Formulation and method of preventing or treating hypocalcaemia

Abstract

A controlled-release Vitamin D3 formulation that provides both an initial burst release as well as sustained release of an active form of Vitamin D3, and its use in preventing or treating conditions such as hypocalcaemia.

Description

Formulation and Method of Preventing or Treating Hypocalcaemia Related Applications

[0001] This application claims priority of Australian Patent Application No.2023900581, filed 6 March 2023, the entire contents of which are incorporated herein by way of reference. Technical Field

[0002] This invention broadly relates to a controlled-release Vitamin D3 formulation that provides both an initial burst release as well as sustained release of an active form of Vitamin D3, and its use in preventing or treating conditions such as hypocalcaemia. Background

[0003] Hypocalcaemia is one of the most commonly recognised metabolic diseases affecting dairy cows during the peripartum period (Liang et al., 2017). Hypocalcaemia often presents as a clinical condition (parturient paresis, milk fever, paresis puerperalis, or parturient apoplexy) but can also be subclinical. The burden of subclinical hypocalcaemia has been found to be higher than the clinical form (Goff, 2008).

[0004] The pathophysiology of hypocalcaemia has been described as a result of excessive calcium demand for colostrum and milk production soon after calving within 24-48 hours postpartum (Horst et al., 2005). In most cases, cows are unable to meet the increase in calcium demand within 24-48 hours postpartum. This results in calcium being sourced from feed as well as from the skeletal system by an increased calcium metabolism (Goff, 2008). Because there is a time delay between the sudden increase in demand for calcium and an increase in supply, this results in a cow failing to maintain calcium homeostasis. The normal reference range for total serum calcium in a dairy cow has been established as 2.1-2.5 mM (8.5-10 mg / dL), whereas 1.38-2 mM (5.5-8 mg / dL) has been described as subclinical hypocalcemia (approximately 50% of older cows and 25% heifers) and a serum level below 1.38 mM (5.5 mg / dL) as clinical hypocalcaemia (Goff, 2008).

[0005] One of the risk factors that have been associated with impairing calcium homeostasis in cows is metabolic alkalosis. Metabolic alkalosis predisposes cows to clinical and subclinical hypocalcaemia (Craige & Stoll, 1947). This is because metabolic alkalosis causes a reduced response to the parathyroid hormone (Goff, 2008; Phillipo et al., 1994). Metabolic alkalosis has been associated with a diet that provides more cations than anions to the blood (Goff, 2008). This condition can be reversed or prevented prepartum through diet supplementation with calcium and magnesium, phosphate and / or Vitamin D (Beede et al., 2001; Goff and Horst, 1997; Barton, 1978). Many postpartum interventions for acute hypocalcaemia include the use of intravenous injectionof calcium salts (calcium borogluconate) (Salgado-Hernández et al., 2014). These salts may contain magnesium, phosphates and glucose in addition (Goff, 2008). Calcium salts have also been used subcutaneously and also as oral calcium salt administrations (pastes and boluses) (Roberts et al.2019; Lawlor et al., 2019; Oetzel & Miller, 2012; Goff & Horst, 1993). The oral calcium salts have been extensively used in the management of hypocalcaemia in cows.

[0006] Although oral and intravenous calcium salts have been used in the prevention / treatment of postpartum hypocalcaemia in cows for many decades (Goff & Horst, 1993), usually these have to be administered to the cow multiple times over a period of days in order to normalizing blood calcium levels.

[0007] It would be desirable to prevent or treat hypocalcaemia or otherwise restore abnormal blood calcium levels with a formulation requiring single administration. Detailed Description of the Invention

[0008] It is an object of one or more embodiments of the present invention to provide a controlled- release formulation of an active form of Vitamin D3 (‘Vitamin D3 active’) which would allow for: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; avoiding insufficient calcium levels in the blood of a cow; or, to provide the public with a useful commercial choice.

[0009] The inventors have found that an initial release (‘initial burst release’) of a Vitamin D3 active (eg. calcitriol) in the first 1-2 days followed by a progressively decreasing release rate over the subsequent few days (‘sustained release’) can be effective in increasing and restoring blood calcium levels, and thus prevent or treat hypocalcaemia.

[0010] The invention therefore, in one or more embodiments, broadly relates to a controlled- release Vitamin D3 formulation that provides both an initial burst release as well as sustained release of a Vitamin D3 active, and its use for: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow.

[0011] According to a first aspect of the present invention, there is provided a controlled-release Vitamin D3 formulation comprising an active form of Vitamin D3 (‘Vitamin D3 active’) and a carrier, said carrier providing both an initial burst release as well as sustained release of the Vitamin D3 active.

[0012] According to a second aspect of the present invention, there is provided a controlled-release Vitamin D3 formulation for: preventing or treating hypocalcaemia in a cow; maintaining normaland / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, said formulation comprising a Vitamin D3 active and a carrier, and said carrier being formulated to provide to the cow both an initial burst release as well as sustained release of the Vitamin D3 active.

[0013] According to a third aspect of the present invention, there is provided a controlled-release Vitamin D3 formulation for use or when used for: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, said formulation comprising a Vitamin D3 active and a carrier, and said carrier being formulated to provide to the cow both an initial burst release as well as sustained release of the Vitamin D3 active.

[0014] According to a fourth aspect of the present invention, there is provided use of a Vitamin D3 active and a carrier in the manufacture of a medicament for: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, said medicament being formulated to provide to the cow both an initial burst release as well as sustained release of the Vitamin D3 active.

[0015] According to a fifth aspect of the present invention, there is provided a method of: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, said method comprising the step of administering to the cow a controlled- release Vitamin D3 formulation comprising a Vitamin D3 active and a carrier, said carrier providing to the cow both an initial burst release as well as sustained release of the Vitamin D3 active.

[0016] According to a sixth aspect of the present invention, there is provided use of a controlled- release Vitamin D3 formulation comprising a Vitamin D3 active and a carrier for: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, said carrier being formulated to provide to the cow both an initial burst release as well as sustained release of the Vitamin D3 active.

[0017] According to a seventh aspect of the present invention, there is provided an injector containing a controlled-release Vitamin D3 formulation comprising a Vitamin D3 active and a carrier for: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, said carrier being formulated to provide to the cow both an initial burst release as well as sustained release of the Vitamin D3 active.

[0018] According to an eighth aspect of the present invention, there is provided a kitset for use or when used in a method of: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, wherein the kitset comprises: an injector capable of administering a controlled-release Vitamin D3 formulation comprising a Vitamin D3 active and a carrier, said carrier being formulated to provide to the cow both an initial burst release as well as sustained release of the Vitamin D3 active.

[0019] Features described below relate to all aspects of the invention described above, context permitting. Features described in respect of formulations / medicaments / compositions may also relate to methods / uses and vice-versa, context permitting. It is to be understood that the terms ‘formulation’, ‘medicament’ and ‘composition’ may be used interchangeably, context permitting.

[0020] Any suitable type or types of Vitamin D3 active can be used in the formulation or in the methods / uses. In some embodiments, the Vitamin D3 active is calcitriol (1,25- dihydroxycholecalciferol, 1alpha,25-dihydroxyvitamin D3, 1,25-dihydroxyvitamin D3, 1α,25- (OH)2D3, 1,25(OH)2D), or a derivative, prodrug or analogue thereof. In some embodiments, the Vitamin D3 active is a calcitriol analogue, such as paricalcitol. In some embodiments, the Vitamin D3 active is a calcitriol derivative, such as calcitriol-glycoside. This type of derivative can be sourced, for example, from dried leaves of the plant Solanum glaucophyllum.

[0021] Typically, the controlled-release Vitamin D3 formulation will be administered to the cow in an injectable form. That is, in some embodiments, the formulation is formulated for injection, or administration of the formulation to the cow comprises the step of injecting the cow with the formulation. In some embodiments, the formulation is injected subcutaneously. In some embodiments, the formulation is injected intramuscularly.

[0022] In some embodiments, the controlled-release Vitamin D3 formulation is a liquid formulation. In some embodiments, the formulation is a solution, suspension, dispersion, emulsion or low viscosity gel, suitable for injection.

[0023] Formulations for injection can be prepared by dissolving or mixing the Vitamin D3 active with the carrier. Preferably, the carrier is veterinary acceptable. The formulation can be sterilised, such as by heat, filtration or irradiation, or prepared aseptically. Formulations for injection may be prepared by methods and techniques known to persons skilled in the art.

[0024] The carrier can comprise one or more of the following types of veterinary acceptable ingredients / excipients: a vehicle; an aqueous or oily diluent; a base; a buffering agent; a pH adjusting agent; a suspending agent; a flocculating agent; a thickener; a viscosity building agent; a gelling agent; a solvent; an organic solvent; a co-solvent; a solvent system; an emulsifier; a stabilizer; a dispersant; a detergent; a solubilizer; a fragrance; a preservative; a surfactant; an acid; a base; an antioxidant; a wetting agent; a chelating agent; a reducing agent; a bulking agent; a protectant; a tonicity adjustor; and, a colorant.

[0025] Examples of tonicity adjustors used in liquid injections / carriers include electrolytes, dextrose, glycerol / glycerin, sodium chloride, and mannitol.

[0026] Examples of preservatives used in liquid injections / carriers include antioxidants, antimicrobials and chelating agents, including ascorbic acid, acetylcysteine, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sulfurous acid salts (bisulfite, metabisulfite), monothioglyercol, phenol, meta-cresol, benzyl alcohol, gallate, parabens (methyl, propyl, butyl), benzalkonium chloride, chlorobutanol, thimerosal, and phenylmercuric salts.

[0027] In some embodiments, the formulation can be stabilised by way of using an oxygen-free environment, such as purging with nitrogen (having a nitrogen headspace).

[0028] Examples of solubilising agents used in liquid injections / carriers include surfactants, solvents and co-solvents, and include water, polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20), lecithin, polyoxyethylene copolymers (pluronics), propylene glycol, glycerin, organic solvents, ethanol, polyethylene glycol (300 and 400), sorbitol, dimethylacetamide and cremophor EL.

[0029] Examples of complexing and dispersing agents used in liquid injections / carriers include cyclodextrins and modified cyclodextrins such as hydroxypropyl-b-cyclodextrin and sulfobutylether-b-cyclodextrin.

[0030] Examples of buffering agents used in liquid injections / carriers include phosphate, citrate, acetate, lactate and tartrate buffers.

[0031] Suspensions may provide more prolonged release of the Vitamin D3 active from the injection site than a comparable solution. Examples of ingredients / excipients used in suspensions include flocculating / suspending agents, viscosity building agents, wetting agents, solvents, solventsystems, preservatives, antioxidants, chelating agents, buffering agents, surfactants, and tonicity adjusting agents.

