AU2020204386A1 - Injectable trace mineral supplement containing vitamin B12 - Google Patents

Abstract

OF THE DISCLOSURE A stable aqueous vitamin B12 trace mineral supplement for grazing animals is disclosed. The stable aqueous vitamin B12 trace mineral supplement comprises vitamin B12, selenium, optionally, at least one trace mineral selected from the group consisting of copper, manganese, and zinc, a pH adjusting agent, optionally, at least one pharmaceutically acceptable excipient, and water. The stable aqueous vitamin B12 trace mineral supplement permits the simultaneous administration of vitamin B12 with the applicable trace minerals for the supplementation of livestock. The pH of the injectable mineral supplement is manufactured between about 4.5 to about 6.5 to stabilize the vitamin B12 in solution for extended periods of time without having to add a stabilizing agent, such as butaphosphan, to the stable aqueous vitamin B12 trace mineral supplement.

Description

INJECTABLE TRACE MINERAL SUPPLEMENT CONTAINING VITAMIN B12

INCORPORATION BY REFERENCE

[0001] The foregoing applications, and all documents cited therein or during their prosecution ("appln cited documents") and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

FIELD OF THE INVENTION

[0002] The invention relates a stable aqueous injectable vitamin B12 trace mineral supplement formulation for grazing animals, such as ruminants, which include bovines, ovines, caprinae, equines, llamas, and camels, comprising vitamin B12 to permit the simultaneous administration of the trace minerals and vitamin B12, wherein the vitamin B12 remains stable for extended periods of time as a result of pH adjustment at manufacture.

BACKGROUND OF THE INVENTION

[0003] Trace mineral deficiencies have been shown to be a relatively common problem of grazing ruminants in areas where the soil is heavily leached; these areas include Australia, New Zealand, the United Kingdom, and the western plains of the United States. A number of areas are known to be deficient in trace metals, such as selenium, copper, zinc, manganese, and cobalt, with clinical deficiencies periodically observed. Subclinical deficiencies may be even more important, leading to production and reproductive losses. The full range of interactions between minerals, pasture and grazing animals are still being elucidated and production gains from mineral supplementation are still being demonstrated in regions and herds not previously designated as being deficient. Mineral injections are routinely administered in grazing systems in these places to address actual or perceived deficiencies in trace minerals and to capitalize on gains seen in livestock production.

[0004] Trace minerals play critical roles in a diverse array of complex biochemical pathways and are key components of many proteins and enzymes essential for a number of physiological processes. Deficiencies in trace minerals can cause severe abnormalities and in some cases death. Even at the subclinical level, deficiencies can result in significant production losses. Conversely, high concentrations of trace minerals can cause acute or chronic toxicity, which can also have severe consequences, including death.

[0005] Trace minerals are chemical elements required by animals in minute quantities for normal functioning and are essential for reproduction, growth, health and immunity. Copper, zinc, manganese, selenium, and cobalt are among the most important trace minerals for livestock. However, copper, cobalt, selenium, and zinc are among the most common mineral deficiencies in cattle and sheep due to the heavily leached soils in parts of Australia, New Zealand, the United Kingdom, and the United States, and manganese is poorly absorbed (<I%) from ruminant diets. Deficiencies, even at the subclinical level, may result in significant production losses. Traditional methods of trace mineral supplementation have relied on the oral route. However, variable feed intake, fluctuating demand cycles, inherently low trace mineral absorption coefficients, and antagonists in feed and water can make achieving optimal trace mineral levels via the oral route challenging.

[0006] In general, the problem with injectable solutions is that there are too low concentrations of the minerals in the solutions. This means that relatively large quantities have to be injected, which in turn can cause tissue damage and also abscesses at the site of injection. Furthermore, it is generally the case that different trace elements seldomly are individually sufficient. This means that two or more trace element solutions have to be provided by way of separate injections.

[0007] However, the injection of highly available forms of the trace minerals and vitamin B12 (the active form of cobalt) avoids enteric inefficiency in uptake and the potential for adverse interactions among minerals and feeds in the gastrointestinal tract. This allows trace minerals and vitamin B12 to be rapidly absorbed into the blood stream and made available for the required function at strategic production times.

[0008] Sub-optimal trace mineral and vitamin B12 status at calving, mating and drying off has been shown to negatively impact production, reproduction, and health. Optimizing trace element status relying solely on oral supplements may fail as a result of variation in individual intake and reduced absorption because of antagonism of other ration components and minerals to these essential trace elements. Using an injectable supplement can improve trace mineral and vitamin B12 status at these critical times, promoting a healthy immune system, which is important in resisting disease, and has a positive impact on reproduction.

[0009] Some of the roles selenium that selenium assists in include preventing cellular oxidation, disease resistance, muscle function, reproduction/fertility, spermatogenesis, embryo survival, and placental retention.

[0010] Some of the roles zinc assists in include cell division, maintaining the integrity of skin and mucous membranes, hair and hooves and in wound healing, lung function, bone and cartilage development, disease resistance, reproduction/fertility, spermatogenesis, placental retention, and udder function.

[0011] Some of the roles copper assists in include iron metabolism, bone development, maintenance of elastic connective tissue, blood formation, disease resistance, reproduction/fertility, and placental retention.

[0012] Some of the roles manganese assists in include protein metabolism, disease resistance, bone, cartilage and connective tissue development, reproduction/fertility, ovulation, spermatogenesis, and embryo survival.

[0013] Vitamin B12, is the active form of cobalt and some roles that vitamin B12 assists in include energy metabolism, DNA synthesis, red blood cell production, nervous system function, and reproduction/fertility. Vitamin B12 has various active forms, which include the vitamers cyanocobalamin, hydroxycobalamin, and methylcobalamin (Mecobalamin).

[0014] Symptoms of deficient copper levels in bovines can include depressed growth, scouring, bone fragility, anemia, and reduced fertility. Ovines exhibit similar symptoms, but also exhibit reduced wool growth, depigmentation of the wool, steely wool and rough coats, and swayback in lambs.

[0015] Ovines can exhibit acute copper toxicity based on supplementation with high concentrations of copper. According to Van Niekerk et al., sheep that were administered high concentrations of copper and subsequently died exhibited liver concentrations of copper of about 198 mg/kg of body weight to about 547 mg/kg of body weight though a number of deceased sheep exhibited liver concentrations of copper that were within a normal range of about 100 mg/kg to about 450 mg/kg. Van Niekerk et al. reported that the exhibited acute copper toxicity in sheep was due to the route of supplementation and that sheep administered a copper edetate resulted in a breakdown of erythrocytes followed by a decrease in hemoglobin concentration and hematocrit ad advises monitoring the copper status of a flock of sheep prior to supplementation. (Van Niekerk et al., An Assessment of the Toxicity of Parenteral Treatment with Copper EDTA and Copper Heptonate in Sheep, Tydskr. S.Afr.vet. Ver. (1994) 65(2):46-51).

[0016] Symptoms of deficient manganese levels in bovines and ovines can include infertility, reduced feed intake, impaired growth, and skeletal abnormalities in young.

[0017] Symptoms of deficient selenium levels in bovines can include reduced growth, subclinical mastitis, diarrhea, white muscle disease characterized by bilateral symmetrical lesions in skeletal muscle, and heart lesions. Ovines with deficient manganese levels can exhibit reduced wool growth, reduced growth, diarrhea, white muscle disease characterized by bilateral symmetrical lesions in skeletal muscle, heart lesions, and increased mortality rates in ewes.

[0018] Symptoms of deficient zinc levels in bovines can include reduced growth rate, reduced reproductive rate, reduced feed intake, rough coats, and dry and/or cracked skin. Ovines with deficient zinc levels can exhibit reduced growth rate, reduced reproductive rate, reduced feed intake, reduced wool growth and loss of crimp, rough coats, and dry and/or cracked skin. (Judson & McFarlane, Mineral disordersin grazing livestock and the usefulness of soil andplantanalysis in the assessment of these disorders, 38 Austl. J. Experimental Agric. 707 (1998)).

