Patent Application
20140342018
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 (Ilich and Kerstetter, 2000; Ilich et al., 2003).
Dietary supplements with calcium were thought to be primary to maintaining bone health in the past 50 years (Seelig et al., 2004). 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 (Heaney 1993b; Heaney, 1993a).
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 (Riis et al., 1987), all showed that calcium had a significant beneficial effect.
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 (Ilich et al., 2003). It has also, however, been found that high levels of calcium intake may be linked to higher incidence of cardiovascular disease (Seelig et al., 2004).
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.
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 (Seelig et al., 2004). The daily requirement of calcium was recently re-evaluated (Hunt and Johnson, 2007). It was found that an average intake of 749 mg of calcium is required, an estimate lower than previously estimated.
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 (Riis et al., 1987).
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 (Bass et al., 2006). 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 mg—lower than that of the RDA of 1000 and 310 mg, respectively.
Physical activity was associated with increase in vertebral bone mineral density (Kanders et al., 1988). 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 (1999). They reported that calcium 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.
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. (2007) 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 zinc retention. Consequently, calcium supplementation in the absence of the administration of other optimized amounts of minerals may further aggravate the severity of osteoporosis (Ilich and Kerstetter, 2000; Lowe et al., 2002; Abrams and Atkinson, 2003). Apart from calcium, magnesium, zinc, manganese and copper deficiencies are linked to osteoporosis (Saltman and Strause, 1993).
Angus et al. (1988b) showed that calcium was not a predictor of bone mineral density in pre- and post-menopausal women. Magnesium 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 (Angus et al., 1988a), while other nutrients such as magnesium, etc. were also deficient.
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 (Abraham and Grewal, 1990). 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 11% increase in bone mineral density, whereas, the other group had a non-significant increase of 0.7%.
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.
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 (Bo-Linn et al., 1984). 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 (Bo-Linn et al., 1984). The average amount absorbed in humans is 24%.
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 (Tsugawa et al., 1995). 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 PTH, do not provide truly accurate comparisons of calcium bioavailability.
Using Ca45 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 mg calcium); however, calcium absorption from calcium carbonate was slightly but significantly better than calcium citrate (Heaney et al., 1999). Heaney (2001) reported that the rates of urinary excretion for three marketed calcium products (marketed calcium carbonate, encapsulated calcium carbonate and marketed calcium citrate) were identical.
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 (Tsugawa et al., 1999).
Bioavailability of calcium acetate was measured using 45Ca (Cai et al., 2004). 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 from that of calcium chloride and calcium phosphate.
Magnesium absorption from 10 organic and inorganic salts was tested in rats (Coudray et al., 2005). 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.
Zinc absorption occurs throughout the small intestine and it is dose dependent in humans (Lee et al., 1989). With respect to zinc, there was no difference in the bioavailability of zinc oxide and zinc sulfate as measured using dual isotope techniques (Abrams et al., 2002); 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. Ten mg of zinc per day is the recommended intake (Record et al., 1985). The recommended daily allowance of zinc was 6 mg (Smith et al., 1983).
The following are inventions and disclosures noteworthy in the art:
U.S. Pat. No. 5,879,698 issued in 1999 for a calcium dietary supplement comprising calcium, magnesium, zinc, etc. (Ellenbogen and Buono, 1999). The calcium to magnesium ratio is high and the range of magnesium used was between 50 to 150 mg. 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.
U.S. Pat. No. 6,716,454, awarded to Meignant and Stenger in 2004, cites a composition which consists of calcium and a vitamin D mixture.
U.S. Pat. No. 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 B12, folate and Vitamin B6. The present application, however, does not include phosphorus.
Mazer et al. were granted U.S. Pat. No. 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.
In another patent, U.S. Pat. No. 5,075,499, issued in 1991, Walsdorf et al. described the synthesis of dicalcium citrate-lactate by mixing stoichiometric mixtures of citrate and lactate salts to produce the calcium salt (Walsdorf et al., 1991).
Krumhar and Johnson designed a diet supplement for bone health, disclosed in U.S. Pat. No. 7,029,703 which issued in 2006, consisting of microcrystalline calcium hydroxyapatite, protein (mostly collagen), phosphorus, fat, and other minerals. It also contains vitamin D3, 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.