[0032] Examples of flocculating / suspending agents include electrolytes, surfactant and hydrophilic colloids, including potassium / sodium chloride, potassium / sodium citrate, and potassium / sodium acetate.

[0033] Examples of viscosity building agents include sodium carboxymethyl cellulose, acacia, gelatin, methyl cellulose, and polyvinyl pyrrolidone.

[0034] Examples of wetting agents include glycerin, alcohol, propylene glycol, lecithin, polysorbate 20, polysorbate 80, pluronic F-68, sorbitan, and trioleate.

[0035] Examples of solvents, including organic solvents, include water, ethanol, glycerin, propylene glycol, n–lactamide, polyethylene glycol (PEG), dimethyl sulfoxide, glycofurol, Solketal, acetone, tetrahydrofurfuryl alcohol, diglyme, dimethyl isosorbide, ethyl lactate, diethylene glycol monoethyl ether (DEGEE) (eg. sold under the trade mark Transcutol), N- methylpyrrolidone (NMP), triacetin, benzyl benzoate, miglyol, propylene carbonate, benzyl alcohol, ethyl lactate, 2-pyrrolidone, propylene glycol, methyl acetate, ethyl acetate, methyl ethyl ketone, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, caprolactam, decylmethylsulfoxide, oleic acid, and 1-dodecyazacycloheptan-2-one.

[0036] In some embodiments the formulation / carrier is in the form of a low viscosity, injectable gel which gels rapidly at the injection site to provide an initial burst release as well as control the release of the Vitamin D3 active over a suitable period of time.

[0037] The formulation can be made to gel in situ (in the cow) in any suitable way. In some embodiments, the carrier can comprise one or more gelling agents. In some embodiments, the carrier comprises one or more solvents or a solvent system, such as one or more organic solvents. In some embodiments, the carrier comprises sucrose acetate isobutyrate (SAIB) and one or more solvents or a solvent system. Once a formulation containing SAIB and solvent / s or solvent system is injected, the solvent / s can diffuse out leaving a matrix that is both adhesive and viscous. The matrix can retain the Vitamin D3 active at the injection site for a prolonged period of time, rather than the Vitamin D3 active dispersing completely almost immediately.

[0038] In some embodiments, the formulation comprises SAIB in the following approximate ranges: 30% to 90%, 35% to 90%, 40% to 90%, 45% to 90%, 50% to 90%, 30% to 85%, 35% to 85%, 40% to 85%, 45% to 85%, 50% to 85%, 30% to 80%, 35% to 80%, 40% to 80%, 45% to 80%, 50% to 80%, 30% to 75%, 35% to 75%, 40% to 75%, 45% to 75%, 50% to 75%, 30% to 70%, 35% to 70%, 40% to 70%, 45% to 70%, or 50% to 70% w / v SAIB, including all numerical values between 30 to 90, including approximately 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 90. In some embodiments, the formulation comprises about 50% w / v SAIB. In some embodiments, the formulation comprises about 70% w / v SAIB.

[0039] Any suitable solvent / s or solvent system can be used provided that it / they produce a low viscosity, injectable gel. Preferred solvents include ethanol, diethylene glycol monoethyl ether (DEGEE) sold under the trade mark Transcutol, N-methylpyrrolidone (NMP), triacetin, benzyl benzoate, miglyol, propylene carbonate, benzyl alcohol, ethyl lactate, glycofurol, 2-pyrrolidone, propylene glycol, acetone, methyl acetate, ethyl acetate, methyl ethyl ketone, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, caprolactam, decylmethylsulfoxide, oleic acid, and 1- dodecyazacycloheptan-2-one, or any mixtures of these. In some embodiments, the solvent / s can comprise a mixture of ethanol, triacetin, and diethylene glycol monoethyl ether in any suitable ratio and quantities. The solvent / s can be present in an amount of between approximately 10% to 50% w / v, preferably approximately 15 to 30% w / v. These ranges include all numerical values between 10 to 50, and 15 and 30 (including approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50).

[0040] In some embodiments, the carrier comprises a surfactant, detergent or emulsifier, to assist with the release of the Vitamin D3 active. Any suitable type of surfactant, detergent or emulsifier can be used. Suitable examples include non-ionic surfactants such as polysorbates. Tween 80 is particularly preferred. The surfactant can be present in an amount of between approximately 0.1% to 10% w / v, preferably approximately 0.4% to 10% w / v, and more preferably approximately 0.5% to 5% w / v. These ranges include all numerical values between 0.1 and 10, 0.4 and 10, and 0.5 and 5 (inclusive of 0.1, 0.2…1, 1.1, 1.2…2, 2.1, 2.2…3, 3.1, 3.2…4, 4.1, 4.2…5, 5.1, 5.2…6, 6.1, 6.2…7, 7.1, 7.2…8, 8.1, 8.2…9, 9.1, 9.2…10). In some embodiments, the formulation comprises about 2% w / v surfactant, preferably Tween 80.

[0041] In some embodiments, the carrier comprises at least one type of preservative, preferably at least one type of antioxidant. Any suitable type of preservative(s) or antioxidant(s) can be used. Examples of suitable preservatives or antioxidants include phenolic antioxidants, such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA). Further examples include esters of 4-hydroxybenzoic acid, such as parabens, such as methyl paraben and propyl paraben. In some embodiments, BHT is preferred. In some embodiments, one or more parabens are preferred. In some embodiments, methyl paraben is preferred. In some embodiments, propyl paraben is preferred. In some embodiments, one or more phenolic antioxidants are used in combination withone or more esters of 4-hydroxybenzoic acids. In some embodiments, BHT is used in combination with one or more parabens, preferably methyl paraben and propyl paraben.

[0042] In some embodiments, the, or each type of, preservative or antioxidant can be present in an amount of at least approximately 0.001% w / v, more preferably at least approximately 0.01% w / v. In some embodiments, each type of preservative or antioxidant can be present in an amount of between approximately 0.001% to 10% w / v, preferably approximately 0.01% to 10% w / v, 0.01% to 5% w / v, 0.05% to 5% w / v, 0.01% to 1% w / v, or 0.01% to 0.1% w / v. These ranges include all numerical values between 0.001 to 10, 0.01 to 10, 0.01 to 5, 0.05 to 5, 0.01 to 1, and 0.01 to 0.1, etc. In some embodiments, the preservative(s) or antioxidant(s) can be present in a total amount of between approximately 0.001% to 10% w / v, preferably approximately 0.01% to 10% w / v, 0.01% to 5% w / v, 0.05% to 5% w / v, 0.01% to 1% w / v, or 0.01% to 0.1% w / v. These ranges include all numerical values between 0.001 to 10, 0.01 to 10, 0.01 to 5, 0.05 to 5, 0.01 to 1, and 0.01 to 0.1, etc. In some embodiments the formulation comprises approximately 0.1% to 1% w / v preservative or antioxidant, such as a phenolic antioxidant and / or ester of 4-hydroxybenzoic acid, including all numerical values between 0.1 to 1. In some embodiments, the formulation comprises approximately 0.01 to 0.1% w / v preservative or antioxidant (including all numerical values between 0.01 to 0.1), more preferably approximately 0.03% w / v, preferably BHT. In some embodiments, the formulation comprises approximately 0.01 to 0.2% w / v preservative or antioxidant (including all numerical values between 0.01 to 0.2), more preferably approximately 0.1% w / v paraben(s), more preferably approximately 0.09% w / v methyl paraben and approximately 0.01% w / v propyl paraben.

[0043] The concentration of the Vitamin D3 active in the formulation is preferably in the range of approximately 0.0001% w / v to 10% w / v depending on the potency of the Vitamin D3 active, including all numerical values between 0.0001 and 10. In some embodiments the Vitamin D3 active is present in an amount of between approximately 0.001% to 1% w / v, preferably approximately 0.005% to 0.05% w / v, including all numerical values between 0.001 and 1, and 0.005 to 0.05. Preferably, the Vitamin D3 active is present in an amount of between approximately 0.005 % to 0.01% w / v, including all numerical values between 0.005 and 0.01. Preferably, the Vitamin D3 active is calcitriol. In some embodiments, the formulation comprises about 0.005 % to 0.01% w / v of Vitamin D3 active, such as calcitriol.

[0044] Any suitable dose of the formulation can be administered to the cow. Approximately 0.01 μg to 10 μg per kg bodyweight of the cow is administered, preferably approximately 0.03 μg to 3.3 μg, more preferably approximately 0.25 μg to 0.5 μg, and even more preferably approximately 0.33 μg per kg bodyweight of the cow.

[0045] The quantity of formulation administered is preferably within the range of approximately 0.05 to approximately 10 ml, including all numerical values between 0.05 and 10. A particularly preferred range is from 0.5 ml to 5 ml, including all numerical values between 0.5 and 5. For example, if the formulation contained 100 μg / ml Vitamin D3 active and the dose of Vitamin D3 active was 0.33 μg / kg, then 1.65 ml can be administered to a 500 kg cow.

[0046] Preferably an injector is used to carry out injection of (administration to) the cow. Preferably an injector suitable for single-handed use is used, so as to allow use of the other hand to steady the cow.

[0047] Particularly preferred formulations are shown below:

[0048] Formulation 1:

[0049] Vitamin D3 active, preferably calcitriol: approximately 0.005% to 0.1% w / v, preferably approximately 0.005% to 0.01% w / v;

[0050] SAIB: approximately 45% to 80% w / v, preferably approximately 50% to 70% w / v;

[0051] Optionally, preservative / s such as antioxidant / s, with a quantity to suit, preferably one or more of BHT, methyl paraben and propyl paraben. For example: BHT - approximately 0.01% to 1% w / v, preferably approximately 0.1% w / v; methyl paraben - approximately 0.009% to 0.9% w / v, preferably approximately 0.09% w / v; propyl paraben - approximately 0.001% to 0.1% w / v, preferably approximately 0.01% w / v;

[0052] Solvent / s, preferably a mixture of ethanol, triacetin and diethylene glycol monoethyl ether (DEGEE): quantity to suit; and

[0053] Optionally, surfactant / s or detergent / s for any SAIB concentration above about 70% w / v, preferably Tween 80: approximately 0.5% to 5% w / v, preferably approximately 2% w / v.

[0054] Formulation 2:

[0055] Vitamin D3 active, preferably calcitriol: approximately 0.005% to 0.01% w / v;

[0056] SAIB: approximately 50 % w / v;

[0057] Optionally, preservative / s such as antioxidant / s, with a quantity to suit, preferably one or more of BHT, methyl paraben and propyl paraben. For example: BHT - approximately 0.01% to 1% w / v, preferably approximately 0.1% w / v; methyl paraben - approximately 0.009% to 0.9% w / v, preferably approximately 0.09% w / v; propyl paraben - approximately 0.001% to 0.1% w / v, preferably approximately 0.01% w / v; and

[0058] Solvent / s: approximately 9.8% w / v ethanol, approximately 8.6% w / v triacetin, and quantity to suit diethylene glycol monoethyl ether (DEGEE).