[0019] Oxidative stress, brought by the production of free radicals and reactive oxygen species through normal metabolic processes, can similarly affect livestock as well as humans. Oxidative stress in sheep, for example, can influence the animal's growth, reproduction, offspring survival and overall health. (D.G. Masters, Practical implications of mineral and vitamin imbalance in grazing sheep, 58 Animal Production Sci. 1438 (2018)). In dairy cattle, oxidative stress can be reduced and the performance of the cattle optimized through supplementing diets with optimal levels of micronutrients with antioxidant capabilities, such as trace elements. (Sordillo & Aitken, Impact of oxidative stress on the health and immune function of dairy cattle, 128 Veterinary Immunology & Immunopathology 101 (2009)).

[0020] U.S. Pat. No. 4,335,116 (Howard) relates to mineral-containing therapeutic compositions containing water soluble organometallic sources for ions of trace elements for parenteral administration. The salts of zinc, copper, and manganese are dissolved in water any complexed with organic metal-ion complexing agents such as EDTA. If mixtures are required, the individual solutions are to be blended..

[0021] ZA 1982/6778 (Laurie) relates to a trace element solution and a method of providing the trace elements to livestock. These trace element solutions include ethylene diamino tetra acetic acid complex of the required mineral in suitable quantities. The trace element solution includes no selenium or selenite compound.

[0022] U.S. Pat. No. 6,638,539 (Laurie et al.) relates to a method of preparing a trace element solution, which includes the steps of providing at least one EDTA-complex, of providing a sodium selenite solution, and of combining the EDTA-complexes and the sodium selenite solution. The method provides production of a trace element solution of about 55 mg/mL.

[0023] US 7,285,292 (Laurie et al) relates to a trace element solution, which comprises at least one metal selected from the group comprising selenium, copper, zinc, manganese, and chromium and which comprises a concentration of the metal(s) of at least 60 mg/mL. The solution further comprises at least one compound selected from the group comprising iodine, potassium iodide, sodium iodide, iron, iron chloride, zinc oxide, manganese sulphate, sodium selenite, copper carbonate, sodium carbonate, anhydrous disodium EDTA, and sodium hydroxide. The trace element solution is prepared by a method providing the steps of preparing a MnCO 3 mixture in a container; adding an EDTA/NaOH mixture to the container and subsequently adding at least one metal compound; and adding Na2SeO3 to the container to obtain the trace element solution. The method also comprises the step of adding CrCl 3 •6H 20 to the trace element solution. The EDTA complex is made in situ, which may reduce the overall yield and effective concentration of the trace metal elements.

[0024] Australian Pat. No. 2012379964 (Smith) relates to a trace element solution, which includes the trace elements zinc, manganese, selenium, and copper. Salts of zinc, manganese, and copper were dissolved in water and complexed with EDTA, followed by the addition of a selenium salt. The trace element solution further includes vitamin B12 and butaphosphan, which is used to stabilize the vitamin B12 in solution. The document reports a ratio of butaphosphan to vitamin B12 per formula unit in solution is approximately 100:1.5, or approximately a 66-fold excess of butaphosphan per formula unit of vitamin B12. The excess concentration of butaphosphan in solution to stabilize vitamin B12 may contribute to increased, and possibly unnecessary costs of manufacture. Additionally, Australian Pat. No. 2012379964 only relates to the visual stability of the solution at 6 M at temperatures of 25 and 40 °C at 60% and 75% relative humidity respectively.

[0025] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

[0026] The present invention includes a stable aqueous injectable vitamin B12 trace mineral supplement comprising about 0.5 to about 20 (g/L) vitamin B12, about 1.0 to about 50 (g/L) selenium, optionally at least one trace mineral selected from the group consisting of about 1.0 to about 50 (g/L) copper, about 1.0 to about 50 (g/L) zinc, and about 1.0 to 50 (g/L) manganese, a pH adjusting agent, optionally, at least one pharmaceutically acceptable excipient, and water, wherein, the vitamin B12 trace mineral supplement has a pH between about 4.5 to about 6.5 and does not contain butaphosphan.

[0027] One non-limiting embodiment of the present disclosure is to provide a stable aqueous injectable vitamin B12 trace mineral supplement comprising about 1 to about 5 (g/L) vitamin B12 and about 1 to about 25 (g/L) selenium.

[0028] A further non-limiting embodiment of the present invention is to provide a stable aqueous injectable vitamin B12 trace mineral supplement comprising about 0.5 to about 20 (g/L) vitamin B12, about 1.0 to about 50 (g/L) selenium, optionally at least one trace mineral selected from the group consisting of about 1.0 to about 50 (g/L) copper, about 1.0 to about 50 (g/L) zinc, and about 1.0 to 50 (g/L) manganese, a pH adjusting agent, optionally, at least one pharmaceutically acceptable excipient, and water, wherein, the vitamin B12 trace mineral supplement has a pH between about 4.5 to about 6.5 and does not contain butaphosphan to permit the simultaneous administration of vitamin B12 and the applicable trace minerals to ruminant animals. Ruminant animals include but are not limited to, for example, bovines, ovines, or caprinae. The stable aqueous injectable vitamin B12 trace mineral supplement intended for ovines may optionally include copper as low as 1.0 (g/L).

[0029] The present invention also includes a method for supplementing the diet of a grazing animal, which comprises injecting said grazing animal with a supplementally effective amount of the stable aqueous injectable vitamin B12 trace mineral supplement, wherein the stable aqueous injectable vitamin B12 trace mineral supplement comprises about 0.5 to about 20 (g/L) vitamin B12, about 1.0 to about 50 (g/L) selenium, optionally at least one trace mineral selected from the group consisting of about 1.0 to about 50 (g/L) copper, about 1.0 to about 50 (g/L) zinc, and about 1.0 to 50 (g/L) manganese, a pH adjusting agent, optionally, at least one pharmaceutically acceptable excipient; and water, wherein, the vitamin B12 trace mineral supplement has a pH between about 4.5 to about 6.5 and does not contain butaphosphan. In one non-limiting embodiment, supplementation can occur via subcutaneous injection.

[0030] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

[0031] It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0032] These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

DETAILED DESCRIPTION OF THE INVENTION

[0033] A "metal amino acid complex" is the product resulting from the reaction of a metal ion from a soluble metal salt with amino acids with a mole ratio of one mole of metal to one to three (preferably two) moles of amino acids to form coordinate covalent bonds. The average molecular weight of the hydrolyzed amino acids must be approximately 150 g/mol and the resulting molecular weight of the chelate must not exceed 800 g/mol.

[0034] A "metal amino acid complex" is the product resulting from complexing of a soluble metal salt with an amino acid(s).

[0035] A "metal polysaccharide complex" is the product resulting from the complexing of a soluble salt with a polysaccharide solution.

[0036] A "metal proteinate" is the product resulting from the chelation of a soluble salt with amino acids and/or partially hydrolyzed protein.

[0037] A "grazing animal" in the context of the present invention is a mammal that is used in animal husbandry for grazing whereby the animals are used to convert grass and other forage into meat, milk, wool and other product. Grazing animals include ruminant animals, including bovines ovines, caprines, and equines. Ruminant animals include cattle, sheep, goats, buffalo, deer, elk, giraffes, gazelles, and camelids.

[0038] The present disclosure relates to a stable aqueous injectable vitamin B12 trace mineral supplement comprising vitamin B12, selenium, optionally at least one trace mineral selected from the group consisting of copper, zinc, and manganese, a pH adjusting agent, optionally, at least one pharmaceutically acceptable excipient, and water, wherein, the vitamin B12 trace mineral supplement has a pH between about 4.5 to about 6.5 and does not contain butaphosphan.

[0039] Vitamin B12 is present at a concentrations of about 0.5 to about 20 (g/L), of about 1 to about 10 (g/L), of about 1 to about 5 (g/L), or about 1 to about 2 (g/L) in solution. Vitamin B12 can be present in any active form, which may include, but not limited to the vitamers cyanocobalamin, hydroxycobalamin, or methylcobalamin.

[0040] Zinc is present at a concentrations of about 1 to about 50 (g/L), of about 2 to about 35 (g/L), of about 10 to about 30 (g/L),or about 25 to about 30 (g/L) in solution. Zinc may be present in the form of any soluble salt, which may include, but not limited to fluorides, chlorides, bromides, iodides, carbonates, oxides, nitrites, nitrates, sulphates, phosphates, acetates, edetates, amino acid chelates, amino acid complexes, proteinates, polysaccharide complexes, gluconate complexes, or glucuronic acid salts. The use of a chelated metal salt versus a non-chelated metal salt for the trace mineral source provides the advantage of providing improved nutrient mobility within the livestock, and prevents nutrient precipitation and nutrient leaching, thus improving the livestock's overall nutrient uptake.

