Patent Description:
Vitamin B<NUM> is a water-soluble vitamin which acts as a coenzyme for various metabolic functions, including fat and carbohydrate metabolism and protein synthesis. It is vital for glucose production in ruminants, which is essential to meet the high energy demands of growth and lactation. Vitamin B<NUM> deficiency results in reduced appetite, growth rates and milk production and can lead to anaemia and death depending on the extent of deficiency.

The supplementation of grazing livestock with vitamin B<NUM> and essential trace elements such as copper, cobalt, zinc and selenium has long been seen as a method of enhancing productivity particularly when these trace minerals are deficient or marginally deficient in their diets.

Grazing ruminants such as sheep, cattle, deer, goats, llamas etc. do not receive any vitamin B<NUM> directly from their diet. They rely on bacteria in the rumen to manufacture it for them. Vitamin B<NUM> manufactured in the rumen is then absorbed by the animal. Cobalt is a vital component of the vitamin B<NUM> molecule and is a trace mineral that is often deficient in the diet of grazing animals. When this occurs manufacture of vitamin B<NUM> by ruminal bacteria is compromised and the animal becomes vitamin B<NUM> deficient.

Cobalt administered by injection is not effective in correcting a vitamin B<NUM> deficiency caused by a cobalt deficiency because, when given this way, it does not find its way into the ruminal fluid allowing the ruminal bacteria to manufacture vitamin B<NUM> for the animal.

Nutritional supplements that provide a vitamin B<NUM> supplement together with one or more trace elements in a single administration are desirable particularly in countries which have pastures deficient in these minerals. Administration of supplements by subcutaneous injection is particularly convenient as injection is a convenient method to administer supplements especially to larger livestock where oral dosing is difficult.

Aqueous injectable concentrate compositions are desirable for tissue compatibility as well as for promoting the ready uptake of the trace minerals but vitamin B<NUM> is labile in aqueous compositions and degrades on storage resulting in a lower effective concentration than originally prepared.

Injectable formulations which combine vitamin B<NUM> and trace elements are particularly problematic as the trace mineral concentration, must be high so that the volume administered by injection is of an acceptable volume.

In the course of conducting experiments to prepare aqueous injectable compositions of vitamin B<NUM> and trace element complexes the present inventor found that the trace element complexes exacerbated the degradation problem of aqueous vitamin B<NUM> for which previously reported strategies for controlling degradation were not particularly effective. There is a need for a stable injectable composition of vitamin B<NUM> with one or more trace elements which is storage stable and convenient to administer.

<CIT> refers to a vitamin composition, particularly for veterinary use, containing minerals and trace elements, more especially intended for correcting or improving the feeding of ruminants. This document does not disclose the presence of EDTA or metal complexes thereof, nor trace elements at a concentration disclosed in the present application.

<CIT> relates to a trace element solution, which includes at least two metals selected from the group comprising selenium, copper, zinc, manganese, iron and chromium and which comprises a concentration of the metals of at least <NUM>/ml. At least one of the metals selected from the group comprising copper, zinc, manganese, iron and chromium may be provided in the form of an EDTA complex. This document does not disclose glycols or vitamin B<NUM> in the trace element solution.

<CIT> refers to compositions that include one or more vitamin B<NUM> compounds and one or more excipients that enhance solubility of the vitamin B<NUM> compounds. In aspects of the invention disclosed in <CIT>, the excipients are alcohols, in particular ethanol, propylene glycol, a polyethylene glycol (PEG), glycerol, mannitol, sorbitol, Tween <NUM>, or dimethylsulfoxide or a combination thereof, and/or a salt former. This document does not disclose the presence of trace elements or an EDTA complex thereof.

<CIT> refers to a trace element solution, which comprises at least the following metals: zinc; manganese; selenium; and copper; and which comprises Vitamin B<NUM>. The solution furthermore comprises butaphosphan to stabilize the Vitamin B<NUM> and the inclusion of butaphosphan may have synergistic activity with the minerals. This document does not disclose the presence of a glycol in the composition.

Accordingly, there is provided an aqueous injectable nutritional supplement composition for livestock, comprising:.

The more preferred water-soluble liquid is propylene glycol.

The one or more trace elements present in the composition typically include <NUM> to <NUM> of zinc per litre and <NUM> to <NUM>, preferably <NUM> to <NUM>, of copper per litre of aqueous injectable nutritional supplement composition. At least one of copper and zinc will typically be in the form of an EDTA complex. Preferably at least one and more preferably both zinc and copper are present as EDTA complexes.