[0059] Formulation 3:

[0060] Vitamin D3 active, preferably calcitriol: approximately 0.005% to 0.01% w / v;

[0061] SAIB: approximately 70% w / v;

[0062] Optionally, preservative / s such as antioxidant / s, with a quantity to suit, preferably one or more of BHT, methyl paraben and propyl paraben. For example: BHT - approximately 0.01% to 1% w / v, preferably approximately 0.1% w / v; methyl paraben - approximately 0.009% to 0.9% w / v, preferably approximately 0.09% w / v; propyl paraben - approximately 0.001% to 0.1% w / v, preferably approximately 0.01% w / v;

[0063] Solvent / s: approximately 9.8% w / v ethanol, approximately 8.6% w / v triacetin, and quantity to suit diethylene glycol monoethyl ether (DEGEE); and

[0064] Surfactant / s or detergent / s, preferably Tween-80: approximately 2% w / v.

[0065] Preferably, the formulation is used for the prevention or treatment of sub-clinical hypocalcaemia, or the prevention or treatment of clinical hypocalcaemia.

[0066] In some embodiments, the formulation is injected before calving.

[0067] In some embodiments, the formulation is injected at the time of calving.

[0068] In some embodiments, the formulation is injected within approximately 12 hours after calving (postpartum).

[0069] Preferably the formulation is administered to periparturient dairy cows.

[0070] Preferably, approximately 198 µg of calcitriol / 600 kg body weight is injected into the cow.

[0071] Preferably, the formulation is effective in increasing serum tCa (total sero-calcium) in approximately the first 7 days postpartum or after injection.

[0072] Preferably, the formulation is effective in achieving a serum calcium level of > / = 2.0 mM within approximately 24 hours of injection.

[0073] Preferably, the formulation is effective in increasing serum calcitriol beginning approximately an hour after injection.

[0074] Preferably, the formulation is effective in maintaining an elevated serum calcitriol concentration until approximately 48 hours after injection.

[0075] Preferably, for controlled release, the Vitamin D3 active is released from the carrier at an ever-decreasing rate.

[0076] In some embodiments, the following approximate Vitamin D3 active doses (% mass) are delivered to the cow, assuming the Vitamin D3 active concentration (preferably being calcitriol) is about 100 µg / ml and a 2 mL dose is administered to a 600 kg cow: by 6 hours, 30-50%, preferably about 40%; by 24 hours, 60-80%, preferably about 70%; by 48 hours, 80-95%, preferably about 90%; and, by 96 hours, preferably about 100%.

[0077] Preferred embodiments of the invention are defined in the paragraphs below:

[0078] 1. A controlled-release Vitamin D3 formulation comprising an active form of Vitamin D3 (‘Vitamin D3 active’) and a carrier, said carrier providing both an initial burst release as well as sustained release of the Vitamin D3 active.

[0079] 2. The formulation of paragraph 1, wherein the formulation is in an injectable form, preferably for subcutaneous or intramuscular injection.

[0080] 3. The formulation of paragraph 1 or paragraph 2, wherein the Vitamin D3 active is present in the range of approximately 0.0001% w / v to 10% w / v, preferably approximately 0.001% to 1% w / v, more preferably approximately 0.005% to 0.05% w / v, and even more preferably approximately 0.005 % to 0.01% w / v.

[0081] 4. The formulation of any one of the preceding paragraphs, wherein the Vitamin D3 active is calcitriol (1,25-dihydroxycholecalciferol, 1alpha,25-dihydroxyvitamin D3, 1,25- dihydroxyvitamin D3, 1α,25-(OH)2D3, 1,25(OH)2D), or a derivative, prodrug or analogue thereof.

[0082] 5. The formulation of paragraph 4, wherein the Vitamin D3 active is calcitriol.

[0083] 6. The formulation of any one of the preceding paragraphs, wherein the formulation is a solution, suspension, dispersion, emulsion or low viscosity gel, suitable for injection.

[0084] 7. The formulation of paragraph 6, wherein the formulation is in the form of a low viscosity, injectable gel which gels rapidly at an injection site.

[0085] 8. The formulation of paragraph 7, wherein the carrier comprises one or more gelling agents.

[0086] 9. The formulation of paragraph 8, wherein the one or more gelling agents comprises sucrose acetate isobutyrate (SAIB).

[0087] 10. The formulation of paragraph 9, wherein the formulation comprises SAIB in the following approximate ranges: 30% to 90%, 35% to 90%, 40% to 90%, 45% to 90%, 50% to 90%, 30% to 85%, 35% to 85%, 40% to 85%, 45% to 85%, 50% to 85%, 30% to 80%, 35% to 80%, 40% to 80%, 45% to 80%, 50% to 80%, 30% to 75%, 35% to 75%, 40% to 75%, 45% to 75%, 50% to 75%, 30% to 70%, 35% to 70%, 40% to 70%, 45% to 70%, or 50% to 70% w / v.

[0088] 11. The formulation of paragraph 10, wherein the formulation comprises about 50% w / v SAIB.

[0089] 12. The formulation of paragraph 10, wherein the formulation comprises about 70% w / v SAIB.

[0090] 13. The formulation of any one of paragraphs 8 to 12, wherein the carrier comprises one or more solvents or a solvent system.

[0091] 14. The formulation of paragraph 13, wherein the one or more solvents is present in an amount of approximately 10% to 50% w / v, preferably approximately 15 to 30% w / v.

[0092] 15. The formulation of paragraph 14, wherein the one or more solvents comprises ethanol, diethylene glycol monoethyl ether (DEGEE), N-methylpyrrolidone (NMP), triacetin, benzyl benzoate, miglyol, propylene carbonate, benzyl alcohol, ethyl lactate, glycofurol, 2-pyrrolidone, propylene glycol, acetone, methyl acetate, ethyl acetate, methyl ethyl ketone, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, caprolactam, decylmethylsulfoxide, oleic acid, or 1- dodecyazacycloheptan-2-one, or any mixture of these.

[0093] 16. The formulation of any one of the preceding paragraphs, wherein the carrier comprises a surfactant, detergent or emulsifier.

[0094] 17. The formulation of paragraph 16, wherein the surfactant, detergent or emulsifier is present in an amount of between approximately 0.1% to 10% w / v, preferably approximately 0.4% to 10% w / v, and more preferably approximately 0.5% to 5% w / v.

[0095] 18. The formulation of paragraph 17, wherein the surfactant, detergent or emulsifier is present in an amount of about 2% w / v.

[0096] 19. The formulation of any one of paragraphs 16 to 18, wherein the surfactant, detergent or emulsifier is at least one type of polysorbate, preferably Tween 80.

[0097] 20. The formulation of any one of paragraphs 1 to 19, wherein the carrier comprises at least one type of preservative, preferably at least one type of antioxidant.

[0098] 21. The formulation of paragraph 20, wherein the, or each type of, preservative or antioxidant is present in an amount of at least approximately 0.001% w / v, more preferably at least approximately 0.01% w / v .

[0099] 22. The formulation of paragraph 21, wherein the formulation comprises approximately 0.1% to 1% w / v preservative or antioxidant, such as a phenolic antioxidant and / or ester of 4- hydroxybenzoic acid.

[0100] 23. The formulation of any one of paragraphs 20 to 22, wherein the at least one preservative or antioxidant comprises one or more of butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), methyl paraben and propyl paraben.

[0101] 24. The formulation of paragraph 1, wherein the formulation comprises (Formulation 1):

[0102] Vitamin D3 active, preferably calcitriol: approximately 0.005% to 0.1% w / v, preferably approximately 0.005% to 0.01% w / v;

[0103] SAIB: approximately 45% to 80% w / v, preferably approximately 50% to 70% w / v;

[0104] Optionally, preservative / s such as antioxidant / s with a quantity to suit, preferably one or more of BHT, methyl paraben and propyl paraben;

[0105] Solvent / s, preferably a mixture of ethanol, triacetin and diethylene glycol monoethyl ether (DEGEE): quantity to suit; and

[0106] Optionally, surfactant / s or detergent / s for any SAIB concentration above about 70% w / v, preferably Tween 80: approximately 0.5% to 5% w / v, preferably approximately 2% w / v.

[0107] 25. The formulation of paragraph 1, wherein the formulation comprises (Formulation 2):

[0108] Vitamin D3 active, preferably calcitriol: approximately 0.005% to 0.01% w / v;

[0109] SAIB: approximately 50 % w / v;

[0110] Optionally, preservative / s such as antioxidant / s with a quantity to suit, preferably one or more of BHT, methyl paraben and propyl paraben; and

[0111] Solvent / s: approximately 9.8% w / v ethanol, approximately 8.6% w / v triacetin, and quantity to suit diethylene glycol monoethyl ether (DEGEE).

[0112] 26. The formulation of paragraph 1, wherein the formulation comprises (Formulation 3):

[0113] Vitamin D3 active, preferably calcitriol: approximately 0.005% to 0.01% w / v;

[0114] SAIB: approximately 70% w / v;

[0115] Optionally, preservative / s such as antioxidant / s with a quantity to suit, preferably one or more of BHT, methyl paraben and propyl paraben;

[0116] Solvent / s: approximately 9.8% w / v ethanol, approximately 8.6% w / v triacetin, and quantity to suit diethylene glycol monoethyl ether (DEGEE); and

[0117] Surfactant / s or detergent / s, preferably Tween-80: approximately 2% w / v.

[0118] 27. A controlled-release Vitamin D3 formulation for: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, said controlled- release Vitamin D3 formulation comprising the formulation of any one of paragraphs 1 to 26.

[0119] 28. A controlled-release Vitamin D3 formulation for use or when used for: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, said controlled-release Vitamin D3 formulation comprising the formulation of any one of paragraphs 1 to 26.

[0120] 29. Use of a Vitamin D3 active and a carrier in the manufacture of a medicament for: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, said medicament being formulated to provide to the cow both an initial burst release as well as sustained release of the Vitamin D3 active, wherein said Vitamin D3 active is asdescribed in any one of paragraphs 1 to 26, and said carrier is as described in any one of paragraphs 1 to 26.

[0121] 30. A method of: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, said method comprising the step of administering to the cow the controlled-release Vitamin D3 formulation of any one of paragraphs 1 to 26.

[0122] 31. Use of a controlled-release Vitamin D3 formulation comprising a Vitamin D3 active and a carrier for: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, wherein said Vitamin D3 active is as described in any one of paragraphs 1 to 26, and said carrier is as described in any one of paragraphs 1 to 26.