[0041] Metal amino acid complexes of zinc include, for example, zinc glutamate, zinc methionine, zinc aspartic acid, and zinc lysine.

[0042] Copper is present at a concentration of about 1 to about 50 (g/L), about 2 to about 25 (g/L), about 5 to about 15 (g/L), or about 6 to about11 (g/L), in solution. Copper may be present in the form of any soluble salt, which may include, but not limited to fluorides, chlorides, bromides, iodides, carbonates, oxides, nitrites, nitrates, sulphates, phosphates, acetates, edetates, amino acid chelates, amino acid complexes, proteinates, polysaccharide complexes, gluconate complexes, or glucuronic acid salts. The use of a chelated metal salt versus a non-chelated metal salt for the trace mineral source provides the advantage of providing improved nutrient mobility within the livestock, and prevents nutrient precipitation and nutrient leaching, thus improving the livestock's overall nutrient uptake.

[0043] In another embodiment of the stable aqueous vitamin B12 trace mineral supplement intended for supplementation in ovines where copper is present, the concentration of copper can be as low as 1.0 (g/L) because ovines can be susceptible to acute copper toxicity via supplementation with high concentrations of copper.

[0044] Metal amino acid complexes of copper include, for example, copper methionine, copper glycinate, copper histidine, and copper cysteine.

[0045] Manganese is present at a concentration of about 1.0 to about 50 (g/L), of about 2 to about 35 (g/L), of about 5 to about 15 (g/L), or about6toabout 10 (g/L), in solution. Manganese may be present in the form of any soluble salt, which may include, but not limited to fluorides, chlorides, bromides, iodides, carbonates, oxides, nitrites, nitrates, sulphates, phosphates, acetates, edetates, amino acid chelates, amino acid complexes, proteinates, polysaccharide complexes, gluconate complexes, or glucuronic acid salts. The use of a chelated metal salt versus a non chelated metal salt for the trace mineral source provides the advantage of providing improved nutrient mobility within the livestock, and prevents nutrient precipitation and nutrient leaching, thus improving the livestock's overall nutrient uptake.

[0046] Metal amino acid complexes of manganese include, for example, manganese methionine.

[0047] Selenium is present at a concentration of about 1.0 to about 50 (g/L), about 1 to about (g/L), about 2 to about 5 (g/L), or about 2 to about 4 (g/L) in solution. Selenium may be present in the form of any soluble salt, which may include, but not limited to fluorides, chlorides, bromides, iodides, carbonates, oxides, nitrites, nitrates, sulphates, phosphates, acetates, edetates, selenites, selenates, amino acid chelates, amino acid complexes, proteinates, polysaccharide complexes, gluconate complexes, or glucuronic acid salts.

[0048] Metal amino acid complexes of selenium include, for example, selenium methionine, selenium glycinate, and selenium cysteine.

[0049] The pH of the stable aqueous injectable vitamin B12 trace mineral supplement is between about 4.5 to about 6.5 or between about 5.5 to about 6.0. The resulting pH of the stable aqueous injectable vitamin B12 trace mineral supplement is achieved through the addition of a pH adjusting agent, which may be a mineral acid, organic acid or a buffer, to the stable aqueous injectable vitamin B12 trace mineral supplement at manufacture to achieve the desired pH. Examples of acids for pH adjustment include, but are not limited to, for example, acetic acid, sulfuric acid, hydrochloric acid, nitric acid, and phosphoric acid. Examples of buffers include acetate, Tris, phosphate, citrate, and other organic acids. The addition of a base may be required. Examples of bases for pH adjustment include, but are not limited to, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium bicarbonate, sodium carbonate, and sodium acetate.

[0050] The provided acidic pH range provides a number of advantages to the injectable trace mineral supplement. The acidic pH improves the stability of the vitamin B12. Stability was observed to be within about 35%, or within about 25%, or within about 10% of its original assay value when stored for about 36 months at 30 °C at about 65% relative humidity. Thus, stability over a period of time can be achieved during the manufacture process through a pH adjustment step with the addition of a pH adjusting agent such as a 1:1 dilution of hydrochloric acid: water and, optionally, a 20% w/v sodium hydroxide solution in water versus the addition and dissolution of a separate stabilizing compound into the manufactured batch such as butaphosphan. The vitamin B12 of the injectable trace mineral supplement of the present disclosure can remain stable without the addition of butaphosphan, whose concentration in solution relative to that of vitamin B12 can be approximately 66-fold higher to maintain the stability of the vitamin B12, driving up the cost of manufacture. Thus, the present invention provides a more economical formulation, whereas the inclusion of butaphosphan to maintain the stability of the vitamin B12 is not necessary and can, for example, reduce the manufacturing cost. Vitamin B12 stability can be achieved via manufacturing the inventive vitamin B12 trace mineral supplement supplement at an acidic pH between about 4.5 to about 6.5. The pH may increase over time and the vitamin B12 maintains stability in solution up to a pH of about 8.0. Accordingly, the injectable mineral supplement may include an overage of the vitamin B12 of about 25% relative to the stated concentration of about 0.6 g/L to about 1.4 g/L to account for the loss of vitamin B12 and subsequently, effectiveness of the supplement.

[0051] In another embodiment, the stable aqueous injectable vitamin B12 trace mineral supplement can further comprise a pharmaceutically acceptable excipient. Acceptable excipients are nontoxic to recipients at the dosages and concentrations employed and include, for example, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol; and chlorocresol); low molecular weight (less than 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; colorants; chelating agents such as EDTA; tonicifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose, or sorbitol; mineral acids; surfactants such as polysorbate; salt-forming counter-ions such as sodium; metal complexes; and/or non-ionic surfactants such as TWEENTM (polysorbate ), PLURONICSTM (polyoxamer), or polyethylene glycol.

[0052] In one embodiment, the pharmaceutically acceptable excipient is a preservative.

[0053] In one embodiment, the preservative is chlorocresol, which serves as an antimicrobial agent. Chlorocresol can be present at a concentration of about 1.0 g/L to about 1.5 g/L in solution.

[0054] In one embodiment of the stable aqueous injectable vitamin B12 trace mineral supplement, at least one of the trace minerals is present and said trace mineral is in the form of an EDTA complex. The EDTA complex can take the form of, for example, a disodium EDTA or a potassium EDTA salt of the respective trace mineral.

[0055] In another embodiment, the copper, zinc, and manganese are present in the stable aqueous injectable vitamin B12 trace mineral supplement in the form of an EDTA complex.

[0056] In another embodiment, selenium is present in the form of an EDTA complex or as sodium selenite.

[0057] In one embodiment, the stable aqueous injectable vitamin B12 trace mineral supplement comprises vitamin B12 at a concentration of about 1.0 to about 2.0 (g/L) in solution, zinc in the form of disodium zinc EDTA at a concentration of about 25 to about 30 (g/L) in solution, copper in the form of disodium copper EDTA at a concentration of about 6 to about 11 (g/L) in solution, manganese in the form of disodium manganese EDTA at a concentration of about 6 to about 10 (g/L) in solution, selenium at a concentration of about 2.0 to about 4 (g/L) in solution, and chlorocresol. The injectable trace mineral supplement in this embodiment can be administered to dairy and beef cattle via subcutaneous injection.

[0058] In another embodiment, the invention provides for the use in the manufacture of a stable aqueous injectable B12 trace mineral supplement, which comprises about 0.5 to about 20 (g/L) vitamin B12, about 1.0 to about 50 (g/L) selenium, optionally at least one trace mineral selected from the group consisting of about 1.0 to about 50 (g/L) copper, about 1.0 to about 50 (g/L) zinc, and about 1.0 to 50 (g/L) manganese, a pH adjusting agent, optionally, at least one pharmaceutically acceptable excipient; and water, wherein, the vitamin B12 trace mineral supplement has a pH between about 4.5 to about 6.5 and does not contain butaphosphan in the manufacture of a medicament for supplementing a diet of a grazing animal.