In one embodiment the aqueous injectable nutritional supplement comprises:.

The aqueous injectable nutritional supplement composition comprises the trace elements in an amount of <NUM> to <NUM> per litre of aqueous injectable composition, preferably from <NUM> to <NUM> per litre of aqueous injectable composition.

In a further aspect there is provided a method of supplementing the nutritional status of livestock comprising administering by subcutaneous injection to the livestock (particularly ruminant livestock) an aqueous injectable nutritional supplement composition described herein.

We have found that the presence of a water-soluble liquid selected from glycols, glycol ethers and mixtures thereof has a very significant effect in stabilising vitamin B<NUM> against degradation in an aqueous solution in the presence of the EDTA complexes of trace elements. The presence of significant amounts of trace element complexes was found to exacerbate degradation of vitamin B<NUM> which could not be effectively controlled by methods normally used in formulation injectable vitamin B<NUM> compositions.

Strategies previously adopted for stabilising vitamin B<NUM> against degradation such as providing an acidic pH, the addition of aluminium sulfate or cysteine hydrochloride and formation of micelles were found to be ineffective in the presence of the high concentration of EDTA complexes of the trace elements.

The present inventor found that glycols and glycol ethers and in particular propylene glycol provided an unexpected stability of the vitamin B<NUM> in the presence of the even high concentration of trace element complexes.

The term "glycol" refers to any of a group of alcohols containing two hydroxyl groups, particularly including propylene glycol (PG) (propane <NUM>,<NUM>-diol) andpropane-<NUM>,<NUM>-diol.

The term "glycol ether" refers to a compound in which one or both of the hydroxyl groups of a diol or condensate thereof are etherified. The term includes both monoglycol ethers and polyglycol ethers. Typically glycol ethers are of formula HO-[A--O]n--R, wherein A represents ethylene or propylene moieties, R represents alkyl moieties of <NUM> to <NUM> carbon atoms, and n can assume values between <NUM> and <NUM>, preferably between <NUM> and <NUM>. Examples of glycol ethers include ethylene glycol monoisopropyl ether, ethylene glycol monobutyl ether, ethylene glycol monoisobutyl ether, ethylene glycol monoallyl ether, diethylene glycol monomethyl ether, diethylene glycol monoisopropyl ether, diethylene glycol monobutyl ether, diethylene glycol monoisobutyl ether, diethylene glycol monobenzyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, diethylene glycol methyl ethyl ether, triethylene glycol monomethyl ether, triethylene glycol monobutyl ether, triethylene glycol dimethyl ether, polyethylene glycol monomethyl ether, propylene glycol monomethyl ether, propylene glycol monopropyl ether, dipropylene glycol monomethyl ether, tripropylene glycol monomethyl ether and <NUM>-methoxy-<NUM>-methyl-<NUM>-butanol.

Preferred glycols and glycol ethers are water-soluble liquids, having molecular weight no more than <NUM>, preferably no more than <NUM> such as no more than <NUM>. The more preferred water-soluble liquids, selected from glycols and glycol ethers, are propylene glycol and polyethylene glycol of molecular weight no more than <NUM>.

Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

The aqueous injectable nutritional supplement comprises an EDTA complex of one or more trace elements. The one or more trace elements comprises at least one of zinc, copper, manganese, selenium and chromium. Preferably the trace elements comprise zinc, copper or mixture thereof. In one embodiment the trace elements comprise zinc, copper and selenium. The amount of the trace element referred to herein is based on the element itself and not the whole trace element complex or mixture of complexes.

The aqueous injectable nutritional supplement composition comprises the trace elements in an amount of <NUM> to <NUM> per litre of aqueous injectable composition. Preferred amounts of trace elements are in the range of from <NUM> to <NUM> such as <NUM>/L to <NUM>/L, <NUM>/L to <NUM>/L, <NUM>/L to <NUM>/L or <NUM>/L to <NUM>/L. Accordingly in one embodiment the trace elements comprise <NUM> to <NUM> per litre of zinc, copper or mixture thereof preferably <NUM> to <NUM> such as <NUM>/L to <NUM>/L, <NUM>/L to <NUM>/L, <NUM>/L to <NUM>/L or <NUM>/L to <NUM>/L. The total trace element content is typically up to <NUM>/L of composition.