[0123] 32. An injector containing a controlled-release Vitamin D3 formulation comprising a Vitamin D3 active and a carrier for: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, wherein said Vitamin D3 active is as described in any one of paragraphs 1 to 26, and said carrier is as described in any one of paragraphs 1 to 26.

[0124] 33. A kitset for use or when used in a method of: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, wherein the kitset comprises: an injector capable of administering the controlled-release Vitamin D3 formulation as defined in any one of paragraphs 1 to 26.

[0125] 34. The controlled-release Vitamin D3 formulation of paragraph 27 or paragraph 28, the use of paragraph 29 or paragraph 31, the method of paragraph 30, the injector of paragraph 32, or the kitset of paragraph 33, wherein approximately 0.01 μg to 10 μg, preferably approximately 0.03 μg to 3.3 μg, more preferably approximately 0.25 μg to 0.5 μg, and even more preferably approximately 0.33 μg per kg bodyweight of the cow is injected.

[0126] 35. The controlled-release Vitamin D3 formulation of paragraph 27, 28 or 34, the use of paragraph 29, 31 or 34, the method of paragraph 30 or paragraph 34, the injector of paragraph 32or paragraph 34, or the kitset of paragraph 33 or paragraph 34, wherein approximately 0.05 to approximately 10 ml of formulation is injected.

[0127] 36. The controlled-release Vitamin D3 formulation of paragraph 27, 28, 34 or 35, the use of paragraph 29, 31, 34 or 35, the method of paragraph 30, 34 or 35, the injector of paragraph 32, 34 or 35, or the kitset of paragraph 33, 34 or 35, wherein the formulation is used for the prevention or treatment of sub-clinical hypocalcaemia, or the prevention or treatment of clinical hypocalcaemia.

[0128] 37. The controlled-release Vitamin D3 formulation of paragraph 27, 28, 34, 35 or 36, the use of paragraph 29, 31, 34, 35 or 36, the method of paragraph 30, 34, 35 or 36, the injector of paragraph 32, 34, 35 or 36, or the kitset of paragraph 33, 34, 35 or 36, wherein:

[0129] the formulation is injected before calving;

[0130] the formulation is injected at the time of calving;

[0131] the formulation is injected within approximately 12 hours after calving (postpartum);

[0132] the formulation is administered to a periparturient dairy cow;

[0133] approximately 198 µg of calcitriol / 600 kg body weight is injected into the cow;

[0134] the formulation is effective in increasing serum tCa (total sero-calcium) in approximately the first 7 days postpartum or after injection;

[0135] the formulation is effective in achieving a serum calcium level of > / = 2.0 mM within approximately 24 hours of injection;

[0136] the formulation is effective in increasing serum calcitriol beginning approximately an hour after injection; or

[0137] the formulation is effective in maintaining an elevated serum calcitriol concentration until approximately 48 hours after injection.

[0138] Having broadly described the invention in its various embodiments, non-limiting examples of preferred embodiments will now be described. Brief Description of the Figures

[0139] Figure 1. The mean values and 95% confidence intervals for mean total serum calcium concentrations of dairy cows receiving various preparations of calcitriol, IVP-A (108 µg, 18 µg, 18 µg, & 12 µg)-green, IVP-B (198 µg)-blue or IVP-C (198 µg)-purple, and a placebo (orange). Values represent means ± SD. Error bars indicate the traditional 95% confidence intervals for the mean. The time on the x-axis was measured hours post time 0.

[0140] Figure 1A. The standardised residuals from the final linear mixed effects model fit by REML suggesting that the residuals did not violate the homogeneity assumption. This alsosuggests that the residuals meet the normality assumption as demonstrated in the quantiles quantile plot (not shown here).

[0141] Figure 2. The mean values and 95% confidence intervals for mean total serum calcium concentrations of dairy cows receiving various preparations of calcitriol, IVP-A (108 µg, 18 µg, 18 µg, & 12 µg)-green, IVP-B (198 µg)-blue or IVP-C (198 µg)-purple, and a placebo (orange) from 24 hrs post time 0. Values represent means ± SD. Error bars indicate the traditional 95% confidence intervals for the mean. The time on the x-axis is in days.

[0142] Figure 3. Violin plots (with median, quartiles, and distribution) showing total serum calcium concentration of dairy cows receiving calcitriol IVP-A (108 µg, 18 µg, 18 µg, & 12 µg)- yellow, IVP-B (198 µg)-orange or IVP-C (198 µg)-red and a placebo (blue). Each treatment group had significantly higher serum calcium than the control group. The box plot elements show the median calcium concentration for cows treated with IVP-C was highest among the other treatment types. The shape of the distribution (extremely skinny on each end and wide in the middle) indicates the calcium concentration of IVP-C treated cows were highly concentrated around the median. The overall treatment effect of calcitriol on cows on serum calcium was significantly higher than control cows (p=0.000).

[0143] Figure 4. Concentrations of calcitriol in serum of dairy cows receiving preparations of calcitriol IVP-A (108 µg, 18 µg, 18 µg, & 12 µg)-green, IVP-B (198 µg)-blue, IVP-C (198 µg)- purple and a placebo-red. Values represent means ± SD. The time on the x-axis was measured in hours post time 0.

[0144] Figure 5. Concentrations of calcitriol in serum of dairy cows receiving preparations of calcitriol IVP-A (108 µg, 18 µg, 18 µg, & 12 µg)-green, IVP-B (198 µg)-blue, IVP-C (198 µg)- purple and a placebo-red. Values represent means ± SD. The time on the x-axis was measured as days post time 0.

[0145] Figure 6. Violin plots with box plots inside of total serum calcitriol concentration of dairy cows receiving calcitriol IVP-A (108 µg, 18 µg, 18 µg, & 12 µg)-yellow, IVP-B (198 µg)-orange or IVP-C (198 µg)-red and a placebo (blue). The overall treatment effect of serum calcitriol treated cows was significantly higher than control cows (p=0.042).

[0146] Figure 7. The mean values and 95% confidence intervals for mean total serum parathyroid hormone concentrations of dairy cows after receiving calcitriol IVP-A (108 µg, 18 µg, 18 µg, & 12 µg)-green, IVP-B (198 µg)-blue or IVP-C (198 µg)-purple and a placebo (orange). Values represent means ± SD. Error bars indicate the traditional 95% confidence intervals for the mean. The time on the x-axis was measured in hours post treatment.

[0147] Figure 8. The mean values and 95% confidence intervals for mean total serum parathyroid hormone concentrations of dairy cows after receiving calcitriol IVP-A (108 µg, 18 µg, 18 µg, & 12 µg)-green, IVP-B (198 µg)-blue or IVP-C (198 µg)-purple and a placebo (orange) from 24 hrs post- time 0. Values represent means ± SD. The time on the x-axis was measured in days.

[0148] Figure 9. Violin plots with box plots inside of total serum parathyroid hormone concentrations of dairy cows receiving calcitriol IVP-A (108 µg, 18 µg, 18 µg, & 12 µg)-yellow, IVP-B (198 µg)-orange or IVP-C (198 µg)-red and a placebo (blue). The p-values indicate the mean differences observed between treatments.

[0149] Figure 10. Release profiles of various calcitriol-SAIB formulations using a rotating basket. Description of Preferred Embodiments

[0150] The inventors postulated that a single injection of a controlled-release formulation containing a Vitamin D3 active such as calcitriol would allow for the prevention of hypocalcaemia in dairy cows when administered peripartum, and could also be used to treat clinical / sub-clinical hypocalcaemia when diagnosed post-calving. To that end, the inventors developed a low viscosity, injectable gel that gels rapidly at the injection site so as to provide an initial release burst as well as provide sustained release of the Vitamin D3 active over a suitable period of time, so as to raise and substantially normalise blood calcium levels.

[0151] Such a formulation would fill a currently unmet need in dairy farming systems, would have a positive impact on animal welfare, and would reduce the negative impacts of hypocalcaemia on productivity.

[0152] Example 1 – Preparation and in vitro testing of various calcitriol-sucrose acetate isobutyrate (SAIB) formulations

[0153] Various low viscosity, injectable gels comprising different calcitriol-SAIB formulations were prepared and protected from sunlight. The release of calcitriol from its SAIB carrier was examined using a rotating basket (USP 1) method.

[0154] Table 1 - Formulations prepared and tested. Formulation Ingredients (g / L) 20% SAIB Calcitriol 0.1 Sucrose acetate isobutyrate 200 Ethanol 98 Triacetin 86 Transcutol V QS (DEGEE)40% SAIB Calcitriol 0.1 Sucrose acetate isobutyrate 400 Ethanol 98 Triacetin 86 Transcutol V QS (DEGEE) 50% SAIB (‘F-2’) Calcitriol 0.1 Sucrose acetate isobutyrate 500 (‘20OTCASR-F-2' in Ethanol 98 Example 3) Triacetin 86 Transcutol V QS (DEGEE) 60% SAIB Calcitriol 0.1 Sucrose acetate isobutyrate 600 Ethanol 98 Triacetin 86 Transcutol V QS 70% SAIB Calcitriol 0.1 Sucrose acetate isobutyrate 700 Ethanol 98 Triacetin 86 Transcutol V QS (DEGEE) 70% SAIB + Tween 80 Calcitriol 0.1 (‘F-1’) Sucrose acetate isobutyrate 700 (‘20OTCASR-F-1' in Tween 80 20 Example 3) Ethanol 98 Triacetin 86 Transcutol V QS (DEGEE) 80% SAIB Calcitriol 0.1 Sucrose acetate isobutyrate 800 Ethanol 98 Triacetin 86Transcutol V QS (DEGEE)

[0155] Preparation of these formulations is essentially as described in Example 2 for F- 1 / 20OTCASR-F-1.

[0156] In vitro release testing: A volume of 0.5 ml of each formulation was injected into a basket (USP 1 rotating basket) while it was submerged in 400 ml of dissolution medium held at 37oC. The aqueous medium contained 4 mg / ml of Tween 20 and 2.5 mg / ml ascorbic acid. The rotation speed of the basket was 50 rpm. At predetermined times, 0.5 ml samples were taken with replacement. Each sample (25 µl) was injected into a HPLC comprising: a C18 column (250X4.6), a mobile phase of acetonitrile:water (65:35), a flow rate of 1 ml / min, UV detection at 265 nm. The release profiles of the SAIB formulations are shown in Figure 10.

[0157] Release from the 70% SAIB formulation was puzzling and the inventors postulated that it was due to binding of the calcitriol to the SAIB or degradation of the calcitriol in the presence of SAIB. Experiments showed that it was not a decomposition problem. It was hypothesised that the equilibrium binding of calcitriol with SAIB could be modified by including a surfactant such as Tween 80 to solubilise the calcitriol (in Tween micelles). Whether or not this is the mechanism involved is not clear but including Tween 80 did alter the release profile (see Figure 10).