Sultenfuss, in U.S. Pat. No. 5,514,382, issued in 1996, described another daily vitamin and mineral supplement for women comprising vitamin A, beta-carotene, niacin, riboflavin, pantothenic 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 10-15/4-6.
A dietary supplement consisting of an extensive list of minerals and vitamins was described in U.S. Pat. No. 5,654,011 (Jackson and Blumberg, 1997). The patent sets forth no quantitative description on the contribution of each component to bone health.
In this invention, a calcium supplement, comprising optimum amounts of acetate salts of calcium, magnesium and zinc, and vitamin D3, is described. The daily dosage of calcium is significantly lower than that of regular calcium supplement. This product was designed using in vitro and in vivo models which are key to determining elemental balance.
The present invention provides a dietary supplement comprising acetate salts of calcium, magnesium, zinc and vitamin D3. This preparation is highly soluble in water, gastric and intestinal fluids. It is also shown that elemental absorption is high and the dosage required for bone health maintenance is approximately a quarter to a third of that of the conventional calcium dose.
The following terms shall be used to describe the present invention. In the absence of a specific definition set forth herein, the terms used to describe the present invention shall be given their common meaning as understood by those of ordinary skill in the art.
As used herein, the expression in vivo refers to in the living organism.
As used herein, the expression in vitro refers to in an artificial environment outside the living organism.
As used herein, the expression RDA refers to recommended daily allowance.
As used herein, the expression RDI refers to recommended daily intake.
As used herein, the expression AI refers to adequate intake.
As used herein, the expression juice composition refers to a composition comprising juice from fruit, fruit drink, a natural juice, or an artificial juice.
The present invention describes a supplement comprising acetate salts of calcium, magnesium, zinc and vitamin D3.
In one embodiment, the composition of the present invention comprises a weight ratio of calcium to magnesium of from 0.5:1 to 2:1, with preferred embodiments of 0.5:1, 1:1, or 2:1. For example, the preferred embodiments of the composition of the present invention may comprise 220 mg of calcium and either 440 mg, 220 mg, or 110 mg of magnesium. In other embodiments, the composition comprises 100 to 300 mg of calcium and 50 to 150 mg of magnesium.
In one embodiment, the composition of the present invention comprises a weight ratio of zinc to calcium ranging from about 0.05:1 to about 0.1:1. In a further embodiment, the weight ratio of zinc to calcium ranges from about 0.05:1 to about 0.20:1.
In one embodiment of the present invention may comprise a daily dose of 10 to 40 mg of zinc.
In one embodiment, the composition of the present invention may comprise a daily dose of 400 to 1200 IU of vitamin D3.
In one embodiment, the composition may comprise 400 to 1200 IU of vitamin D3 per 100-300 mg of calcium.
The composition of the present invention may comprise a daily dose of vitamin D3 of at least 1200 to 3000 IU.
In another embodiment, the composition of the present invention may comprise a daily dose of vitamin D3 that is 4000 IU.
In a further embodiment, the composition of the present invention may comprise a daily dose of vitamin D3 that is 5000 IU.
In still another embodiment, the composition of the present invention may comprise a daily dose of vitamin D3 that is 6000 IU.
In still another embodiment, the composition of the present invention may comprise a daily dose of vitamin D3 that is 10,000 IU.
In one embodiment, the present invention provides a composition comprising calcium or synthetic calcium in the form of acetate salt, wherein the composition is further fortified with magnesium, zinc and vitamin D3. In one embodiment, the composition before fortification is an extract from pearl, coral, oyster, or natural mines.
In one embodiment, the present composition comprises magnesium in the form of acetate salt. In another embodiment, the composition comprises zinc in the form of acetate salt.
In an embodiment, the source of magnesium is synthetic.
In another embodiment, the source of magnesium is an extract from other magnesium compounds such as magnesium oxide.
In one embodiment, the present composition is more soluble at pH 7 than calcium carbonate.