In one embodiment the content of <NUM> to <NUM> of zinc per litre, <NUM> to <NUM> of copper per litre such as <NUM> to <NUM> copper per litre or <NUM> to <NUM> of zinc per litre and <NUM> to <NUM> of copper per litre such as <NUM> to <NUM> copper per litre.

The vitamin B<NUM> may be in a range of known forms. For example, the vitamin B<NUM> may comprise at least one of cyanocobalamin, hydroxocobalamin, hydroxocobalamin acetate, methylcobalamin and aquacobalamin and mixtures thereof. The amount of the vitamin B12 is typically <NUM> to <NUM> such as <NUM> to <NUM> per litre of aqueous injectable composition or <NUM> to <NUM> per litre of aqueous injectable composition.

The water-soluble liquid selected from glycols, glycol ethers and mixtures thereof is typically present in the aqueous injectable composition in an amount of <NUM> to <NUM> per litre preferably <NUM> to <NUM> per litre, more preferably <NUM> to <NUM> per litre such as <NUM>/L to <NUM>/L or <NUM> to <NUM>/L of the aqueous injectable composition.

In one set of embodiments the aqueous injectable supplement composition comprises:.

The aqueous injectable nutritional supplement composition comprises an EDTA complex of one or more trace elements. The one or more EDTA complexes may be formed with any of a range of EDTA forms such as di-sodium EDTA, di-potassium EDTA, di-alkyl ammonium EDTA, di-ammonium EDTA or combination of two or more thereof. Typically, we have found it convenient to use the di-sodium EDTA in forming the trace element complexes.

Selenium deficiency in many pastures in countries such as Australia can severely inhibit growth of livestock and even result in death. The aqueous injectable nutritional supplement composition preferably comprises <NUM> to <NUM> selenium per litre of aqueous injectable composition such as <NUM> to <NUM> per litre.

In a further embodiment the aqueous injectable nutritional supplement composition comprises:.

The pH of the aqueous injectable nutritional supplement composition is not narrowly critical and does not have a significant effect on the degradation of the vitamin B<NUM> in the presence of the trace element complexes. The pH is preferably the range of <NUM> to <NUM> such as <NUM> to <NUM>, <NUM> to <NUM> or about <NUM>.

In embodiments of the invention in which the pH control is to provide a tight range the composition may be buffered using a suitable buffer such as a citrate, phosphate, trimethamine (also called tris buffer) or acetate buffer. Phosphate buffers, such as sodium phosphate, potassium phosphate and mixtures thereof, are preferred.

The water content of the aqueous injectable nutritional supplement composition in one set of embodiments is <NUM>% to <NUM>% v/v.

In a further embodiment the invention provides a method of supplementing the nutritional status of livestock comprising administering by subcutaneous injection to the livestock the aqueous injectable nutritional supplement composition as hereinbefore described.

The method is particularly useful in providing nutritional supplementation to ruminant animals especially bovine animals.

The invention will now be described with reference to the following examples. It is to be understood that the examples are provided by way of illustration of the invention and that they are in no way limiting to the scope of the invention.

A formulation, the same as a purportedly stable, commercially available and registered solution of vitamin B<NUM> plus selenium was prepared.

The formulation contained the components in the amounts shown in Table <NUM>.

To the formulation of Table <NUM>, water-soluble disodium Copper EDTA and disodium Zinc EDTA, were successfully added at the rate of <NUM>/L and <NUM>/L respectively and then topped up with water to the required volume.

This provided a final formulation shown in Table <NUM>.

The formulation was fully dissolved with no evidence of precipitate.

This formulation was sent to Chemical Analysis, and analysed at <NUM> time and placed on stability storage at <NUM> and <NUM> (<NUM>%RH).

The results were as shown in Table <NUM>.

Due to the obvious lack of stability of the formulation the trial was abandoned.

Formulations shown in Table <NUM> were prepared with single or combinations of chelated minerals (disodium EDTA) were formulated with Cyanocobalamin (CNCBL) and subjected to screening stability studies.

The results shown in Table <NUM> demonstrate that in the presence of <NUM>/L of Zinc (as disodium zinc EDTA), <NUM>/L of Copper (as disodium copper EDTA) and <NUM>/L of selenium (as selenium selenate) (in formulations <NUM>, <NUM> and <NUM>), which are the levels necessary to provide enough trace mineral in a single injection to livestock, sufficient levels of cyanocobalamin could not be maintained over time. Contrary to reported findings in the literature lowering the pH did not enhance the stability of the cyanocobalamin.