[0158] Example 2 – Controlled-release calcitriol-SAIB formulations and stability testing

[0159] Based on these release profiles and preliminary stability data, the following formulations in Table 2 were prepared and packed in single dose vials.

[0160] Table 2 - Controlled-Release Calcitriol-SAIB Formulations. Formulation Ingredients (g / L) w / v % 70% SAIB T80 (F-1) Calcitriol 0.1 0.01 (20OTCASR-F-1) Sucrose acetate isobutyrate 700 70 Tween 80 20 2 Ethanol 98 9.8 Triacetin 86 8.6 Transcutol V QS QS (DEGEE) 50% SAIB (F-2) Calcitriol 0.1 0.01 (20OTCASR-F-2) Sucrose acetate isobutyrate 500 50 Ethanol 98 9.8Triacetin 86 8.6 Transcutol V QS QS (DEGEE)

[0161] Preparation of 70% SAIB T80: 14 g of SAIB was warmed to 50oC and 0.4 g Tween 80 was added. An ethanol and triacetin mixture was prepared at a ratio of 63:37 v / v.0.002 g of calcitriol was weighed and dissolved in 10 ml of the ethanol triacetin mixture. The calcitriol solution was filtered through a 0.22 µm sterile syringe filter and added to the SAIB / Tween 80 mixture. The empty calcitriol solution bottle was rinsed with 1 g of Transcutol (DEGEE) and filtered through the same 0.22 µm sterile syringe filter. The mixture was shaken overnight at ambient conditions until it formed a homogenous solution. The solution was transferred into a 20 ml volumetric flask and made up to 20 ml by adding Transcutol.2.1 ml of the formulation was transferred into single dosing vials and sealed.

[0162] Preparation of 50% SAIB: Prepared in a similar manner to 70% SAIB except that Tween 80 was omitted from the formulation.

[0163] Stability: The stability of calcitriol was tested in the ethanol triacetin mixture. This was preliminary testing (24 h at 40oC) and suggested stability sufficient for the period of the animal trial (see below).

[0164] Stability of formulations during the animal trial: Some of the vials of the two trial formulations that were prepared were stored under ambient conditions (20-25°C) and protected from light. These were analysed by HPLC assay approximately 6.5 months later.

[0165] HPLC Method: Approximately 0.5 g of a formulation was weighed into a 10 ml volumetric flask. Ethanol was added and shaken before making up to final volume. The ethanolic solution was diluted ten times with an aqueous solution of 4 mg / ml Tween 20 and 2.5 mg / ml and ascorbic acid to precipitate the SAIB. The mixture was vortexed and centrifuged at 14000 rpm for 5 min. The supernatant was taken and injected into the HPLC. A standard was prepared as follows: 0.4 g of SAIB was weighed in a 10 ml volumetric flask and spiked with a volume of a stock solution of calcitriol prepared in methanol. Then calcitriol was extracted as above before injecting the supernatant into the HPLC.

[0166] Results: The second column of Table 3 below shows the percentage of calcitriol remaining in the two formulations after 6.5 months.

[0167] Table 3 - Calcitriol remaining after 6.5 months.Formulation Calcitriol Calculated calcitriol Calculated calcitriol remaining at 6.5m remaining after 1w (% of remaining after 4w (% of initial) initial) (% of initial) 70% SAIB T80 69 98.6 94.5 (F-1) 50% SAIB (F-2) 39 96.4 86.5

[0168] The formulations were stored <25°C and used within 4 weeks (70% SAIB T80 – F-1) and 1 week (50% SAIB – F-2) by the CRO. The percentages remaining calculated assuming first order degradation of the calcitriol are highlighted in underline above in Table 3.

[0169] Based on these calculations it is concluded that the experimental formulations contained >90% of label claim.

[0170] Future work on stability: It is clear from the preliminary stability work that the formulations will have to be stabilised in order to achieve an acceptable shelf-life of at least two years (see Table 3 above for degradation at 6.5 m). This can be achieved by:

[0171] ^ Including one or more preservatives such as one or more antioxidants in the formulation, eg. BHT (eg.0.1% w / v), or a combination of BHT (eg.0.03% w / v), methyl paraben (eg.0.09% w / v) and propyl paraben (eg.0.01% w / v).

[0172] ^ If necessary, flushing vials with nitrogen before closure.

[0173] ^ Selecting an appropriate rubber bung (ie impervious to oxygen and does not absorb calcitriol.

[0174] With these approaches, it should be possible to achieve a 2-year shelf-life without the need for refrigeration.

[0175] Example 3 – Evaluating the effect of three formulations of calcitriol administered subcutaneously on the prevention of clinical and subclinical hypocalcaemia in dairy cows

[0176] Summary

[0177] The objective of this work was to assess the pharmacokinetics and efficacy of a singular slow release versus a multiple short acting calcitriol formulation in postpartum dairy cows on maintaining normocalcemic and thus preventing clinical or subclinical hypocalcemia when compared to a negative control. Normocalcaemia for the purposes of this study was defined as serum calcium levels > / = 2.0 mM whereas 1.38-2 mM (5.5-8 mg / dL) has been described as subclinical hypocalcemia (approximately 50% of older cows and 25% heifers) and a serum level below 1.38 mM (5.5 mg / dL) as clinical hypocalcaemia (Goff, 2008). Serum concentrations ofcalcitriol, total sero-calcium (tCa), and parathyroid hormone (PTH) of dairy cows receiving three different calcitriol formulations (IVP A, B, & C) and controls were determined.

[0178] In phase one, prepared randomisation lists of pregnant cows were used to assign cows to one of two treatment (calcitriol) groups (IVP-A [multiple short-acting], n=9 and IVP-B [singular slow release], n=8) or the control (n=8). In phase two, a single treatment group (IVP-C [singular slow release], n=8) was enrolled. The independent variables were group, treatment type, time of treatment, body condition score, and age. The dependent variables were the serum concentrations of calcitriol, tCa, and PTH. Prior to treatment, all cows were blood sampled and a cow-side test was performed to determine ionised calcium (iCa) sero-calcium (Horiba iCa Checker). Cows were sampled for blood within 12 hr of giving birth (0 hr=before treatment) and at times after treatment (1, 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240 and 264 hr). Cows with whole blood iCa ≤1.10 mM using the cow side test were enrolled and were retained in the study if the total sero-Ca was ≤2.10 mM using a confirmatory laboratory test. Control cows received a single dose of a placebo. Treatment group IVP-A received four doses (108 µg, 18 µg, 18 µg, and 12 µg-short acting), in a descending dose rate, at times 0, 24, 48 and 72 hrs. Cows receiving regimen IVP-B and IVP-C received a single dose (198 µg-burst + slow release) at time 0.

[0179] The tCa (treatment groups) increased significantly from 6 hr post treatment to 24 hr and remained elevated above baseline up to 144 hr or (days 1, 2, 3, 4, 5, 7, and 9) compared with the control cows. The mean serum concentrations of tCa for each treatment group was (control =2.34 vs IVP-A=2.48 vs IVP-B=2.45 vs IVP-C=2.55 mM). There was no significant difference in tCa concentrations among treatment groups (p > 0.05). However, tCa in IVP-C (198 µg) was significantly higher (p = 0.046) than other (0, 108, 126, 144, & 156 µg) dosages at 72 hr post treatment. Moreover, cows treated with IVP-C (198 µg) achieved a higher concentration of serum tCa by 12 hr post treatment and also had a higher serum calcitriol at 12 hours post treatment compared with cows treated with IVP-A and B. Overall, all treated cows had a higher concentration of tCa compared with control cows.

[0180] All cows in the treatment groups showed an increase in calcitriol within 12 hr of treatment compared with controls. Calcitriol concentration was highest in IVP-C treated group compared with controls (p< 0.008). The serum PTH concentration in control cows was significantly higher from 12 hr to 168 hr (days 1-8) post-partum than treatment groups but no difference was observed beyond this time frame. The IVP-C group had the lowest PTH sero-concentration from 12 -168 hr compared with control cows (p<0.05). Cows that received IVP-C did not experience any subclinical or clinical hypocalcaemia after 12 hr study period. There were no cases of clinicalhypocalcaemia reported or treated in the 12 days study period for all study cows. However, 1 cow in IVP-A did record a serum tCa level of 1.26 mM on day 10, but had no clinical signs.

[0181] This work demonstrates that all three calcitriol formulations for the treatment of cows within 12 hr post-partum were safe and effective in increasing serum tCa in the first 7 days post- partum. This study suggests that IVP-C formulation could provide a better treatment and preventative option for hypocalcemia in cows because no cow in this group experienced subclinical / clinical hypocalcaemia. Our findings provide important guidance for effective calcitriol dosage determination.

[0182] Study Objective

[0183] The objective of this study was, firstly, to assess both the pharmacokinetics and the efficacy of a multiple slow release versus singular long-acting formulations of calcitriol in post-partum dairy cows. A second objective was to determine whether treated dairy cows with calcitriol would maintain serum calcium levels that can reduce the incidence of clinical and sub-clinical hypocalcaemia.

[0184] Materials and Methods

[0185] Study design

[0186] Type of study and overall study design

[0187] This study was a randomised, negatively controlled, study. The study was divided into two separate phases. In phase 1, prepared randomisation lists of pregnant cows were used to assign cows to any of two treatment groups (IVP-A, n=9 and IVP-B, n=8) or the control group (n=8) on each day. In phase 2, a single group was treated with IVP-C (n = 8).

[0188] Table 4: Descriptive parameters of enrolled cows. Species / Breed: Dairy cows (Bos taurus) Breed: Commercial dairy breeds including Jersey, Friesians and Crossbred animals Gender: Female Age: Mixed (3+ years) Weight: Open (approx.450-650 kg) Health Status: Systemically healthy, non-pregnant, recently calved (within 6 hr)

[0189] Exclusion criteria

[0190] - Animals showing current signs of illness.

[0191] - Animals showing clinical signs of systemic or concurrent disease that may affect the outcome of the trial.

[0192] - Treatment with antibiotics (systemic or intramammary), or corticosteroids within the last 14 days.

[0193] - Animals that have been treated with injectable or oral calcium following calving, prior to enrolment; or within 14 days prior to calving.

[0194] - Cows with clinical hypocalcaemia.

[0195] - Heifers (first calving 2-year old animals).

[0196] Post-inclusion removal criteria

[0197] - Post enrolment validated laboratory serum tCa levels assessed at >2.10mM / L from 1st blood sample at enrolment on Day 0.

[0198] - Animals that developed concomitant disease, resulting in systemic illness, euthanasia or death.

