) Zinc: requirements, bioavailabilities and recommended dietary allowances. Prog Clin Biol Res 129:147-169. 28. Ellenbogen L & Buono LC (1999) Offic USPaT. 29. Walsdorf NB, Alexandrides G, & Pak CYC (1991) Office USPaT. 30. Jackson SD & Blumberg JB (1997) Office USPaT. 31. Li J & Li X (1995) P.R.C. SIPOoT. 32. Weiss LA Barrett-Connor E, & von Muhlen D (2005) Ratio of n-6 to n-3 fatty acids and bone mineral density in older adults: the Rancho Bernardo Study, The American journal of clinical nutrition 81(4):934-938. 33. Anderson JT, et al. (1975) Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton) 15th Ed. 48

Claims (10)

1. A method of preparing tablets comprising calcium acetate, magnesium acetate, zinc acetate and vitamin D 3 , the method comprises the steps of: (i) blending a first calcium composition comprising calcium acetate, magnesium acetate, and zinc acetate with a composition comprising vitamin D 3 ; and (ii) blending the composition obtained from (i) with a second calcium composition comprising calcium acetate, magnesium acetate, and zinc acetate; (iii) performing one or more rounds of blending wherein in each blending a composition obtained from a previous step is blended with yet another calcium composition comprising calcium acetate, magnesium acetate, and zinc acetate, thereby obtaining composition comprising calcium acetate, magnesium acetate, zinc acetate and vitamin D 3 for preparing tablets.

2. A tablet produced by the method of claim 1.

3. A method of preparing soft gel capsules comprising calcium acetate, magnesium acetate, zinc acetate, oil and vitamin D 3 , the method comprises the steps of: (i) dissolving vitamin D 3 in oil comprising omega 3 or omega 3-6-9; and (ii) blending the composition obtained from (i) with a calcium composition comprising calcium acetate, magnesium acetate, and zinc acetate, thereby obtaining composition comprising calcium acetate, magnesium acetate, zinc acetate, oil and vitamin D 3 for preparing soft gel capsules.

4. The method of claim 1 or 3, wherein the calcium composition comprises at least 10 percent by weight of calcium acetate, at least 5 percent by weight of magnesium acetate, and at least 0.2 percent by weight of zinc acetate.

5. The method of claim 3, wherein the oil is fish oil or flaxseed oil.

6. The method of claim 3, wherein the oil to calcium acetate blend ratio is selected from the group consisting of 1:1, 1.5:1 and 2:1. AMENDED SHEET (ARTICLE 19)

7. The method of claim 3, wherein the oil to calcium ratio is selected from the group consisting of 1:0.14, 1.5:1 and 2:1.

8. The method of claim 3, wherein the oil to magnesium ratio is selected from the group consisting of 1:0.07, 1.5:0.07 and 2:0.07.

9. The method of claim 3, wherein the oil to zinc ratio is selected from the group consisting of 1:0.007, 1.5:0.007 and 2:0.007.

10. A soft gel capsule produced using the method of claim 3. AMENDED SHEET (ARTICLE 19)