[0059] The stable aqueous injectable B12 trace mineral supplements of the present invention are suitable for all types of ruminant animals. The determination of a dose and the dosing regimen for a particular animal is well within the skill level of a person of ordinary skill in this art. Typically a dose ranges between about 0.005 ml/kg about 0.060 ml/kg of animal body weight, between about 0.010 or to about 0.015 ml/kg of animal body weight; about 0.015 ml/kg to about 0.030 ml/kg; or about 0.010 ml/kg to about 0.030 ml/kg of animal body weight, depending upon, for example the type, age, and condition of the animal.

[0060] For example, for dairy and beef cattle up to 1 year old, the dosage is about 1.0 mL to about 1.5 mL per 50 kg of body weight. For dairy and beef cattle between 1 and 2 years old, the dosage is about 1.0 mL to about 1.5 mL per 75 kg of body weight. For dairy and beef cattle over 2 years old, the dosage is about 1.0 mL to about 1.5 mL per 100 kg of body weight.

[0061] For bulls, supplementation can occur 3 months before joining and at joining.

[0062] For beef cows, supplementation can occur 4 weeks before mating and 4 weeks before calving.

[0063] For dairy cows, supplementation can occur 4 weeks before calving, 4 weeks before mating or insemination, and 4 weeks before drying off.

[0064] For calves, supplementation can occur at marking and at weaning.

[0065] For heifers, supplementation can occur every 3 months and 4 weeks before mating.

[0066] For all cattle, supplementation can occur every 2 months where the cattle exist in wet conditions.

[0067] Cattle typically should not be resupplemented for a minimum of 8 weeks following the previous treatment. Additionally, the injectable trace mineral supplement should not be used on cattle that are severely debilitated, suffering from liver disease, or those animals that are thirsty or exhausted.

[0068] Because the concentration of copper can be varied and even omitted from the stable aqueous injectable vitamin B12 trace mineral supplement, the stable aqueous injectable vitamin B12 trace mineral supplement is also appropriate for all ovines. For sheep, the dosage is based on the liveweight of the sheep at a rate of 0.2 mL to about 0.3 mL per 10 kg of body weight per supplementation.

[0069] Sheep that have a liveweight between about 15 and 17 kg typically should be administered about 0.25 mL to about 0.5 mL of the injectable trace mineral supplement per supplementation.

[0070] Sheep that have a liveweight between about 18 and 20 kg typically should be administered about 0.36 mL to about 0.60 mL of the injectable trace mineral supplement per supplementation.

[0071] Sheep that have a liveweight between about 21 and 24 kg typically should be administered about 0.4 mL to about 0.7 mL of the injectable trace mineral supplement per supplementation.

[0072] Sheep that have a liveweight between about 25 and 30 kg typically should be administered 0.5 mL to about 0.9 mL of the injectable trace mineral supplement per supplementation.

[0073] Sheep that have a liveweight between about 31 and 40 kg typically should be administered about 0.60 mL to about 1.2 mL of the injectable trace mineral supplement per supplementation.

[0074] Sheep that have a liveweight between about about 41 and 50 kg typically should be administered about 0.70 mL to about 1.5 mL of the injectable trace mineral supplement per supplementation.

[0075] Sheep that have a liveweight between about 51 and 60 kg typically should be administered about 1.0 to about 1.8 mL of the injectable trace mineral supplement per supplementation.

[0076] Sheep that have a liveweight between about 61 and 70 kg typically should be administered about 1.2 mL to about 2.1 mL of the injectable trace mineral supplement per supplementation.

[0077] The dosage for sheep that have a liveweight greater than about 70 kg typically should be at the rate of about 0.2 mL to about 0.3 mL per 10 kg of body weight.

[0078] For rams, supplementation can occur about 4 times per year.

[0079] For ewes, supplementation can occur 4 weeks before mating and 4 weeks before lambing.

[0080] For replacement (maiden) ewes, supplementation can occur 4 times per year and 4 weeks before mating.

[0081] For lambs, supplementation occurs at weaning.

[0082] Sheep typically should not be resupplemented for a minimum of 8 weeks following the previous supplementation. Additionally, the injectable trace mineral supplement should not be used on cattle and sheep that are severely debilitated, suffering from liver disease, or those animals that are thirsty or exhausted.

[0083] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

[0084] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.

Examples

Example 1

[0085] An example of the manufacture of 1000 L of the injectable trace mineral supplement comprising vitamin B12 in the form of cyanocobalamin at a concentration of about 1.4 g/L in solution, zinc in the form of disodium zinc EDTA at a concentration of about 26.7 g/L in solution, copper in the form of disodium copper EDTA at a concentration of about 10 g/L in solution, manganese in the form of disodium manganese EDTA at a concentration of about 6.7 g/L in solution, and selenium in the form of sodium selenite at a concentration of about 3.3 g/L in solution is provided.

[0086] 600 L of a batch volume of water for injection were loaded into a main manufacturing vessel followed by the addition of 1.25 kg of chlorocresol. The solution was stirred at ambient temperature or at an elevated temperature between 45 and 55 °C until complete dissolution of the chlorocresol was achieved. Complete dissolution occurred in approximately 2 hours at ambient temperature or less than 15 minutes when the temperature was between 45 and 55 °C.

[0087] Following dissolution of the chlorocresol, 26.7 kg of disodium zinc EDTA was added to the main manufacturing vessel with stirring until complete dissolution of the zinc salt was achieved.

[0088] Following dissolution of the zinc salt, 6.7 kg of disodium manganese EDTA was added to the main manufacturing vessel with stirring until complete dissolution of the manganese salt was achieved.

[0089] Following dissolution of the manganese salt, 3.33 kg of sodium selenite was added to the main manufacturing vessel with stirring until complete dissolution of the selenium salt was achieved.

[0090] Following dissolution of the selenium salt, 10 kg of disodium copper EDTA was added to the main manufacturing vessel with stirring until complete dissolution of the copper salt was achieved.

[0091] 200 L of water for injection were added to a separate vessel and heated to a temperature between 30 and 40 °C. 1752 g of cyanocobalamin were added to the separate vessel with stirring until complete dissolution of the cyanocobalamin was achieved. Upon complete dissolution of the cyanocobalamin in the separate vessel, the 200 L solution of water for injection and cyanocobalamin was added to the main manufacturing vessel. The pH of the resulting combined solution was subsequently adjusted to be between 5.5 and 6.5 with the addition of a 1:1 dilution of hydrochloric acid: water for injection with stirring. If an excess amount of the 1:1 dilution of hydrochloric acid: water for injection was used such that the pH of the resulting solution was less than 5.7, the pH solution could be further adjusted to be more basic with a 20% w/v sodium hydroxide solution in water for injection until achieving the desired pH level.

[0092] Following pH adjustment additional water was added to bring the total volume to 1000 L and the entire volume was stirred until a homogeneous solution was obtained. The solution appeared as a clear, purple solution. The final concentrations of the constituents in solution at the final volume of 1000 L are listed in Table 1. Table 1

Constituent Concentration (% w/v) Chlorocresol 0.125 Zinc 2.67 Manganese 0.67 Selenium 0.333 Copper 1.00 Cyanocobalamin 0.17521 1 Reported concentration takes into account a 25% overage in the formulation. The effective concentration of cyanocobalamin in the injectable trace mineral supplement is 0.14% w/v, or 1.4 g/L.

[0093] The homogeneous solution was subsequently pre-filtered through an appropriate pre filter, which could be a Pall Supor EKV (hydrophilic polyethersulfone membrane), Pall Profile II (polypropylene membrane), or other comparable pre-filter.

[0094] Following pre-filtration, a final filtration step was performed using sterilizing grade filters having a pore size of 0.2 m, which could be a Pall Supor EKV (hydrophilic polyethersulfone membrane) or Pall Fluorodyne® III DFL (hydrophilic polyvinylidene fluoride membrane). The injectable trace mineral supplement solution was filtered into sterile containers. Example 2

[0095] All mineral actives (copper, manganese, zinc and selenium) were determined by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). The samples were digested in concentrated hydrochloric acid and nitric acid and then diluted to a suitable concentration prior to analysis. The method was validated for selectivity, robustness, linearity (r > 0.99, n = 5, covering at least 50-100% of nominal for each mineral), repeatability precision (%

RSD = 0.72-0.74, n = 6 @ 100% for each mineral), intermediate precision (RSD < 2%, n=6 injections @100% for each mineral for each analyst (n = 2)) and accuracy (spiked placebo, recovery for all mineral: 96.0-104.8% (meeting the criteria: 95.0-105.0%), n = 3 @ 80, 100 and 120% of nominal).