[0199] - Non-permissible concomitant therapy (i.e. systemic antibiotics) that could bias study results.

[0200] - Animals that developed clinical hypocalcaemia that required treatment within 24 hours post -enrolment.

[0201] For both phases, following selection, the field process was identical. At visit 1, all eligible cows that had calved 0-12 hours prior to being brought to the shed were available for enrolment. Prior to treatment, all cows were blood sampled and a cow-side test was performed to determine ionised serum Ca (Horiba iCa Checker). Serum samples were sent to the reference laboratory to analyse for total serum calcium (tCa) status within 24 h after treatment. Cows with whole blood iCa ≤1.10 mM on the cow side test were enrolled if the serum total calcium level was ≤2.10 mM on laboratory (confirmatory) test. Cows showing clinical signs of hypocalcaemia were excluded from the study.

[0202] For phase 1, prepared randomisation lists of pregnant cows were assigned cows to one of each treatment of three groups and the dose was set for a 600kg cow (Table 9).

[0203] Table 9: Dosing regimen. Group 1 Group 2 Group 3 Group 4 Active Negative control IVP-A IVP-B IVP-C Treatment Day 1 4.3ml (Vieira- 4.3ml (108μg) 2ml (198μg) 2ml (198μg) Neto’ solution without calcitriol) Treatment Day 2 0.7ml (18μg) Treatment Day 3 0.7ml (18μg) Treatment Day 4 0.5ml (12μg) Required sample 8 (actual n=8) 8 (actual n=9) 8 (actual n=8) 8 (actual n=8) size with complete data (cows) (and actual used)

[0204] For Group IVP-A, treatment was administered by subcutaneous injection in descending doses at time 0 hr for each of study days (SDs) 1-4 (Table 9). The dose rate was descending, beginning with 108, 18, 18, 12 µg / cow respectively for days 1-4. The calcitriol concentration in the formulation was 25 µg / mL and the dose was set for a 600 kg cow. Therefore, the volume given per cow per day was as follows: Day 14.3 ml; Day 20.7 ml; Day 30.7 ml; and Day 40.5 ml.

[0205] For treatment group IVP-B and IVP-C, a single dose was given at time 0 hr on SD1. Dose rate was calculated as: 0.33 µg / kg, equates to 198 µg / 600 kg cow (~ 2ml).

[0206] Cows in the control group received a single subcutaneous injection of placebo (formulation according to Vieira-Neto et al. (2017) without calcitriol) on SD1 at 0 hours (Table 9). Cows in the treatment group IVP-A received four doses of calcitriol (1α,25(OH)2 vitamin D3), formulation according to Vieira-Neto et al. (2017) subcutaneously (SC); formulation IVP-A (Table 6) every 24 hours from time 0 on SD1, in a descending dose rate (Table 9).

[0207] Table 5: Control product (CP) composition. Identification on Certificate of Provisional Calcitriol Injection control Registration (ACVM) Name on label Calcitriol injection control Active ingredient Not applicable Dosage route Subcutaneous injection Dose 4.3ml Packaging 50ml amber glass vials

[0208] Table 6: Investigational Veterinary Products (IVP-A) composition. Identification on Certificate of Provisional IVP-A Registration (ACVM) Name on label Calcitriol injection A Active ingredient Calcitriol (25 μg / mL) (1α, 25(OH)2vitamin D3) Dosage route Subcutaneous injection Dose Formulation A (Group 1): Once a day injections for 4 days w / descending dose rates of 108, 18, 18, 12 μg / dose respectively (equivalent to 4.3, 0.7, 0.7 and 0.5mls respectively) Packaging 50 ml amber glass vials

[0209] Cows in treatment group IVP-B (Table 7) received a single slow-release dose of calcitriol (1α, 25(OH)2 vitamin D3), SC (Table 5); as outlined in (Table 9).

[0210] Table 7: IVP-B composition. Identification on Certificate of Provisional IVP-B Registration (ACVM) Name on label 20OTCASRF-1 Active ingredient Calcitriol (100 μg / mL) (1α, 25(OH)2vitamin D3)Dosage route Subcutaneous injection Dose IVP-B 0.33g μg / kg (2mls / 600kg) Packaging 2.3ml glass vials

[0211] Table 8: IVP-C composition. Identification on Certificate of Provisional IVP-C (ACVM) Name on label 20OTCASRF-2 Active ingredient Calcitriol (100 μg / mL) (1α, 25(OH)2vitamin D3) Dosage route Subcutaneous injection Dose Formulation C (Group 3): 0.33g μg / kg (2mls / 600kg) Packaging 2.3ml glass vials

[0212] Formulation IVP-C (Table 8) was only used during phase 2 as outlined previously.

[0213] Assessment of subclinical hypocalcaemia

[0214] A case of subclinical hypocalcaemia was defined as any cow that had just calved but did not exhibit any clinical signs associated with acute clinical hypocalcaemia. The cow also met the laboratory diagnostic criteria of (subclinical) hypocalcaemia e.g. tCa <2.10 mM of serum calcium ion concentration within a few hours post-partum.

[0215] Analysis and testing

[0216] Blood (serum) samples collection

[0217] Individual blood samples for biochemistry were collected from each cow included in the trial: On the day of calving (first day of lactation, visit 1 (V1)), a veterinarian or trained veterinary technician collected pre-treatment whole blood (for iCa in cowside test) and serum samples (for tCa, calcitriol and PTH testing) from all cows included in the study at time = 0 hrs. Following sample collection on V1, a veterinarian or trained veterinary technician administered the appropriate treatment to each cow.

[0218] The subsequent blood samples of serum were drawn at times 1, 2, 4, 6, 8 and 12 hr; by a veterinarian or trained veterinary technician. On subsequent study days (2-12), further blood samples were drawn at time = 0 hr (+ / - 2 hr) until the end of the study on SD12. Cases were defined as completing the study if they have completed treatment and were followed through until the last blood sample was drawn as per the protocol, or if they exit the study due to hypocalcaemia.

[0219] Blood samples were collected using evacuated glass tube (~10 mL) was collected from the coccygeal vessels of all study cows. Concentrations of 1α, 25- dihydroxyvitamin D3 in serum were analysed by ELISA [1α, 25(OH)2Vitamin D3 ELISA; IBL-America, Minneapolis, MN] according to the manufacturer’s instructions and as described by others (Vieira-Neto et al., 2017). PTH was assayed using a Roche Elecsys ECL (electrochemilluminesence immunoassay) run on a Cobase411 analyser (Cobas, 2011). Concentrations of tCa in serum were analysed by atomic absorption (AAnalyst, 200; Perkin-Elmar Inc.) as previously described (Martinez et al., 2012).

[0220] Methods for computing and calculating the product effect

[0221] The primary outcome variable for this study was:

[0222] 1. The proportion of cows normocalcaemic at 24 hrs (binomial, all tests after 24 h are within normocalcaemic range). Normocalcaemia for the purposes of this study was defined as serum calcium levels > / = 2.0 mM.

[0223] In addition, secondary outcomes of the primary outcome were:

[0224] 2. The proportion of cows normocalcaemic after 48 hrs (binomial, all tests after 48 h are within defined range).

[0225] 3. The proportion of cows normocalcaemic after 72 hrs (binomial, all tests after 72 h are within normocalcaemic range).

[0226] 4. The mean of serum calcium concentration in each group at one / all time points >24 h post treatment.

[0227] 5. The incidence of clinical cases / treatment of hypocalcaemia in study period after treatment until study end (day 12).

[0228] 6. The incidence of clinical cases (treatments) of mastitis in study period after treatment until study end (day 12).

[0229] In addition, the concentration of both calcitriol and PTH was measured as an outcome for each treatment group. In project 1, a total of 25 cows (control (n=8), IVP-A (n=9), and IVP-B (n=8)) were enrolled in this study. In project 2, a total of 8 cows were assigned to treatment group IVP- C. This is reported under Other Outcomes (these were secondary and third outcomes).

[0230] This was ascertained by the following:

[0231] Success was defined as normocalcaemic (serum calcium levels > / = 2.0m / L) at all sampling time points after 24 hr post treatment, and assuming that 10% of cows in the control group and 70% of cows in the treatment groups would meet these criteria. To achieve a power of 80 % (1- β=0.80) and a level of significance (α =0.05 / 3 or 0.017, two sided) for detecting this as a true difference, 8 cows were required to enroll per group (3 treatment groups) or 24 cows total.16 cows allows for drop out of cows who are ineligible for inclusion based on post-initial enrolment criteria of serum tCa levels >2.10mM / L, carried out within 24 h after treatment. The calculations were achieved using methods described by Machin and others (2009) using the MedCalc statistical software version 19.4.1.

[0232] The dependent variables were the serum concentrations of total sero-calcium (tCa, primary outcome), and parathyroid hormone (PTH, secondary outcome) and calcitriol (third outcome). Theindependent variables were group, treatment type, and time of treatment, body condition score, and age. The dependent variables were the serum concentrations of calcitriol, tCa, and PTH. However, other potential influencing variables were assessed by univariable analyses, including:

[0233] - Age (3, 4 to 7, 7+ years).

[0234] - Previous season yield (binary, dichotomised around the farm mean).

[0235] - Body condition score at enrolment (using methods described by Roche and others (2004).

[0236] - Cow group.

[0237] - Treatment type.

[0238] - Time after treatment (hours and / days).

[0239] - Weight.

[0240] Where a variable was associated (i.e. p<0.2) with the outcome, it was added to a multivariable model, but only left in the final model if it was significant (p<0.05), where the model goodness of fit improved or when inclusion of the variable results in a change of >10% in the treatment coefficient. Biologically plausible interactions were investigated among independent variables. The interaction term was significant p<0.05 it was kept in the model otherwise it was dropped.

[0241] The cow was the unit of analysis. To account for the repeated measurements on a cow, a linear mixed effects (LME) model with factors cow (subject) nested within dose used as a random intercept. All standard model fitting and diagnostics for linear mixed models were employed to arrive at the optimal model using standard statistical tools (Bates et al., 2014). The first step in model building using the LME from R package NLME was used to assess whether the mixed effects model (Maximum Likelihood Estimation, ML) was better than the ordinary linear regression model that was fit with the GLS function without the random intercept. The random intercept was specified using the varIdent variance structure as determine by the Akaike information criterion, −2 log likelihood (model deviance). The next step was to determine the fixed effect structure using the ML. The final step was to refit the final model from the ML estimation with the restricted maximum likelihood (REML).

[0242] Model diagnostics of residuals was performed on the REML model to examine if residuals did not violate the homogeneity criteria. This was further verified by plotting the quantile-quantile plot to examine if the assumption of normality was achieved. Finally, the assumption of normality was examined for the random effects. The study day and an interaction term of dose and study day and the random effect of cow nested within dose were the optimal terms fitted in the linear mixed- effects model using lme4 R package for the primary outcome.