It has been claimed that the addition of Aluminium sulphate may enhance the stability of vitamin B<NUM> in aqueous solutions.

The solutions of Table <NUM> were formulated and subjected to accelerated stability testing at <NUM>.

It was concluded that the addition of Aluminium sulphate did not enhance the stability of Cyanocobalamin in solutions containing high levels of trace minerals.

Micelle formulations have been used to enhance the stability of molecules that are soluble in organic solvents but break down in aqueous solutions. Solutions including organic solvents singularly or in combination and a suitable surfactant were formulated and tested for stability of the cyanocobalamin in accordance with Table <NUM>.

GFormal* - Glycerol Formal; ECT80 ** - Polyoxyethylene sorbitan monooleate (Tween <NUM>); PG - propylene glycol.

It was concluded that these attempts to stabilise the vitamin B<NUM> in a micelle formulation were not successful.

In the process of combining organic solvents into the aqueous formulations of vitamin B<NUM> and high concentrations of trace minerals (during attempts to produce micelles containing the vitamin B<NUM>) it was surprisingly discovered that by adding propylene glycol alone increased the stability of vitamin B<NUM> in a solution containing high amounts of trace minerals.

The formulations and stability results are shown in Table <NUM>.

The following Trial formulations were initially studied. T80 - Polyoxyethylene sorbitan monooleate (Tween <NUM>).

A further study mimicking commercial production and packaging was implemented and is reported below:
Method of manufacture:.

Once manufactured the formulation was placed in stability testing at <NUM>. The formulation is shown in Table <NUM> and the stability testing results are shown in Table <NUM> below.

A composition in accordance with the invention was prepared by combining premixed compositions identified as Part A and Part B below:.

Note: DOWINOL DPM is dipropylene glycol monomethyl ether (DOWINOL is a trademark of the Dow Chemical Company).

Part B was added to Part A to provide a total combined batch weight of <NUM> grams.

The composition was subject to stability testing at <NUM> for <NUM> days and the analytical results are shown in Table <NUM> below.

The results show no loss of Vitamin B12 after <NUM> days at <NUM>.

Part B was added to Part A to provide a total combined batch weight of <NUM>.

The composition was subject to stability testing at <NUM> and the analytical results are as shown in Table <NUM>.

The results show virtually no loss of Vitamin B12 after <NUM> days at <NUM>.

The composition was subject to stability testing at <NUM> and the analytical results are as shown Table12.

The results show Vitamin B12 stable after <NUM> days at <NUM>.

Some laboratory scale formulations may differ in performance and stability to larger scale formulations manufactured under commercial conditions in a commercial manufacturing plant.

A composition in accordance with the invention in a commercial scale facility was manufactured by combining premixed compositions identified as Part A and Part B below. The composition was then filtered through a <NUM>µ filter and aseptically filled into <NUM> HDPE/LDPE flow-pack containers.

After <NUM> months at ambient temperature the composition was subjected to storage at <NUM>.

The analytical results are as shown in Table <NUM>.

The results show that a composition in accordance with the invention manufactured in a commercial scale facility under commercial conditions including sterilization by filtration and packaging remained stable with respect to Vitamin B12 for <NUM> months at <NUM>.

It may be advantageous to ensure that the pH of a composition in accordance with the invention was held between a specified narrow range. Accordingly, a phosphate/potassium buffer was added to the above composition between month <NUM> and month <NUM> in an amount calculated to hold the pH between pH <NUM> and pH <NUM>.

Claim 1:
An aqueous injectable nutritional supplement composition for livestock, comprising:
an EDTA complex of one or more trace elements;
vitamin B<NUM>; and
a water-soluble liquid selected from glycols, glycol ethers and mixtures thereof,
wherein the one or more trace elements are in an amount of <NUM> to <NUM> per litre of aqueous injectable composition, wherein the one or more trace elements comprise at least one of zinc, copper, manganese, selenium and chromium, wherein the amount of trace element is based on the element itself and not the whole trace element complex or mixture of complexes,
wherein the water-soluble liquid is selected from the group consisting of propylene glycol, glycol ethers of molecular weight no more than <NUM>, polyethylene glycol of molecular weight no more than <NUM>, dipropylene glycol monomethyl ether and mixtures thereof,
wherein the water-soluble liquid is present in the aqueous injectable composition in an amount of <NUM> to <NUM> per litre.