[0096] The cyanocobalamin content of the samples was determined using an HPLC method with UV (361 nm) detection. The method was validated for selectivity, linearity (r = 0.9998, n = 7, 40-160% of nominal), precision (% RSD = 0.5, n = 6 @ 100%) and accuracy (spiked placebo, Ave: 100.7-101.2%, n = 3). Representative diluent, standard and sample chromatograms were provided. The active constituent eluted at 3.2 minutes, while no interfering peaks were observed to elute in this region of the chromatograms of the blank or placebo solution.

[0097] The chlorocresol content of the samples was determined using an HPLC method with UV (230 nm) detection. Sufficient raw data was provided to allow the calculations to be checked. The method was validated for selectivity, robustness, linearity (r = 0.9996, n = 9, 20-180% of nominal), method precision (% RSD = 0.2, n = 6 @ 100%) and accuracy (spiked placebo, Ave: 99.2-102.6%, n = 6). Representative diluent, placebo, standard and sample chromatograms were provided. The chlorocresol eluted at 2.7 minutes, while no interfering peaks were observed to elute in this region of the chromatograms of the blank or placebo solution.

[0098] The results from the chemical analysis of the injectable trace mineral supplement are shown in Table 2. Table 2

Test Method Specifications Results Identification - Selenium ICP-OES The sample solution exhibits Complies an absorption maxima at approximately 196 nm when prepared and tested as directed in the assay Selenium Assay ICP-OES 0.26 - 0.40 % w/v 0.32 % w/v Identification - ICP-OES The sample solution exhibits Complies Manganese an absorption maxima at approximately 258 nm when prepared and tested as directed in the assay. Manganese Assay ICP-OES 0.54 - 0.80 % w/v 0.65 % w/v Identification - Zinc ICP-OES The sample solution exhibits Complies an absorption maxima at approximately 206 nm when prepared and tested as directed in the assay. Zinc Assay ICP-OES 2.14 - 3.20 % w/v 2.63 % w/v Identification - Copper ICP-OES The sample solution exhibits Complies an absorption maxima at approximately 325 nm when prepared and tested as directed in the assay. Copper Assay ICP-OES 0.80 - 1.20 % w/v 0.98 %w/v Identification - HPLC 1. The retention time of Complies Cyanocobalamin with UV cyanocobalamin in the sample (361 nm) should match that of the detection standard within ±5%.

2. The UV spectrum of the cyanocobalamin peak in the sample should closely match the authentic spectrum of cyanocobalamin. Cyanocobalamin Assay HPLC 0.1663 - 0.1838 % w/v 0.1755 % w/v with UV (361 nm) detection Identification - HPLC 1. The retention time of Chlorocresol with UV chlorocresol in the sample (230 nm) should match that of the detection standard within ±5%.

2. The UV spectrum of the chlorocresol peak in the sample should closely match the authentic spectrum of chlorocresol. Chlorocresol Assay HPLC 0.1125 - 0.1375 % w/v 0.1214 % w/v with UV (230 nm) detection

Example 3

[0099] The safety of the injection site and reactivity to four embodiments of the mineral injections for cattle were studied.

[00100] Thirty cattle of less than 12 months old were recruited: 12 heifers and 18 steers ranging from 222 to 359 kg and from a range of different beef breeds (Angus, Hereford, Angus-cross and

Hereford-cross). Cattle were identified by individual ear tags. They were moved to a single site and allowed 14 days' acclimation to adjust to the trial site and the other animals. The cattle were kept as a single group, housed in a single open grazing paddock for the duration of the study. They had ad lib access to native and improved pasture and were watered from either troughs or dams.

[00101] All cattle were weighed the day before treatment. They were clinically examined to confirm overall good health. An injection site on the left-hand side of the neck was clipped of hair.

[00102] Cattle were ranked on body weight, sequentially blocked and randomly allocated from within each block to one of 6 treatment groups, which comprised 5 animals each. The Investigational Veterinary Pharmaceutical Products (hereinafter "IVPP") used in the evaluation of each group are listed in Table 3. Table 3

Group Treatment 1 Negative control: 0.9% Saline, 1 mL/50 kg BW 2 IVPP 1: Element Injectable Trace Mineral with Vitamin B12 for Cattle, 1.5 mL/50 kg BW 3 IVPP 2: Element Injectable Trace Mineral, Copper free for Cattle & Sheep, 1.5 mL/50 kg BW 4 IVPP 3: Element Injectable Trace Mineral, Selenium free for Cattle, 1.5 mL/50 kg BW 5 IVPP 4: Element Injectable Selenium and Vitamin B12 for Cattle & Sheep, 1.5 mL/50 kg BW 6 Positive control: Multimin@ Injection for Cattle (APVMA No. 59628), 1 mL/50 kg BW

[00103] On the administration day, cattle were weighed and treated according to Table 3. Cattle were secured in a crush for the administration of treatment which was injected subcutaneously. Blood samples were collected from Groups 2 and 6 immediately prior to treatment. Blood samples were also taken from Groups 2 and 6 on Day 1, 7, and 21. Two replicates per blood sample were taken, including whole blood and serum. Samples were stored at -20 °C pending dispatch for analysis. One replicate was sent for analysis on ice bricks via overnight dispatch to the testing laboratory for mineral, vitamin B12 and enzyme analyses. The second replicate, whole blood and serum, was stored at -20 °C as a backup. Laboratory analysis included blood/serum concentrations of copper, zinc, selenium, manganese, Glutathione peroxidase (GSHPx), caeruloplasmin, and vitamin B12.

[00104] Injection site were assessed immediately after administration and approximately 8 hours post-treatment. Blood samples were taken from Groups 2 and 6 at 8 hours post-treatment and processed as above.

[00105] Injection sites and pain response were assessed, along with the animal's overall health on Days 1, 3, 5, 7, 14, and 21. Injection site assessment included observations/reactions (if any) immediately post-treatment such as vocalization, head movements, bucking, skin twitching, kicking, pawing, flight speed from the crush, and/or unusual behavior once released from the crush (with regard to normal behavior for beef cattle confined in a crush and treated with an injectable substance).

[00106] Injection sites were initially observed for visible lesions and then from Day 1 for palpable lesions as well. If lesions were palpable they were measured in 3 dimensions using electronic calipers.

[00107] Pain scores at the injection site were assessed with the injection site assessments from Day 1, based on animal reaction to palpation. Pain was scored on a system of 0 (nil) to 3 (severe), based on vocalization, head movements, kicking, other reactions, and flight speed from the crush.

[00108] Statistical analysis: Data including body weight, injection site lesion size and blood mineral, enzyme, and vitamin B12 concentrations were tabulated and the equality of variances between groups assessed using Levene's test to determine if data be analysed either untransformed or log-transformed. Where variances were unequal, the Kruskal-Wallis test was used; otherwise parametric ANOVA was used. Where there was a significant difference using ANOVA, post-hoc Tukey's test was used to assess the level of difference between more than two means. For Kruskal Wallis tests, Dunn's pairwise comparison test was used to assess the level of difference between more than two means. Appropriateness of the study or trial design and experimental conditions

[00109] The study was carried out under field conditions that the products would be used. It would be expected that cattle treated with the IVPP would be injected in a crush and returned to their paddock together with their conspecifics after treatment.

[00110] The study investigated all four IVPPs for which approval is being sought and allows for direct comparison both between products and comparison of all four IVPPs with both the negative control and the reference product Multimin, which acted as the positive control.

[00111] The management of the animals both for the experimental procedures and general husbandry was sound, as were the measurements taken and the time points designated.

[00112] As the IVPP contained zinc, copper, manganese, selenium and vitamin B12, it is pertinent that blood concentrations of these substances be measured and compared to the positive control. The measurement of GSHPx and caeruloplasmin are also standard for determining if trace elements such as these are incorporated into the body to increase antioxidant enzymes and demonstrate increased copper uptake. However, there was no reference product included to compare plasma vitamin B12 concentrations. Analysis of data and interpretation

[00113] No animals were withdrawn from the study and no adverse effects evident. For the injection site assessments, head shaking immediately post-injection was a relatively consistent response across all treatment groups, including the negative control. Other signs not seen in the negative control were observed across the other treatment groups, in particular, pawing the ground, but no clear tendency was observed to be associated with any particular treatment.