In one embodiment, the present composition comprises more bioavailable calcium per unit weight than calcium carbonate. For example, the present composition may comprise at least 11 percent by weight of calcium in the form of calcium acetate, at least 5 percent by weight of magnesium in the form of magnesium acetate, and at least 0.5 percent by weight of zinc in the form of zinc acetate. In another embodiment, the composition may comprise at least 7 percent by weight of calcium in the form of calcium acetate, at least 7 percent by weight of magnesium in the form of magnesium acetate, and at least 0.3 percent by weight of zinc in the form of zinc acetate. In yet another embodiment, the composition may further comprise at least 400 IU of vitamin D3.
The present invention also provides a use of the composition disclosed herein for the preparation of medicament for alleviating or treating symptoms of osteoporosis. In one embodiment, the composition comprises between 100 to 300 mg of calcium.
The present invention also provides a juice composition comprising the compositions described herein.
The present invention also provides a use of the composition disclosed herein for the preparation of medicament for increasing bone mineral density. In one embodiment, the composition comprises between 100 to 300 mg of calcium.
The present invention also provides a method of preparing tablets comprising calcium acetate, magnesium acetate, zinc acetate and vitamin D3, comprising the steps of: (i) blending a calcium composition comprising calcium acetate, magnesium acetate, and zinc acetate with a composition comprising vitamin D3; and (ii) blending the composition obtained from (i) with a calcium 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 14 percent by weight of calcium acetate, at least 7 percent by weight of magnesium acetate, and at least 0.7 percent by weight of zinc acetate.
The present invention also provides a tablet produced by the method described above.
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.
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.
A pearl extract was prepared by adapting the patented method reported by Li and Li (1995). 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:
This extract, A1, 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:
aIn-house Data. ***p < 0.001
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 A1, A4 through A6 could also be achieved by mixing appropriate amounts of acetates salts of calcium, magnesium and zinc.
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.
The solubility of calcium in the four formulas in an artificial gastric (pH=1) and intestinal fluid (pH=7) was tested using a method developed for ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometer) (PerkinElmer Optima 4300DV). Two commercial samples, Caltrate™ and calcium acetate, were also tested in parallel for comparison. The results are shown in Table 3.
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, A1, 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 Caltrate™. Therefore, unlike Caltrate™, solubility of acetate salts should not be an issue in gastrointestinal tract fluids because the acetate salts will still be in solution.
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 Caltrate™ is low.
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 pH and environmental conditions is less drastic (Table 5).
This set of experiments thus leads to the conclusion that acetate salts are preferred salts in the disclosed formulations for their high solubility.
In this example, the effects of pH (ranging from 1 to 9) on the solubility of three elements of the four pearl formulas (A1, A4, A5 and A6), a commercial product (Caltrate™) and a synthetic compound (Calcium Acetate, Ca ACE) were investigated. Solution pH was adjusted using appropriate amounts of acetic acid (AcOH), nitric acid (HNO3) or ammonium hydroxide (NH4OH). Saturated solutions were prepared by dissolving each preparation in a solution with a final pH value ranging from 1 to 9. The resultant mixtures were incubated in a water bath at 37° C. 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.
Throughout the pH range tested, both A1 and calcium acetate showed significantly higher calcium content in solution than the other preparations. Caltrate™ had the lowest calcium content (p<0.05). A1 and calcium acetate have the highest solubility at pH 1 (Table 6)
Magnesium has a negative effect on the content of calcium in solution; the rank order in terms of solubility is A5>A4>A6. Except for Caltrate™, calcium acetate and A1, which are more soluble at pH 1, pH has no effect on the solubility of magnesium in solution (Table 7).
Similarly, the amount of zinc in solution correlated well with the zinc content in different formulations (A5>A4>A1>A6) (Table 8). For all four acetate formulas tested, pH values higher than 5 were associated with higher solubility of zinc than that at pH 2 and 3.
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™ under such pH conditions. These results are consistent with those reported in Table 3.
Experimental Data on Solubility in the Presence of Common Gastric and Intestinal Anions and Cations
The following analyses using anions which are present in abundance in gastro-intestinal tract fluids were performed on the four test formulas (A1, A4, A5 and A6), Caltrate™ 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.
aValues were cited from TheDigestiveSystem (ISBN 0443062455).