[0243] The statistical analysis of all outcomes (projects 1 and 2, tCa) was performed using the per protocol dataset, which was defined at cow level and included cows that met the following criteria:

[0244] - received the allocated treatment.

[0245] - completed the first 24 hr of the study (all sample points to 24 hr are complete).

[0246] - available biochemistry results for all sample points.

[0247] Results

[0248] Enrolments

[0249] A total of 74 cows were assessed for eligibility for inclusion into the study. There was a total of 28 cows that was excluded from the study pre-enrolment. A total of 46 cows met all the inclusion criteria and none of the exclusion criteria and were enrolled in the study. Of the 46 cows enrolled, 13 cows were subsequently excluded for meeting one or more of the post-inclusion removal criteria meaning 33 cows completed the study. Of the 33 cows, controls (n=8), IVP-A (n=9), IVP-B (n=8) and IVP-C (n=8).

[0250] Primary outcome

[0251] Proportion of cows normocalcaemic after 24 hours

[0252] Summary

[0253] All cows in all treatment groups (IVP-A, IVP-B and IVP-C) were normocalcaemic at 24 hours; in the control group, 25% of cows (2 / 8) were subclinically hypocalcaemic (Table 15).

[0254] Table 15: Proportion of dairy cows that were normocalcaemic, subclinical or with clinical hypocalcaemia in relation to total serum calcium (tCa) concentration (mmol / L, mM) after 24hr or study day 2 post treatment with any of the three formulations of calcitriol (IVP- A, B, & C). Study day (DMI) Treatment allocation 2 3 4 5 6 7 8 9 10 11 12 Gand Total Calcium (mmol / L) Clinical status Control normal 6 7 8 8 8 8 7 8 7 7 8 82 subclinical 2 1 1 1 1 6 Control total 8 8 8 8 8 8 8 8 8 8 8 88 IVP-A clinical 1 1 normal 9 9 9 9 9 9 9 9 8 8 9 97 subclinical 1 1 IVP-A Total 9 9 9 9 9 9 9 9 9 9 9 99 IVP-B normal 8 8 8 8 8 8 8 7 8 5 7 83 subclinical 1 3 1 5 IVP-B Total 8 8 8 8 8 8 8 8 8 8 8 88 IVP-C normal 8 8 8 8 8 8 8 8 8 8 8 88 IVP-C Total 8 8 8 8 8 8 8 8 8 8 8 88 Grand Total 33 33 33 33 33 33 33 33 33 33 33 363

[0255] *Normocalcemic was defined as serum concentration as greater than or equal to 2.0 mM as described by Goff (2008).

[0256] #Subclinical hypocalcaemia was defined as a serum concentration of calcium less than 2.00 mM.

[0257] Clinical hypocalcaemia was defined as serum concentration of total calcium less than 1.38 mM.

[0258] Cows that received a single dose of 198 µg calcitriol IVP-B or IVP-C or a multiple dose of IVP-A in descending concentrations had increased sero-tCa within 6 hr of treatment, (Table 14, p<0.05) and this remained for the first 7 days of treatment compared to cows that received a placebo (Figure 1).

[0259] Table 14: Parameter estimates, standard error (std. error), statistics of a linear mixed effects model evaluating the primary outcome, total sero-calcium concentration (tCa) in treated dairy cows with three formulations of calcitriol (IVP-A, n=9, B, n=8 &C, n=8) within 12 hr of calving compared with control cows (n=8)#.

[0260] # In the final linear mixed effects model, study day and the interaction terms of dose and study day (SD) were the parameters that were considered optimal. The interaction term between dose and SD was significant on days 1, 2, 3, 4, 6, and 8, (p<0.05) with Ca concentrations being higher in the treatment groups than control. Significant variables are represented by an asterisk*. The random intercept factors used were cows (subject) nested within dose.

[0261] Detail

[0262] Concentration of tCa in serum did not significantly differ between the treated cows to the controls by study day 9 (Figure 1, 216 hr). Overall, the calcitriol treated cows exhibited a higher concentration of tCa on days 1, 2, 3, 4, 5, 6, and 8 post treatment compared with the control cows that received a placebo (Table 14). The mean serum concentrations of tCa for each treatment group across the study period was (control =2.34 vs IVPA=2.48 vs IVPB=2.45 vs IVPC=2.55 mM).

[0263] Compared with control cows, cows enrolled into treatment group IVP-A had a slightly higher mean concentration of tCa at time = 0 hrs (Figure 1). A similar trend was observed in cows that were treated with IVP-B, although the mean tCa in this group was marginally lower than that observed in the IVP-A group at time=0 hr (Figure 1 and Table 16).

[0264] Table 16: The mean total serum calcium (tCa) concentration (mmol / L, mM) in each group of dairy cows at all sampling time points 24hr post treatment. Study day (DMI) Treatment Status 2 3 4 5 6 7 8 9 10 11 12 Gand allocation Total Control normal 2.26 2.40 2.43 2.42 2.36 2.42 2.28 2.36 2.44 2.41 2.34 2.38 subclinical 1.79 1.91 1.98 1.89 1.89 1.88 Control total 2.14 2.34 2.43 2.42 2.36 2.42 2.24 2.36 2.37 2.35 2.34 2.34 IVP-A clinical 1.26 1.26 normal 2.50 2.57 2.54 2.71 2.57 2.64 2.57 2.42 2.38 2.31 2.25 2.50 subclinical 1.95 1.95 IVP-A Total 2.50 2.57 2.54 2.71 2.57 2.64 2.57 2.42 2.25 2.27 2.25 2.48 IVP-B normal 2.37 2.56 2.54 2.57 2.70 2.50 2.48 2.48 2.38 2.29 2.28 2.48 subclinical 1.96 1.96 1.76 1.92 IVP-B Total 2.37 2.56 2.54 2.57 2.70 2.50 2.48 2.42 2.38 2.16 2.22 2.44 IVP-C normal 2.61 2.57 2.70 2.67 2.62 2.63 2.51 2.54 2.47 2.42 2.32 2.55 IVP-C Total 2.61 2.57 2.70 2.67 2.62 2.63 2.51 2.54 2.47 2.42 2.32 2.55 Grand 2.41 2.51 2.55 2.59 2.56 2.55 2.46 2.43 2.36 2.30 2.28 2.46 Total

[0265] *Normocalcemic was defined as serum concentration as greater than or equal to 2.0 mM as described by Goff (2008).

[0266] #Subclinical hypocalcaemia was defined as a serum concentration of calcium less than 2.00 mM.

[0267] Clinical hypocalcaemia was defined as serum concentration of total calcium less than 1.38 mM.

[0268] In the treatment group IVP-C, the mean tCa at time=0 hr was lower than the control group. At time (8 hr) the mean tCa was higher in each of the treatment groups than the control cows and this remained so up to time 120 hr (study day 6). The overall treatment effect of calcitriol on serum calcium increase was significantly higher than observed in control cows (p=0.000, Figure 3). The final mixed effects model for tCa change in treated and control cows is shown in Table 14. Therewas an interaction between dose and SD on days 1, 2, 3, 4, 6, and 8, (p<0.05) with Ca concentrations being higher in treatment groups than control cows (Figure 1). The model performance was optimal as it met the standard diagnostics including the normality assumption of residuals as shown in Figure 1A.

[0269] Secondary outcomes (2-3): Proportion of cows with normal calcaemic status post treatment at time 48 and 72 hrs

[0270] After 24 hr (study day 2), 48 hr and 72 hr of treatment, all cows in the IVP-A (n=9 / 9), IVP- B (n=8 / 8) and IVP-C (n=8 / 8) were normocalcaemic (Table 15). In contrast, the control group had (75%, n=6 / 8) normocalcaemic cows in the same time frame.

[0271] Secondary outcome 4: The mean of serum calcium concentration in each group at one, or all time points >24h post treatment

[0272] The mean serum calcium concentration in each treatment group at one / all-time points 24 hr post treatment are shown in (Table 16) and (Figure 1). On study day 2, the treatment group IVP- C had the highest mean of tCa (2.61 mM), followed by IVP-A (2.50 mM / L), IVP-B (2.37 mM) and control cows (2.26 mM).

[0273] After day 2, the highest mean in the control group at any time point was 2.42 mM at day 5; and the lowest was 2.26 mM at day 2. The highest mean in the treatment group IVP-A was achieved on day 5 (2.71 mM) and the lowest mean on day 12 (2.25 mM). The highest mean of serum calcium concentration in the treatment group IVP-B was achieved on day 6 (2.70 mM) and the lowest mean 2.28 mM was achieved on day 12. The highest mean of serum calcium concentration in the treatment group IVP-C was achieved on day 4 (2.70 mM) and the lowest mean 2.32 mM was achieved on day 12.

[0274] Secondary outcome 5 - The incidence of clinical cases / treatment of hypocalcaemia in the study period after treatment until the end of the study (day 12)

[0275] There were no cases of clinical hypocalcaemia reported or treated in the 12 days study period for all study cows. However, 1 cow in IVP-A did record a serum tCa level of 1.26 mM on day 10, but did not exhibit any clinical signs.

[0276] Secondary outcome 6 - The incidence of clinical cases / treatment of mastitis in the study period after treatment until the end of the study (day 12)

[0277] There were no cases of clinical mastitis reported in the 12 days study period for all study cows.

[0278] Other Outcomes

[0279] The control group had four new incident cases (cows) of subclinical hypocalcaemia on days 2, 3, 8, 10 and 11 (4 / 74 (5.4%)) cumulative incidence of hypocalcaemic cases in 12 daysstudy period (6 events). The IVP-A treatment group had one (n=1 / 99 (~1.0%)) new incidence case per 12 study days (period)) on day 11 or the prevalence of subclinical hypocalcaemia (n=1 / 9 cows, 11.1%); while IVP-B (n=4 / 87 new incidence cases per 12 study days (period)) or the prevalence (n=4 / 8, 50% in cows), on days 9, 11 and 12. The cows that received IVP-C did not have any hypocalcaemia cases. IVP-A had one clinical hypocalcaemic tCa (1.26 mM) (n=1 / 99 new incidence case per 12 study days (period)) on study day 10 as discussed previously and as shown in Table 16.

[0280] Mean serum calcitriol concentration

[0281] The calcitriol treatments increased serum calcitriol (1α, 25(OH)2vitamin D3) concentrations compared with control cows beginning an hour up to 72 hrs after which there was no difference between treatment and control cows (Figure 7).

[0282] There was a significant difference between means of serum calcitriol in dairy cows treated with IVP-C compared with control cows (p=0.008). There was an overall significance difference between all cows treated with calcitriol compared with control cows (p=0.042, Figure 9).