[00114] Cattle treated with IVPP 1, 2, or 4 demonstrated significantly larger injection site lesions than animals treated with IVPP 3 or the negative control (no lesions observed) through to and including 5 days' post-treatment. After this, the difference in lesion sizes across all groups did not vary significantly through to 21 days' post-treatment. Injection site lesions seen with IVPP 1, 2, or 4 were similar to those resulting from the positive control at all time points assessed.

[00115] Cattle treated with IVPP 4 demonstrated the largest lesions the day after treatment which then subsequently reduced. Animals treated with IVPP 2 demonstrated a steady increase in lesion size to Day 5, recording the largest lesions overall on Day 5, but these resolved to similar size to the other groups by Day 7. Lesions in IVPP1-treated animals had smaller injection site lesions than those treated with IVPP 2, IVPP 4, or the reference product. Lesions associated with IVPP 3 treatment were minimal. Lesions associated with IVPP 2, IVPP 4, and the reference product took longer to resolve, but all were resolved by Day 21.

[00116] Despite the presence of injection site lesions, pain associated with these was not a predominant feature. Animals with palpable lesions demonstrated a mild pain score on Day 1. In some animals, this had slightly increased by Day 3, but pain on palpation was largely resolved by Day 7. Pain responses were observed in greater proportions of IVPP 1, IVPP 2, IVPP 4, and the positive control than the IVPP 3 treated cattle. Negative control treated animals did not demonstrate any pain response.

[00117] Bodyweights of all groups increased over the study period, as the animals were growing, with the mean change in bodyweight during this period similar across all treatment groups. This demonstrates no systemic, adverse, or unwanted effects on the animals due to treatments and no disadvantage of the addition of vitamin B12 in the IVPP.

[00118] Blood copper, caeruloplasmin, selenium, GSHPx, manganese, and zinc concentrations were similar in both the IVPP 1 and the positive control at each time point. This was to be expected given that the dose rates were the same. The IVPP 1 treated animals had significantly higher vitamin B12 concentrations than the positive control animals up to Day 7. This was to be expected as the positive control did not contain vitamin B12.

[00119] Blood concentrations were compared with the analytical laboratory's reference range. Blood concentrations of selenium and zinc remained within the reference range at all measurement points in the IVPP 1 treated cattle. Blood copper concentrations were slightly above the reference range at 8-hours post treatment but remained within the reference range thereafter. Blood manganese were above the reference range at 8 hours and Day 1 post-treatment but within normal ranges thereafter. Similar findings were apparent in the positive control treated animals. Blood concentrations of vitamin B12 were well above the reference range at 8 hours and Day 1 post treatment in the IVPP 1 treated group as would be expected, but returned to normal ranges thereafter. Conclusions

[00120] Administration of IVPP 1, 2, and 4 induced injection site lesions significantly larger than the negative control and IVPP 3 treated animals up to and including 5 days post treatment. Injection site lesions associated with IVPP 1, 2, and 4 were similar to those resulting from the administration of the positive control treatment at all time points. The proportions of animals demonstrating injection site pain reactions were consistent between IVPP 1, 2, 4, and the positive control groups through to 7 days' post injection. Proportions of animals treated with IVPP 3 demonstrating any pain response was minimal but more than that of the negative control. Assessments of blood copper, caeruloplasmin, selenium, GSHPx, manganese, and zinc in the IVPP 1 treated animals were similar to the positive control/reference product treated animals and were either within laboratory reference ranges or if above, returned to the reference range without demonstrating any apparent signs of toxicity. Treatment with IVPP 1 resulted in significantly increased blood vitamin B12 concentrations for up to 7 days post-treatment and were well above reference ranges. However, there were no safety concerns regarding toxicity as this has not been reported in the scientific literature, despite regular and routine parenteral administration of vitamin B12 in animal production systems.

[00121] From these results one may concluded that the IVPP 1, 2, 3, and 4 are safe to use in cattle.

Example 4

[00122] The safety of the injection site and the reactivity to four embodiments of the mineral injection for sheep was studied.

[00123] Forty-two sheep, consisting of 21 merinos and 21 crossbreds of mixed sex (22 females, males, less than 18 months old) from two flocks, were recruited. The crossbred sheep were allowed 14 days acclimation and the merino sheep allowed 8 days acclimation to the study site. Sheep were individually identified using ear tags, drenched and vaccinated with 5 in 1 on the right hand side of the neck. Injection sites on the left hand side of the neck were shorn down to skin level.

[00124] The sheep were kept as a single group, housed in a single open grazing paddock for the duration of the study. They had ad lib access to native and improved pasture and were watered from either troughs or dams.

[00125] Sheep were weighed the day before treatment for allocation into treatment groups and clinically examined to confirm overall good health. Sheep were ranked on body weight, sequentially blocked and randomly allocated from within each block to one of 7 treatment groups, which comprised 6 animals each. The treatments are listed in Table 4. Table 4

Group Treatment 1 Negative control: 0.9% Saline, 1 mL/50 kg BW 2 IVPP 1: Element Injectable Trace Mineral with Vitamin B12 for Cattle, 1.5 mL/50 kg BW 3 IVPP 2: Element Injectable Trace Mineral, Copper free for Cattle & Sheep, 1.5 mL/50 kg BW 4 IVPP 3: Element Injectable Trace Mineral, Selenium free for Cattle, 1.5 mL/50 kg BW

5 IVPP 4: Element Injectable Selenium and Vitamin B12 for Cattle & Sheep, 1.5 mL50 kg BW 6 Positive control 1: Multimin© Injection for Cattle (APVMA No. 59628), 1 mL/50 kg BW 7 Positive control 2: Multimin© Copper Free Injection for Cattle and Sheep (APVMA No. 61724), 1 mL/50 kg BW

[00126] On the administration day, sheep were weighed and treated according to Table 4. Sheep were secured in a race for the administration of treatment which was injected subcutaneously. Blood samples were collected from Groups 2 and 7 immediately prior to treatment. Blood samples were also taken from Groups 2 and 7 on Day 1, 7, and 21. Two replicates per blood sample were taken, including whole blood and serum. Samples were stored at -20 °C pending dispatch for analysis. One replicate was sent for analysis on ice bricks via overnight dispatch to the testing laboratory for mineral, vitamin B12 and enzyme analyses. The second replicate, whole blood and serum, was stored at -20 °C as a backup. Laboratory analysis included blood/serum concentrations of copper, zinc, selenium, manganese, Glutathione peroxidase (GSHPx), caeruloplasmin, and vitamin B12.

[00127] Injection site were assessed immediately after administration and approximately 8 hours post-treatment. Blood samples were taken from Groups 2 and 7 at 8 hours post-treatment and processed as above.

[00128] Injection sites and pain response were assessed, along with the animal's overall health on Days 1, 3, 5, 7, 14, and 21. Injection site assessment included observations/reactions (if any) immediately post-treatment such as swelling, vocalization, head movements, bucking, skin twitching, kicking, pawing/scratching, sitting/lying down, unusual behavior once released from the race (with regard to normal behavior for sheep confined in a race and treated with an injectable substance).

[00129] Injection sites were initially observed for visible lesions and/or swelling and then from Day 1 for palpable lesions as well. If lesions were palpable they were measured in 3 dimensions using electronic calipers.

[00130] Pain scores at the injection site were assessed with the injection site assessments from Day 1, based on animal reaction to palpation. Pain was scored on a system of 0 (nil) to 3 (severe), based on vocalization, head movements, kicking, other reactions when released from the race.

[00131] Statistical analysis: Data including body weight, injection site lesion size and blood mineral, enzyme, and vitamin B12 concentrations were tabulated and the equality of variances between groups assessed using Levene's test to determine if data be analysed either untransformed or log-transformed. Where variances were unequal, the Kruskal-Wallis test was used; otherwise parametric ANOVA was used. Where there was a significant difference using ANOVA, post-hoc Tukey's test was used to assess the level of difference between more than two means. For Kruskal Wallis tests, Dunn's pairwise comparison test was used to assess the level of difference between more than two means. Appropriateness of the study or trial design and experimental conditions

[00132] The study was carried out under field conditions that the products would be used. It would be expected that sheep treated with the IVPP would be injected in a race and returned to their paddock together with their conspecifics after treatment.