The following analyses using anions which are present in abundance in gastro-intestinal tract fluids were performed on the four test formulas (A1, A4, A5 and A6), Caltrate™ 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 (HCO3− and PO43−) 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 pH 1 and pH 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.
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™ at pH 1 (Tables 10-12). At a Cl− concentration of 200 mM, the solubility of calcium was the highest. The maximum magnesium and zinc 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 Cl− concentration (p<0.05).
At pH 7, the solubility of calcium in Caltrate™ 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™ 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.
In the presence of chloride, pH has less of an effect on magnesium solubility (compare values between Tables 11 and 14). In general, the solubility of magnesium at pH 7 is slightly lower for all formulas and the chloride effect is not pronounced.
In the presence of chloride, the solubility of zinc in Caltrate™ 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 solubility to increase with the increase of chloride concentration. Maximum zinc solubility is reached at 120 mM chloride when Caltrate™ was evaluated. For the acetate formulas, maximum zinc solubility occurred when chloride concentration reached 200 mM.
The solubility of calcium in Caltrate™ 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%.
The solubility of magnesium in Caltrate™ increased with bicarbonate concentration (Table 17). Bicarbonate effect was minimal for the acetate formulas.
The solubility of zinc in Caltrate™ 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.
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.
Magnesium solubility decreased as phosphate concentration increased (Table 20). The reduction (80%) is most significant for the magnesium in Caltrate™. For the other formulas, the maximum reduction was approximately 50%. Again, the effect of phosphates was not that significant considering the range of concentration tested.
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 maximum reduction was approximately 70%. Considering the range of phosphate concentration tested, again, the effects of phosphates were not that significant.
The following analyses using cations which are present in abundance in gastro-intestinal tract fluids were performed on the four test formulas (A1, A4, A5 and A6), Caltrate™ and calcium acetate in order to assess the solubility and subsequently, their rate of absorption and bioavailability.
The effects of Na+ concentration on the solubility of the three elements in the four formulations (A1, A4, A5, and A6), Caltrate™ and CaACE were investigated at gastric pH (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 solubility of all formulations were observed. Solubility of zinc in Caltrate™ 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).
Tables 25-27 show the effects of sodium ion at pH 7. Na+ has no significant effects on calcium, magnesium and zinc solubility in general. It is interesting to note that all three elements in Caltrate™ could be not detected in the presence of Na+ at pH 7.
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<0.05).
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™, (p<0.05). However, the magnitude of increase in inconsequential when compared to that of A4, A5 and A6.
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
There was a tendency for the solubility of calcium to increase with an increase in potassium concentration, however, the difference is not significant, p>0.05 (Table 31). No calcium could be detected in preparations using Caltrate™.
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 magnesium and zinc was reported for preparations using Caltrate™.
The objectives of the balance studies were to evaluate the effects of dietary conditions and formulations on calcium, magnesium and zinc balance.
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:
Male Sprague-Dawley rats (about 6-7 weeks), with an initial weight between 220 g to 250 g, 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.
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™, Calcium Acetate (Ca ACE), A1, 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 libitum 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 for magnesium and 0.017 to 2.5 mg/kg/day for zinc. Vitamin D3, 1.06 μg/kg/day (42.512 IU/kg/day; IU=0.025 μg), 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.
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° 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.
Daily calcium balance was calculated using equation 1:
Ca Balance=total Ca intake(dose and dietary intake)−Ca excreted in urine−Ca excreted in feces (1)
While, percentage of Ca balance was determined using equation 2:
% Ca balance=Ca balance/(total Ca intake)×100% (2)
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). Cumulated elemental balance and % cumulated net elemental balance were calculated in a similar fashion as described above.
In general, urinary excretion accounted for less than 5% of fecal excretion. Therefore, fecal excretion practically determines the quantity of elemental balance.
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.
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.
$P < 0.05, compared with Caltrate ™;
+P < 0.01, compared with A1;
&P < 0.05, compared with A4;
%P < 0.001, compared with A5;
#P < 0.001, compared with A4 + Vit D;
@P < 0.05, compared with A5 + Vit D.
