NZ582005A - Calcium and vitaminB12 based formulation for the treatment of milk fever in animals - Google Patents

Abstract

Disclosed is an injectable composition including: (a) at least three different calcium salts; and (b) vitamin B12 or a derivate thereof. The calcium salts may be selected from the group including calcium halide, calcium levulinate, calcium lactate, calcium chlorate, calcium acetate, calcium propionate, calcium mannoate, calcium saccharate and calcium gluconate, wherein calcium lactate, calcium chloride and calcium levulinate are the preferred calcium salts.

Description

James & Wells Ref: 234997/75 PATENTS FORM NO. 5 Fee No. 4: $250.00 PATENTS ACT 1953 COMPLETE SPECIFICATION IMPROVED FORMULATION FOR THE TREATMENT OF MILK FEVER WE BOMAC RESEARCH LIMITED, 102 Wiri Station Road & Hobill Avenue, Manukau City, Auckland, New Zealand, a New Zealand 20 company hereby declare the invention for which We pray that a patent may be granted to us, and the method by which it is to be performed to be particularly described in and by the following statement: 1 James & Wells Ref: 234997/75 IMPROVED FORMULATION FOR THE TREATMENT OF MILK FEVER TECHNICAL FIELD This invention relates to an improved formulation for the treatment of milk fever. 5 More specifically the invention relates to an improved formulation containing calcium and vitamin B12 and its use in the treatment of milk fever in animals.

BACKGROUND ART Milk fever (also known as hypocalcaemia, parturient paresis, periparturient paresis or parturient apoplexy) is an afebrile disease of mature dairy cows and ewes. It 10 occurs most commonly at or soon after calving and is usually associated with the sudden onset of profuse lactation and is accompanied by a decrease in serum calcium levels.

This decrease in serum calcium levels can exhibit itself in a number of ways, including paresis, staggering and cold ears, and in serious cases, coma followed 15 by death. The majority of the incidences of milk fever occur within the first 24 hours following calving.

Milk fever is most commonly treated with calcium borogluconate, either intravenously or subcutaneously. Calcium borogluconate is particularly soluble and stable and permits high levels of calcium dosing without great risk to the animal. It 20 has proven to be an effective method of delivering high levels of calcium safely to animals without the risk of toxicity. 2 James & Wells Ref: 234997/75 NZ 272317 discloses a composition containing calcium borogluconate together with vitamin B12 for treating milk fever and/or vitamin B12 or cobalt deficiency in cattle and sheep.

Vitamin B12 is a cobalt containing vitamin required by cells throughout the body for 5 conversion of ribose nucleotides into deoxyribose nucleotides, a major step in the formation of deoxyribonucleic acid (DNA). Thus it is an essential nutrient for nuclear maturation and cell division. Adult ruminants are not dependent on a dietary source of vitamin B-|2 because bacteria within the rumen synthesise all the vitamin B12 needed. However, cobalt is required by the ruminal micro-organisms to 10 synthesise this vitamin.

With interest growing in reducing environmental impact and avoiding unnecessary introduction of potentially toxic elements to both animals and the environment, there has been an interest in new methods for the treatment of milk fever that do not include the use of boron. High concentrations of boron in soil can result in poor 15 overall growth performance of plants, and therefore a reduction in the levels of boron excreted by animals is becoming increasingly in demand in the animal health field.

A number of different formulations have been suggested for use in delivering calcium to animals in need thereof that do not contain boron.

US 5,393,535 discloses an orally administrable calcium supplement for cattle including a calcium ion containing composition having a dispersed aqueous phase and a continuous oil phase. The oral gels exemplified in US 5,393,535 use calcium chloride as the calcium source in each of the formulations. The gel formulation of this invention was developed to produce a palatable calcium supplement that is 25 willingly taken by cows without being so viscous that it may be detrimental to 3 James & Wells Ref: 234997/75 aspiration of the animal. While suspension of calcium salts within a suspension or emulsion is suitable for oral administration to cattle, parenteral administration of a boron-free calcium supplement is preferable as it allows for greater control over the administration of the calcium and therefore greater success in treating incidences 5 of milk fever.

It has also been found that delivering high levels of calcium in the form of a single salt such as calcium chloride can have toxic effects on the animal if administered in too concentrated a solution.