[00133] The study investigated all four IVPPs for which approval is being sought and allows for direct comparison both between products and comparison of all four IVPPs with both the negative control and two reference products Multimin@ (APVMA No. 59628) and Multimin@ Copper Free (APVMA No. 61724), which acted as the positive controls for the trace minerals. There was no positive control reference product to compare the vitamin B12 component of the IVPP. The allocation of treatments and the blinding of personnel involved was suitable.

[00134] The management of the animals both for the experimental procedures and general husbandry was sound, as were the measurements taken and the time points designated.

[00135] As the IVPP contained zinc, copper, manganese, selenium, and vitamin B12, it is pertinent that blood concentrations of these substances be measured and compared to the positive control. The measurement of GSHPx and caeruloplasmin are also standard for determining if trace elements such as these are incorporated into the body to increase antioxidant enzymes and demonstrate increased copper uptake. However, there was no reference product included to compare plasma vitamin B12 concentrations. Analysis of data and interpretation

[00136] Administration of the negative control and IVPP 4 did not demonstrate any responses in the treated sheep. Immediate swelling was observed in one third of sheep receiving IVPP 3. Head shaking, head nodding, bucking, pawing, scratching, tail twitching, sitting and lying down were observed in a proportion of sheep receiving IVPP 1, 2, 3, and both the positive controls.

[00137] Injection site lesions observed, palpated and measured from Day 1 were only found in sheep treated with either IVPP 2 or 4. Lesions associated with IVPP 2 were observed up to and including Day 14 post-treatment. Lesions resulting from IVPP 4 were observed up to and including Day 21 post-treatment. The palpable lesions were quite small and there were no significant differences detected in lesion size between treatment groups at any of the assessment time points as a result.

[00138] Pain responses were not detected on palpation of the injection site lesions in any animal, regardless of treatment at any of the assessment time points in the study.

[00139] There was no change in mean body weight either within or between groups over the 21 day treatment period, indicating no effect of any product on body weight gain.

[00140] Blood mineral, enzyme, and vitamin B12 assessments for IVPP 1 and the positive control 2 treatments were compared. Blood copper, caeruloplasmin, selenium, GSHPx, manganese and zinc concentrations are reported as comparable in both IVPP 1 and the positive control 2 treated groups at each assessment point. Conclusions

[00141] There were no significant treatment effects noted with respect to volumetric injection site lesions at any time point of the study. Small injection site lesions were only observed in some animals treated with IVPP 2 or 4. Pain responses were not elicited from palpation and measurement of injection site lesions after the administration day. Pain-indicating behaviour immediately after treatment was comparable between IVPP 1, 2, 3, and the two positive controls. There was minimal to no pain reactions observed with the negative control or IVPP 4. Assessments of serum copper, selenium, manganese, caeruloplasmin, zinc, and blood GSHPx were similar to the copper free positive control and within reference ranges of the analysing laboratory. The investigators noted that the copper and caeruloplasmin concentrations measured in the IVPP 1 and copper free positive control group were similar despite the absence of copper in the positive control group. Treatment with IVPP 1 demonstrated significant increases in blood vitamin B12 concentrations for up to 7 days' post treatment.

[00142] From these results one may concluded that the IVPP 1, 2, 3, and 4 are safe to use in sheep. Example 5

[00143] The safety of two aqueous mineral injectable products when administered via subcutaneous injection at 1x, 3x, and repeated doses to cattle was studied.

[00144] One hundred and twenty weaner beef cattle comprising heifers and steers aged 3 months old were recruited on a commercial cattle farm in the Northern Tablelands of NSW. The cattle ranged in body weight from 80 kg to 185 kg and were predominately Angus. The cattle were kept in four mobs consisting of similar numbers of animals from each treatment group in paddocks for the duration of the study. They had ad lib access to native and improved pasture and were watered from either troughs or dams.

[00145] Study animals were ranked based on Day -5 bodyweights from highest to lowest. Blood samples were also taken at this time. Animals were sequentially blocked into sixes (20 blocks) and randomly allocated from within each block to 6 treatment groups, shown in Table 5, each of 20 animals. Allocation was such that each group had a similar group mean bodyweight and range of bodyweights within the group.

[00146] Prior to treatment, injection sites were clipped to assist in post treatment injection site assessments. Treatment doses were administered subcutaneously, injected high on the neck behind the ear (left hand side). Cattle were carefully restrained in a head bail during application. Treatment doses were administered up to a maximum volume of 10 mL per injection site. All animals in Group 2 (3x dose level) were treated with lx dose of Test Item 1 on the left hand side and 2x dose on the right hand side. Table 5

Group Treatment & dose volume Dose Tx Day level 1 IVPP 1: Element Injectable Trace Mineral with Vitamin B12 lx Day 0 for Cattle, 1.5 mL/50 kg BW 2 IVPP 1: Element Injectable Trace Mineral with Vitamin B12 3x Day 0 for Cattle, 1.5 mL/50 kg BW 3 IVPP 1: Element Injectable Trace Mineral with Vitamin B12 lx Day 0 &

for Cattle, 1.5 mL/50 kg BW Day 56 4 IVPP 2: Element Injectable Trace Mineral, Copper free for 1x Day 0 Cattle & Sheep, 1.5 mL/50 kg BW 5 Positive control: Multimin©Injection for Cattle (APVMA No. lx Day 0 59628), 1 mL/50 kg BW 6 Negative control: 0.9% Saline, 1 mL/50 kg BW 1x Day 0 &

Day 56

[00147] All animals were observed at approximately 3, 7, and 30 hours post-treatment for abnormal behaviour and lesions resulting from the treatment. Blood samples were taken from all animals except those in Group 4 on Day 1, 3, and 21. Animals in Groups 3 and 6 were also blood sampled on Day 57 and 59, after their second injection on Day 56 and on Day 77. Blood samples were analysed for routine haematology and biochemistry. Observations of injection site reactions were recorded at these times and any measurements on palpable injection sites made. Study animals were weighed on Day -5, 0, 21, 35, 56, and 77 (Groups 3 and 6 only). Detailed clinical examinations were performed for all Groups on Days -5, 1, 3, 10, 21, 35, and 56, and for Groups 3 and 6 only on Days 57, 59, 66, and 77.

[00148] In addition, Reactions immediately post-treatment (if any) observed both during restraint in the head bail and for approximately 30 seconds following release were recorded. Any abnormal behaviour (beyond normal behaviour for cattle confined in a race or head bail and treated with an injectable compound) such as; vocalisations, head movements, bucking, skin twitching, kicking, pawing, flight speed, unusual or pain-indicating behaviour once released from the crush and any other observation as appropriate was recorded. Injection sites were observed for visible lesions and then palpable lesions. Palpation and measurements commenced on Day 1 (Groups 1 6) and Day 57 (Groups 3 & 6) with assessments on Day 0 or Day 56 respectively limited to observation only to prevent loss of test/reference item as a result of palpation. Palpable lesions were measured in 3 dimensions using electronic callipers and the dimensions.

[00149] Statistical analysis: Data including body weight, clinical parameters, injection site lesion dimensions and the proportion of lesions within each group and blood analysis were tabulated and the equality of variances between groups assessed using Levene's test to determine if data be analysed either untransformed or log-transformed. Where variances were unequal, the Kruskal-Wallis test was used; otherwise parametric ANOVA was used. Where there was a significant difference using ANOVA, post-hoc Tukey's test was used to assess the level of difference between more than two means. For Kruskal-Wallis tests, Dunn's pairwise comparison test was used to assess the level of difference between more than two means. Appropriateness of the study or trial design and experimental conditions

[00150] The study was carried out under field conditions that the products would be used. It would be expected that sheep treated with the IVPP would be injected in a race and returned to their paddock together with their conspecifics after treatment.