The addition of magnesium and zinc to a formula promotes the retention of calcium. A1, a composition with miniscule amounts of magnesium and zinc, has a lower calcium retention (17%, 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.
The addition of vitamin D3 increases calcium retention significantly (
$P < 0.05, compared with Caltrate ™;
+P < 0.05, compared with A1;
#P < 0.05, compared with A4 + Vit D.
Magnesium appears to be required in order to maintain magnesium balance (i.e. to avoid magnesium depletion) (Table 37). Formulas (Caltrate™, CaACE and A1) that have miniscule amounts of magnesium caused a net loss of magnesium (
The addition of vitamin D3 has no significant effect on the retention of magnesium. The cumulative net percentage of magnesium did not change significantly after vitamin D3 was added to A4 and A5 (
$P < 0.05, compared with Caltrate ™;
+P < 0.05, compared with A1.
The retention of zinc is highly variable; it is particularly true with formulas such as Caltrate™, calcium acetate and A1 that contain minute amounts of zinc (Table 38). The results also show that zinc balance became negative when the amount of zinc is low.
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 (
However, the addition of vitamin D3 to A4 and A5 made zinc balance positive (
$P < 0.05, compared with Caltrate ™;
&P < 0.05, compared with A4
Rats that received normal diet gained weight (Table 39). Elemental treatments have no significant effect on weight gain (p>0.05).
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 calcium 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 mg 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™ has minimal effect. It is not surprising because Caltrate™ contains only calcium carbonate. The treatment with A5 has the most pronounced effect (
$P < 0 .05, compared with Caltrate ™;
&P < 0 .05, compared with A4
Average dietary intake of magnesium by the study animals was approximately 35 mg. Magnesium balance for all study groups was positive (
$P < 0.05, compared with Caltrate ™;
%P: < 0.05, compared with A5
There were no statistical differences among elemental treatments in terms of zinc balance (
Contrary to the calcium free diet study (Table 38), zinc balance was positive in this study (Table 42). This was achieved without vitamin D3 (
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 D3 supplementation are necessary if the diet in deficient in these nutrients.
%P < 0.05, compared with A5
H. Results: Calcium Free Diet with Daily Consumed Doses of Calcium
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-ionized water ad libitum (DI Water group), were fed normal calcium diet. Animals, substituting their daily calcium intake by A1 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.
Contrary to the results obtained from the normal and calcium free diet studies, magnesium has a minor effect in enhancing calcium retention (
@P < 0.05 m when compared to A1
Consistent with the calcium free diet study described above, magnesium was required to maintain a positive magnesium balance (
@P < 0.05 m when compared to A1
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. (
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.
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 D3 enhances calcium retention of A5 when the subject is deficient in dietary elements (Table 36).
@P < 0.05, when compared to A1
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.
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.
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 (A1, A4, A5 and A6) and Caltrate™ were investigated in the present study. The Caltrate™ 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 IU/kg/day (equivalent to 800 IU/70 kg man/day) and 1.71 μg/kg/day (equivalent to 120 μg/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 μg/kg/2-week) was injected subcutaneously on the back of the rats once every two weeks.
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 collected from blood samples centrifuged at 1500 g for 15 min. Plasma concentrations of calcium, magnesium, and zinc were measured using ICP-OES.
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+vitamin D and A4+vitamin D+vitamin K (p<0.05).
Body weight changes for different treatment groups are shown in
The effects of test substances on bone mineral density (BMD) are shown on
The BMD results of A1 are similar to that of A5+vit D. This is not surprising because A1 animals were fed normal calcium diet which contains a significant amount of magnesium.
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).
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.
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.
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.
Small quantities of calcium, magnesium and zinc were found in orange, grape and carrot juice (Tables 47, 50 and 53). Temperature and 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).
Similarly, temperature has no effect on the recovery of A5 elements in distilled water (Table 52).
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).
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 calcium, 150 mg of magnesium and 5.6 mg of zinc. 500 mg of A5 is intended to provide a serving of these elements in the functional food format.
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:
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.