US 4,185,093 discloses an aqueous solution of dissolved calcium containing 10 calcium chloride, calcium lactate and calcium levulinate. While addressing the problem of providing a boron free calcium composition, it does not go any way to addressing deficiencies in cobalt and hence vitamin B12. These deficiencies may become particularly apparent (and detrimental to the animal) during times of stress or increased energy requirements, such as with the onset of lactation, with or 15 without the animal being calcium deficient.

It is therefore an object of the present invention to provide a treatment that addresses the foregoing problems or at least provides the public with a useful choice.

All references, including any patents or patent applications cited in this 20 specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not 25 constitute an admission that any of these documents form part of the common 4 James & Wells Ref: 234997/75 general knowledge in the art, in New Zealand or in any other country.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DISCLOSURE OF THE INVENTION According to a first aspect of the invention there is provided a composition including; • at least three different calcium salts; and • vitamin B12 or a derivate thereof.

According to another aspect of the invention there is provided a method of treating 10 or preventing milk fever in animals, characterised by the step of administering to the animal a composition including at least three different calcium salts and vitamin B12 or a derivative thereof.

Preferably, the animal to be treated is a ruminant.

As most ruminants have a plant based diet their daily calcium intake is often less 15 than carnivorous animals, making them more susceptible to milk fever.

Additionally, almost all the glucose produced by the breaking down of the cellulose and hemicellulose in plants is used by microbes in the rumen, and as such ruminants usually absorb little glucose from the small intestine. Rather, ruminants' requirement for glucose (for brain function and lactation) is made by the liver from 20 propionate, one of the volatile fatty acids made in the rumen. Adequate levels of vitamin B12 are therefore especially important in ruminants to maintain healthy glucose levels.

James & Wells Ref: 234997/75 In preferred embodiments of the invention the three different calcium salts are selected from the group including calcium halide, calcium levulinate, calcium lacate, calcium chloride, calcium acetate, calcium propionate, calcium gluconate, calcium mannoate and calcium saccharate.

In more preferred embodiments the composition includes calcium lactate, calcium chloride and calcium levulinate. The combination of these three calcium salts allows the composition to be loaded with effective amounts of calcium for the treatment of milk fever, without risking the calcium toxicity which is sometimes found with administration of high levels of a single calcium salt, particularly calcium 10 chloride.

Calcium levulinate is known to be less irritating to an animal than calcium chloride, and calcium lactate may be converted to biocarbonate which further provides a buffering capability to the compositions. The particular combination of these three salts allows for higher levels of calcium to be administered to an animal without 15 risking toxicity.

In further preferred embodiments the composition includes between 15.0 - 35.0 g/L calcium.

More preferably, the composition includes 25.0 - 30.0 g/L calcium. This level of calcium has proven to be particularly effective in the treatment of milk fever using 20 previously known calcium borogluconate treatments. Such levels provide the animal with an effective amount of calcium to recover from milk fever, without causing calcium toxicity which may occur from the rapid administration of high levels of calcium, particularly calcium chloride.

In further preferred embodiments of the invention, the composition includes vitamin 25 B12 in the form of in the form of cyanocobalamine or hydroxocobalamine. Both 6 James & Wells Ref: 234997/75 cyariocobalamine and hydroxocobalamine are particularly suitable as they are highly stable which is necessary for the production of a formulation with a long shelf life.

More preferably, the amount of cyanocobalamin or hydroxocobalamin in the 5 composition is between 0.001 - 0.2 g/L. Levels of cyanocobalamine above 0.2 g/L may result in toxicity due to the presence of increased cyanide levels within the animal.

In ruminants, vitamin B12 is essential in the conversion of the propionic acid into glucose. A deficiency of B12 can therefore cut this important metabolic pathway for 10 getting energy to cells. Ruminants rely on the conversion of propionic acid to glucose for 70% of their energy requirements.

During times of increased energy requirements, such as lactation, any deficiencies in vitamin B12 will decrease the ability of the animal to cope with the increased energy demands. When the animal is under additional stress, for example when 15 there is a deficiency of calcium leading to milk fever, the animal may become even further compromised if there is a disruption to their glucose metabolism. The supplementation of vitamin B12 or cyanocobalamin to the animal ensures that the animal has sufficient cobalt to produce enough vitamin B12 in the gut to meet the requirements for effective propionic acid conversion to glucose.