[00151] The study investigated all four IVPPs for which approval is being sought and allows for direct comparison both between products and comparison of all four IVPPs with both the negative control and two reference products Multimin® (APVMA No. 59628) and Multimin Copper Free (APVMA No. 61724), which acted as the positive controls for the trace minerals. There was no positive control reference product to compare the vitamin B12 component of the IVPP. The allocation of treatments and the blinding of personnel involved was suitable. Analysis of data and interpretation

[00152] There were no significant differences in body weights between any groups at any of the time points. Statistical analysis of the Group heart rates, respiratory rates and rectal temperatures found some statistically significant differences between the groups for these clinical parameters. However, there was no evidence of a dose titration effect and these statistical differences are not considered to be clinically significant.

[00153] Complete serum biochemistry and haematology results were reported and of these 10 key biochemical and 8 key haematological parameters were identified. The biochemical parameters were: glucose, urea, creatinine, total protein, albumin, globulin, alkaline phosphatase, aspartate aminotransferase, creatine kinase, and glutamate dehydrogenase. The haematological parameters were: red blood cells, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, and white blood cells.

[00154] Statistical analysis of the haematological parameters found similar trends over time for the different treatment groups (with the exception of white blood cells). Some significant differences between the groups at the time points were observed but these were judged to be clinically non-significant due to the lack of a dose titration effect. However, the white blood cell count was found to increase on Day 1 for the 3x Treatment Group. This change was statistically significant and possibly clinically significant.

[00155] Statistical analysis of the biochemical parameters found similar trends over time for most of the parameters for the treatment groups. Some significant differences between the groups at the time points were observed but these were judged to be clinically non-significant due to the lack of a dose titration effect. Glutamate dehydrogenase (GLDH) was found to be elevated in the Group 3 test group on two occasions and this was statistically significant. But given that GLDH remained within the reference range and there was no dose titration effect the clinical meaning of this result is uncertain.

[00156] The main reaction to the treatments was movement in response to the injection. The largest response (20%) was seen in the IVPP 3x group, given that they received a total of 2 injections. This behavior was also seen in the IVPP lx group (10%), IVPP (repeat doses) (15%) and the positive control (5%). Head shaking was seen in the IVPP lx group (10%), IVPP (repeat doses) (10%), IVPP 2 (5%), and the negative control (5%). There was limited vocalization, kicking and flight response. No bucking, twitching, or pawing was evidenced.

[00157] For the repeat dose on Day 56, 15% of animals receiving the IVPP demonstrated head shaking with no other aversion behavior demonstrated.

[00158] The number of animals showing palpable and measurable site reactions was between 5 and 12% on Day 1, 3 and 10 for those treated with IVPP lx, IVPP 3x, and the positive control doses. The number of animals demonstrating an injection site reaction for the IVPP copper free ranged from 15 to 45%, with the highest occurrence occurring 3 days post treatment. All site reactions were essentially resolved by Day 21 in all groups. The repeated dose on Day 56 elicited a site reaction from 5 out of 20 animals with no site reactions evidenced in the negative control group. Conclusions

[00159] In conclusion, treatment with the IVPP at the proposed dose level (Ix), at an increased dose level (3x) and when repeat dosed at an 8 week interval had no adverse effects on treated animals, as assessed by repeated examination of a range of key clinical, hematological and biochemical parameters. All treated animals finished the study, with no animals requiring removal or concurrent medication during the study.

[00160] From these results one may conclude that treatment with the IVPP is safe for use in the target animal, resulting in minimal injection site reaction and pain reactions in either the short or long term.

[00161] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims (26)

WHAT IS CLAIMED IS:

1. A stable aqueous injectable mineral supplement comprising:

(a) about 0.5 to about to about 20 (g/L) vitamin B12;

(b) about 1.0 to about 50 (g/L) selenium;

(c) optionally at least one trace mineral selected from the group consisting of about 1.0 to about 50 (g/L) copper, about 1.0 to about 50 (g/L) zinc, and about 1.0 to about 50 (g/L) manganese;

(d) a pH adjusting agent;

(e) optimally, at least one pharmaceutically acceptable excipient; and

(f) water;

wherein, the vitamin B12 trace mineral supplement has a pH between about 4.5 to about 6.5 and does not contain butaphosphan.

2. The stable aqueous vitamin B12 trace mineral supplement according to claim 1, which comprises about 1 to about 5 (g/L) vitamin B12 and about 1 to about 25 (g/L) selenium.

3. The stable aqueous vitamin B12 trace mineral supplement according to claim 2, which comprises at least one trace mineral selected from the group consisting of about 2 to about (g/L) copper, about 2 to about 35 (g/L) zinc, and about 2 to about 35 (g/L) manganese.

4. The stable aqueous vitamin B12 trace mineral supplement according to claim 2, which comprises at least one trace mineral selected from the group consisting of about 5 to about (g/L) copper, about 10 to about 30 (g/L) zinc, and about 5 to about 15 (g/L) manganese.

5. The stable aqueous vitamin B12 trace mineral supplement according to claim 1, which comprises about 1 to about 2 (g/L) vitamin B12 and about 2 to about 5 (g/L) selenium.

6. The stable aqueous vitamin B12 trace mineral supplement according to claim 5, which comprises at least one trace mineral selected from the group consisting of about 6 to about 11 (g/L) copper, about 25 to about 30 (g/L) zinc, and about 6 to about 10 (g/L) manganese.

7. The stable aqueous vitamin B12 trace mineral supplement according to claim 1, wherein copper, zinc, and manganese are present.

8. The stable aqueous vitamin B12 trace mineral supplement according to claim 1, wherein at least one of the trace minerals is present and said trace mineral is in the form of an EDTA complex.

9. The stable aqueous vitamin B12 trace mineral supplement according to claim 8, wherein the EDTA is disodium EDTA or potassium EDTA.

10. The stable aqueous vitamin B12 trace mineral supplement according to claim 6, wherein the copper, zinc, and manganese are in the form of an EDTA complex.

11. The stable aqueous vitamin B12 trace mineral supplement according to claim 1, wherein at least one of the trace minerals is present and said trace mineral is in the form of an amino acid complex or a glucuronic acid salt.

12. The stable aqueous vitamin B12 trace mineral supplement according to claim 1, wherein the selenium is in the form of an EDTA complex sodium selenite, or sodium selenate.

13. The stable aqueous vitamin B12 trace mineral supplement according to claim 12, wherein the EDTA is disodium EDTA or potassium EDTA.

14. The stable aqueous vitamin B12 trace mineral supplement according to claim 1, wherein the pH is about 5.5 to about 6.0.

15. The stable aqueous vitamin B12 trace mineral supplement according to claim 1, wherein the pH adjusting agent is a mineral acid, an organic acid, or a buffer.

16. The stable aqueous vitamin B12 trace mineral supplement according to claim 1, wherein at least one pharmaceutical excipient is present.

17. The stable aqueous vitamin B12 trace mineral supplement according to claim 15, wherein the at least one pharmaceutical excipient is an antioxidant, a preservative, a tonicifier, a carbohydrate, a colorant, a surfactant, or a combination thereof.

18. The stable aqueous vitamin B12 trace mineral supplement according to claim 16, wherein the pharmaceutically acceptable excipient is a preservative.

19. The stable aqueous vitamin B12 trace mineral supplement according to claim 16, wherein the preservative is chlorocresol.

20. The stable aqueous vitamin B12 mineral supplement according to claim 1, wherein the vitamin B12 comprises vitamer cyanocobalamin.

21. The stable aqueous vitamin B12 mineral supplement according to claim 1, wherein the vitamin B12 comprises vitamers cyanocobalamin, hydroxocobalamin, and methylcobalamin.

22. The stable aqueous injectable vitamin B12 trace mineral supplement according to claim 1, which comprises:

(a) about 1.0 to about 2.0 (g/L) vitamin B12;

(b) about 2.0 to about 4 (g/L) selenium;

(c) about 6 to about 11 (g/L) copper in the form of disodium copper EDTA;

(d) about 25 to about 30 (g/L) zinc in the form of disodium zinc EDTA;

(e) about 6 to about 10 (g/L) manganese disodium manganese EDTA; and

(f) chlorocresol.

23. A method for supplementing the diet of a grazing animal, which comprises injecting said grazing animal with a supplementally effective amount of the stable aqueous injectable vitamin B12 trace mineral supplement according to claim 1.

24. The method of claim 23 wherein the grazing animal is a ruminant.

25. The method of claim 23 wherein the grazing animal is a bovine or an ovine.

26. The method of claim 24, wherein the bovine is cattle and the ovine is a sheep or goat.