The Dry Vitamin D3 100 GFP/HP composition (as mentioned in the certificate of analysis provided by BASF) is as follows: 3
Assay value: 100,000 IU Vitamin D3/g (=2500 microgram cholecalciferol/g). The target weight of Vitamin D3 per tablet is 2.5 mg. 30% extra Vitamin D3 has been added per tablet as overage. The manufacturer assay value is 100000 IU/g i.e. 100 IU/mg. Since 2.5 mg (3.25 mg with 30% overage) has been used each tablet has ˜250 IU of Vitamin D3.
The tablets were created according to the following steps:
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 D3 and Kollidon Va 64.
Step 2: Blending 01: 6.5 g of dispensed Dry Vitamin D3 100 GFP/HP 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.
Step 3: Blending 02: 250 g of dispensed Calcium Acetate blend (Blend 01*3.49) prepared in Step 1 was mixed with Blend 01 prepared in Step 2 for 5 minutes to produce Blend 02 (using tumble blender at 25-30 rpm).
Step 4: Blending 03: 250 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).
Step 5: Blending 04: 600 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).
Step 6: Blending 05: 5.86 g of dispensed Magnesium Stearate was mixed with Blend 04 prepared in Step 5, for 2 minutes.
Step 7: The final blend prepared above was dispensed using a Rotary table press with target tablet weight of 588.7 g.
The following formulation was also created in tablet form:
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.
The Dry Vitamin D3 100 GFP/HP composition (as mentioned in the certificate of analysis provided by BASF) is as presented above for the composition in Table 54.
Assay value: 100,000 IU Vitamin D3/g (=2500 microgram cholecalciferol/g). The target weight of Vitamin D3 per table is 5 mg. 30% extra Vitamin D3 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 mg (6.5 mg with 30% overage) has been used each tablet has ˜500 IU of Vitamin D3.
The tablets were created according to the following steps:
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 D3 and Kollidon Va 64.
Step 2: Blending 01: 13 g of dispensed Dry Vitamin D3 100 GFP/HP 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.
Step 3: Blending 02: 500 g of dispensed Calcium Acetate blend (Blend 01*3.49) prepared in Step 1 was mixed with Blend 01 prepared in Step 2 for 5 minutes to produce Blend 02 (using double cone blender at 25-30 rpm).
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).
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).
Step 6: Blending 05: 11.72 g of dispensed Magnesium Stearate was mixed with Blend 04 prepared in Step 5, for 2 minutes.
Step 7: The final blend prepared above was dispensed using a Rotary table press with target tablet weight of 1.17 g.
The size of these two formulations has proven to be acceptable to a test population.
The objective of this example is to design an elemental formula which would provide an optimal mix of vitamin D3 and acetate salts of calcium, magnesium and zinc for supporting bone health.
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., 2007). Vitamin D3 enhances calcium absorption (Christakos et. al., 2011) and therefore, is an important component of an ideal elemental formula.
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.
A formula containing calcium, magnesium, zinc and vitamin D3 may not work because the form of the elements and the amount of vitamin D3, 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 D3 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.
Using the acetate salts of calcium, magnesium and zinc with the appropriate addition of vitamin D3, an optimum calcium supplement is designed. The ratio of calcium to magnesium is generally 2:1, the ratio of magnesium to zinc is 20:1 and the daily dosage of vitamin D3 ranges from 500 to 1000 IU.
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 Caltrate™.
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 mg 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.
This application is a continuation-in-part application of U.S. application Ser. No. 12/845,301, filed Jul. 28, 2010, which is a continuation-in-part application of International Application PCT/IB2009/005042, filed Jan. 28, 2009, which claims benefit of U.S. Provisional Application No. 61/023,997, filed Jan. 28, 2008. The entire contents and disclosures of the preceding applications are incorporated by reference into this application. 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.
| Number | Date | Country | |
|---|---|---|---|
| 61023997 | Jan 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13193194 | Jul 2011 | US |
| Child | 14231744 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12845301 | Jul 2010 | US |
| Child | 13193194 | US | |
| Parent | PCT/IB2009/005042 | Jan 2009 | US |
| Child | 12845301 | US |