It is also well known in the art that administration of calcium supplements to ruminants has the effect of decreasing appetite. It is therefore very important to ensure that the animal's stores of glucose are readily available and not compromised by deficiencies in vitamin B12.

In further embodiments of the invention the composition is administered via 25 injection. Administration via injection has been shown to most readily introduce 7 James & Wells Ref: 234997/75 calcium ions and vitamin B12 (or derivatives thereof) into the blood stream of the animal, providing a rapid response to the effects of calcium deficiency. Parenteral administration also allows for greater control over the dosing regime.

The absence of boron in the composition provides a treatment with less toxicity 5 than commonly used treatments. Reduced or nil amounts of boron in compositions is appealing both for the health and well being of the animal and also for economic reasons. Consumers are more and more aware of the treatments they are using on their animals, and there is an increased demand for low toxicity, environmentally friendly treatment solutions.

The inclusion of vitamin B12 within the composition addresses any cobalt and/or vitamin B12 deficiencies in the animal. In animals where there is no vitamin deficiency, any excess of the vitamin will be excreted and is not know to be toxic or detrimental to the animal in any way. In animals where there is a deficiency of some degree, addressing this problem in conjunction with the administration of 15 calcium supplements provides the animal with an improved chance of responding to the supplementation of calcium and not succumbing to milk fever.

The combination treatment also provides the obvious advantage in terms of time and cost that comes with a single administration of a product instead of administering two separate treatment programs.

Although treatment of milk fever and vitamin B12 deficiency are described, the formulation may be applicable to other diseases and other animals.

DETAILED DESCRIPTION The present invention provides an injectable formulation including calcium chloride, calcium lactate and calcium levulinate calcium salts, together with vitamin B12 or a 8 James & Wells Ref: 234997/75 derivative thereof. Also provided for is the use of the composition for the treatment and/or prevention of milk fever in animals.

The preferred formulation of the invention and the method of manufacturing this formulation is described below: Formulation 1 Active Ingredients Concentration (%w/v) Calcium lactate pentahydrate 4.34 Calcium chloride dihydrate 3.1 Calcium levulinate dihydrate .78 Cyanocobalamin 0.003 Excipients Methyl paraben 0.100 Hydrochloric acid (AR) q.s Water for injection q.s The formulation provided above can be manufactured using the following method: Step 1 • 70% of water for injection is loaded into a clean manufacturing vessel and heated to 50 - 55°C.

Step 2 *10% of water for injection is loaded into a second vessel and heated to 80 -90°. 9 James & Wells Ref: 234997/75 • Methyl paraben is added to the second vessel and dissolved while stirring. The mixture is maintained at 80 - 90°C.

Step 3 • In a third clean manufacturing vessel 2% of water for injection is loaded and 5 heated to 50-55°C.

• Cyanocobalamin is added and dissolved with stirring.

Step 4 • The mixture of step 2 is added to the mixture of step 1 and stirred well. The temperature is maintained at 50 - 55°C.

• Calcium lactate is added and dissolved with stirring.

• Calcium levulinate is added and dissolved with stirring.

• Calcium chloride is added and dissolved with stirring.

• The mixture from step 3 is added to the main bulk and mixed well.

• The bulk is then cooled to 25-30°C and the pH adjusted to 4.5 - 6 with HCI 15 solution. This pH range has been proven to provide optimum stability for the composition. The volume is made up to q.s with water for injection.

While this formulation exemplifies a preferred composition of the invention, the invention is not intended to be limited by the ingredients listed. For example, formulations containing different combinations of calcium salts in combination with 20 vitamin B12 or derivatives are also envisaged, but not exemplified.

Accordingly, different excipients may also be employed to provide a stable, injectable composition as would be understood by a person skilled in the art.

James & Wells Ref: 234997/75 Animal Trials A cross over study was carried out to evaluate and compare the safety and efficacy of the composition of the present invention (herein after referred to as Calpro BF), a metabolic solution containing three sources of calcium plus Vitamin B12, with that 5 of Calpro 375. Calpro 375 is the subject composition of NZ 272317 which contains calcium borogluconate and Vitamin B12 and is a standard reference product for boron containing milk fever treatments. Calpro 375 has been shown in the past to effectively treat milk fever in cattle as well as providing a source of vitamin B12 to the animal. It is the aim of the below study to demonstrate statistically similar 10 efficacy between the original Calpro 375 and the boron free composition Calpro BF of the present invention in the treatment of milk fever. This study confirms the current novel compositions are equally efficacious, while providing the added advantage of being boron free.

Calpro 375 and Calpro BF were tested in cattle, which is the primary species they 15 are indicated for. The study was carried out on a property in South Auckland in November 2007, and involved 10 Friesian X, dry-dry cows that were being fed maintenance levels of pasture.

Methods and Materials In this trial, 10 dairy (Friesian X) cows, not in calf and culled after peak seasonal 20 production were run on spring pasture fed at maintenance levels. The cows were weighed so the dose rate each received could, subsequently, be calculated. On successive days of the study, four to six cows (selected randomly as they came into the race) were treated with either the reference or the test product.

Calpro BF, containing calcium at a level of 28,2g/L, was administered to 10 cows. 25 After a washout period of 4-7 days, the same 10 cows were then given Calpro 375 11 James & Wells Ref: 234997/75 (A7110), which has a similar level of calcium (28.1g/L). The test arid reference products were each administered as a single intravenous infusion of 500mL.

Immediately before treatment, the cows were blood sampled and the heart rate was recorded. In addition, rectal temperatures were recorded and the nature and 5 character of the respiration and the demeanor of the cow were evaluated. Immediately after treatment and at the times indicated, the same clinical assessments (rectal temperature, respiration, cow demeanor) were made and blood samples were collected. Thus, these activities were carried out at the following times relative to the time of treatment: TO (immediately pre-treatment), T1 (immediately post-treatment), T2 (0.5 hr after beginning treatment), T3 (3 hours post-treatment), T4 (1 day after treatment), and T5 (2 days after treatment).

Blood levels of the minerals of primary interest (Ca, Mg, CI, P) were determined for each time point. Also, a wide range of biochemical and haematological parameters 15 were tested at TO, to confirm initial conformance to normal ranges. The random assignment to treatment group for each animal (Calpro BF or Calpro 375 reference) was validated by showing no significant differences (ANOVA p>0.05 on selected parameters) between the TO clinical variables for the animals in the two treatment groups.

Treatment groups and treatment structure Cumulatively over four treatment days, a group of 10 cows were given each of the following treatments: Treatment 1: Calpro BF, Test item (Containing calcium chloride, calcium levulinate and calcium lactate) 12 James & Wells Ref: 234997/75 • Dose rate: 500mL per cow of (containing 28.2 g/L calcium) by intravenous infusion.

Treatment 2: Calpro 375, Reference item (Containing calcium borogluconate) • Dose rate: 500mL per cow (containing 28.1 g/L calcium) by intravenous infusion.

The study was performed in two phases, with each involving the treatment of the 10 cows that had been recruited for the study.

In phase 1, which involved the administration of the treatments over a period of two 10 days, five were treated with the test and five with the reference product.

In phase 2, which was the cross over part of the study, the five cattle that had been treated with the test product seven days previously were dosed with the reference product and the test product was administered to the cows that had been treated with the reference product seven days earlier. Thus over the course of phases 1 15 and 2 both treatments were given to each cow. Treatments were given once only to each cow, in each phase of the study. The weights of cows ranged from 419 to 562 kg prior to treatment.

Results Serum calcium levels As shown in the graph of Figure 1, when first screened at TO, serum levels of calcium were all within the adequate range (2.0 - 2.6 mmol/L). At T1 (immediately after treatment and generally about 5-10 minutes after the beginning of intravenous infusion), serum calcium levels were elevated for all animals, as 13 James & Wells Ref: 234997/75 expected, with means of 5.4 and 4.9 mmol/L for the reference and test treatments, respectively.

The Ca levels were still above the adequate range at T2, (0.5 hour after the administration of the treatments had begun) with means of 4.3 and 3.8 mmol/L for 5 the reference and test treatments, respectively.

The data points where the selected mineral levels were clearly elevated were statistically analysed using ANOVA to determine if any of the differences between groups were significant.

The small (but not significant) mean differences in favour of the reference 10 treatment shown at the first two time points did not occur at subsequent points for serum calcium. Thus, no significant difference, and no consistent trend of difference in level of serum Ca between the two treatments was seen.

At T3 (3 hours after treatment), serum calcium levels were still above the adequate range, except in one animal in each treatment group.

From T4 (24 hours days after treatment) mean serum calcium levels had all returned to lie within the adequate range.

No significant differences between treatments were found for serum Ca at any time point.

The main finding of this study was that immediately after treatment, serum calcium 20 levels were elevated in all animals, as expected, with means of 5.4 and 4.9 mmol/L for reference and test treatments, respectively. The calcium levels were still above the adequate range 0.5 hours after the administration of the treatments had been completed, with means of 4.3 and 3.8 mmol/L respectively; and also at 3 hours after administration, with means of 2.9 and 3.0mmol/L, respectively. 14 James & Wells Ref: 234997/75 Serum chloride levels As shown in Figure 2, at TO mean serum chloride levels were 98.6 and 99.3 for reference and test groups, respectively, which are both near the lower limit of the adequate range (97 - 111 mmol/L).

The mean level for the reference treatment dropped slightly below adequate levels at T1, and returned to levels that were within the adequate range at T3 (3 hours after treatment). Serum chloride levels remained within the adequate range throughout the period of the study in the test product group, although they dropped very slightly immediately after dosing and had risen to pre-treatment levels by T4 10 (24 hours after treatment).

Significant differences between treatments were found for serum CI at T1 and T2 but these were not thought to be of any clinical importance because of their transitory nature and because they quickly returned to pre treatment levels.

Additionally, after treatment serum magnesium, phosphate and chloride levels 15 were not elevated, and in fact they were slightly decreased, and it is thought that this might be related to a dilution effect from the infused solution.

Summary Overall, it was found that there were no significant differences (ANOVA p>0.05) between treatments for serum levels of calcium, magnesium and phosphorus. 20 There were some transient differences in chloride levels between the treatment groups at the T1 and T2 samplings. However, these were not thought to have been of clinical importance. Clinical examinations were also carried out at five designated times following the administration of the treatments and no significant adverse effects of the two treatments were recorded.

James & Wells Ref: 234997/75 Thus, from the data obtained, no significant differences in tolerance were found between the two products. Additionally, their potential to correct hypocalcaemia appears to be equivalent.

The novel compositions of the present invention have been show to be as effective 5 in raising serum calcium levels in cattle as calcium borogluconate products currently available. Additionally, due to the absence of boron, the compositions are less toxic than known calcium supplements containing vitamin B12, to both animals and the environment.

Aspects of the present invention have been described by way of example only and 10 it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims. 16

Claims (10)

James & Wells Ref: 234997/75 WHAT WE C|_A|M |S.

1. An injectable composition including: a. at least three different calcium salts; and b. vitamin B12 or a derivate thereof.

2. The injectable composition of claim 1, wherein the calcium salts are selected from the group including calcium halide, calcium levulinate, calcium lacate, calcium chlorate, calcium acetate, calcium propionate, calcium mannoate, calcium saccharate and calcium gluconate.

3. The injectable composition of claim 1 or claim 2, wherein the calcium salts are calcium lactate, calcium chloride and calcium levulinate.

4. The injectable composition of any one of the preceding claims wherein the amount of calcium in the composition is 25.0 - 30.0 g/L calcium.

5. The injectable composition of any one of the preceding claims, wherein the composition includes vitamin B12 in the form of cyanocobalamin or hydroxycobalamin.

6. The injectable composition of claim 5, wherein the amount of cyanocobalamin or hydroxycobalamin in the composition is 0.001 - 0.2 g/L.

7. The injectable composition of any of the preceding claims, wherein the composition is injectable.

8. A method of treating or preventing milk fever in non-human animals, characterised by the step of administering to the animal an injectable 17 James & Wells Ref: 234997/75 composition including at least three different calcium salts and vitamin B12 or a derivative thereof.

9. The method of claim 8, wherein the injectable composition includes as the three calcium salts calcium lactate, calcium chloride and calcium levulinate.

10. The method of claim 8 or claim 9, wherein the non-human animal is a ruminant. Bomac Research Limited by their authorised agents James & Wells Intellectual Property 18