[0283] Treatment with calcitriol (1α,25(OH)2vitamin D3) reduced the serum concentration of PTH compared with control cows starting at times 12 hr-168 hr after treatment (Figure 5). After the 168 hr time period, there was no observed difference between treatment cows and controls. The mean PTH for control cows on day 1 was 118.7 pg / mL compared with (63.02, 102.09, & 71.76 [pg / mL]) for IVP-A, B and C respectively. On day 3, the mean PTH in control cows was 48.99 pg / mL compared with (9.80, 7.59 & 3.19 [pg / mL]) for IVP-A, B and C respectively. There was a significant difference (p<0.05) in the PTH serum concentration for B compared with control cows but not cows treated with IVP-A and IVP-C (p>0.05, Figure 6). The overall effect of any treatment on the PTH serum concentration compared to control cows was significantly different (p=0.0034, Figure 6).

[0284] The box plot (Figure 6) shows the median PTH concentration for cows treated with IVP- C lower than for other treatment types. The shape of the distribution (extremely thin on each end and broad in the middle) indicates the PTH of IVP-C treated cows were highly concentrated around the median. The IVP-B treated cows had a bi-modal distribution of PTH concentration. The overall treatment effect of serum calcitriol treated cows was significantly higher than control cows (p=0.0003).

[0285] Discussion

[0286] This is the first study to demonstrate the beneficial effect of calcitriol treatment on pasture fed dairy cows treated within 12 hrs of calving. Cows receiving treatment had an increased serum concentration of tCa and serum calcitriol within 6 hrs post treatment. Dairy cows that receivedtreatment had increased serum tCa concentrations as early as 1 hr postpartum and that lasted well over 144 hrs (7 days in milk [DIM]) (Figure 1). The findings in this study provide new evidence of the rapidity with which a cow’s Ca status may respond to calcitriol.

[0287] This study demonstrated that treatment with any of the 3 formulations of calcitriol increased the proportion of normocalcaemic cows at times 24, 48 and 72 hrs; and increased mean serum tCa from the time of treatment until day 3. Treatment also reduced the incidence of sub- clinical hypocalcaemia when compared to control cows. No effect was determined on clinical hypocalcaemia because neither treatment nor control groups observed any cases of clinical hypocalcaemia. There was only a case in one IVP-A treated cow that had a low serum tCa which was subsequently classified as hypocalcaemia, although no clinical signs were observed. No effect was determined on clinical mastitis because neither treatment nor control groups observed any cases of clinical mastitis.

[0288] Administration of a single dose (198 µg) of calcitriol (IVP-C) to cows within 12 hr of parturition was more efficacious than the use of IVP-B single dose (198 µg) or IVP-A multi-dose (108, 18, 18, & 12 µg) in preventing clinical and subclinical hypocalcaemia (Table 15 and Table 16). The cows that received a single dose of IVP-C had the most rapid increase of the tCa concentration from 6 hr post treatment up to 12 days in milk (DIM) (Figure 1 and Table 16). Although the mean concentration tCa in cows treated with IVP-C was not significantly different from the other two formulations after 12 hr, it appears that the minor difference observed was clinically important (Table 15). The other evidence supporting the efficacious nature of the IVP- C is the consistently high serum concentration of calcitriol compared to cows treated with either IVP-A or B (Figure 7). Dairy cows receiving IVP-C had the lowest serum PTH between 12-168 hr post treatments suggesting that they did not experience low serum tCa necessitating upregulation of PTH compared to the other treatment groups (Figure 4). The results from this study show that the three formulations had similar pharmacokinetic profiles in that they all increased the serum tCa, even though only IVP-C formulation significantly reduced or prevented subclinical hypocalcaemia. Our overall findings in this study suggest that a single dose (198 µg) led to statistically higher levels of calcitriol (p<0.05, Figures 7 and 9) than control cows and those animals receiving a multi-dose formulation IVP-A.

[0289] In the first 12-48 hr period post treatment, the incidence of subclinical hypocalcaemia in the treatment groups was 0% compared with the control group with 25% (n=2 / 8) subclinical hypocalcaemia (Table 15). In this study, the prevalence of subclinical hypocalcaemia was significantly lower in the treatment group IVP-C (n=0 / 8 cows, 0%) after 12 hr and in that of cows in the IVP-A treatment group (n=1 / 9 cows, 11%).

[0290] Three occasions of subclinical hypocalcaemia were detected on the days 2 and 3 in the control group, and again on days 8, 10 and 11. In the IVP-A group, a single occasion of ‘clinical’ hypocalcaemia (as per our protocol definition) was detected on day 10, but no clinical signs were observed, and an occasion of subclinical hypocalcaemia on day 11; in IVP-B, cases of subclinical hypocalcaemia were detected on days 9, 11 and 12. No cases were detected in IVP-C.

[0291] These findings in the control group were interesting. It is well established that under regular conditions the risk of subclinical hypocalcaemia usually increases immediately post calving (Goff, 2008). Because of the increased demand of calcium soon after parturition, the natural metabolic response to increase total calcium concentration in serum is mainly by increasing intestinal absorption and urinary reabsorption to achieve normocalcaemia (Hernandez-Castellano et al., 2020). However, not all cows may experience a tCa reduction in the first three days post-calving (Martinez et al., 2012). This ability to avoid a reduction in tCa has been described in normocalcaemic cows (~90%) than those experiencing subclinical hypocalcaemia (~25%) (Martinez et al., 2012).

[0292] Delayed subclinical hypocalcemia, as evidence of maladaptation of the Ca homeostatic system after treatment (McArt and Neves, 2020), has been reported before for other available therapeutic and dietary strategies aiming to reduce the risk of hypocalcemia in cows, e.g. for Ca I.V. infusion 36-48 hr after treatment (Blank et al., 2014) and for oral Ca salt boli 3-4 d after treatment (Martinez et al., 2016). Similar to this current work, Vieira-Neto et al. (2017) reported a high average subclinical hypocalcaemia prevalence in control cows (56%) vs (14%) in calcitriol treatment group on the first day post calving, but also detected some cases on days 9 and 12 post calving (1% prevalence in control cows vs 14.4% in calcitriol treated). Some of the differences observed in our findings compared with those of others may be attributed in part to the calcitriol formulations. The other difference could be attributed to the different sampling schemes (Chapinal et al., 2011; Reinhardt et al., 2011). Our study had a similar sampling scheme to Vieira-Neto et al. (2017) and (Roberts & McDougall, 2019) but with more sampling time points (n=18 vs 16 vs 10). The addition of multiple time points <24hrs post-partum adds to the body of work around post- partum calcium homeostasis.

[0293] The prevalence of subclinical hypocalcaemia in the treatment groups IVP-B and the control cows were both 50% after 12 hr which is comparable to the New Zealand reported average cow level prevalence (52%, 3 days in milk) (Roberts & McDougall, 2019) and abroad (Vieira-Neto et al., 2017; Reinhardt et al., 2011; Goff, 2008; DeGaris & Lean 2008; Oetzel, 1988). The daily prevalence of subclinical hypocalcaemia was significantly lower in this study for all treatmentgroups and controls (Table 11 and Table 12) compared with that reported by others who used a similar treatment product and sampling scheme (Vieira-Neto et al., 2017, 2021).

[0294] Table 11: Age distribution of cows by treatment allocation. Age (years) Treatment allocation Unknown 3 4 5 7 Grand Total Control 8 8 IVP-A 4 2 1 2 9 IVP-B 4 4 8 IVP-C 4 1 3 8 Grand Total 16 10 2 3 2 33

[0295] Table 12: Body weight (kg) distribution of cows by treatment allocation. Weight (kg) Treatment allocation Unknown 450 475 500 550 600 Grand Total Control 4 4 8 IVP-A 3 1 1 3 1 9 IVP-B 5 3 8 IVP-C 6 1 1 8 Grand Total 13 7 1 1 7 4 33

[0296] The reasons for the lower prevalence of subclinical cases in this study are unclear and may be multifactorial. The enrolled cows’ body condition score (BCS) was nearer the recommended target BCS for calving in New Zealand (5.0 – 5.5 NZ-scale) in this study, and this may have reduced the risk of subclinical hypocalcaemia. These cows were also calving later in the season than the majority of seasonally calving cows in New Zealand, and New Zealand cows are typically of lower yield than those from North America or Europe. Potential differences in diet and environmental exposure to UV light in New Zealand compared to studies abroad may also explain some of these differences observed (Vieira-Neto et al., 2017). The most plausible explanation for the differences in prevalence of hypocalcaemia in this study compared to the studies abroad is likely the differences in the dosage as well as the calcitriol formulation used in the present study. These findings in the present study are supported by the high tCa concentration reported in our study (Figures 1, 2 and 3, Table 16) compared to the two studies in the USA (Vieira-Neto et al., 2017, 2021).

[0297] Calcitriol administration increased serum calcitriol in dairy cows beginning an hour after treatment and the serum calcitriol concentrations remained elevated until 48 hr (day 3). This was more marked in cows treated with IVP-C than A and B. The actions of calcitriol are well documented as they predominantly act through the vitamin D receptors, which in turn upregulate Ca transport and Ca binding proteins in the intestines and kidney cells (Dusso et al., 2005). Thisin turn increases the release of Ca and phosphorus from the bone through osteoclast activity (Hernandez-Castellano et al., 2020). It is plausible that the joint actions of calcitriol in the intestines, kidneys and bone cells accounted for the increased Ca concentrations in calcitriol treated cows. Calcitriol treated cows had a reduced concentration of PTH (p=0.0034, Kruskal- Wallis test, Figures 4 and 6). PTH has been documented to stimulate bone remodelling (Hustmyer et al., 1995). Additionally, we did not examine the bone resorption markers in the treatment and control cows. The bone markers are important to determine the source of Ca in dairy cows postpartum. Although this was not done in this study, others who have looked at this did not find increased levels of bone resorption markers in treated cows, suggesting that Ca increase was as a result of calcitriol treatment by enhanced intestinal absorption (Vieira-Neto et al., 2017).

[0298] There was an interaction between dose and SD on days 1, 2, 3, 4, 6, and 8, (p<0.05) with Ca concentrations being higher in treatment groups than control cows (Figure 1). The findings from this study suggests that the increase in tCa concentrations observed with calcitriol treatment were likely as a result of increased intestinal absorption and not bone resorption as others have found using a similar protocol (Vieira-Neto et al., 2017, 2020).

[0299] The body condition score (BCS) for cows in this study was consistent and ranged from 4.5 to 5.5 for both treatment and control cows (Table 10) using methods described elsewhere (Roche et al., 2004).

[0300] Table 10: Body condition score distribution of cows by treatment allocation. BCS (body condition score) Treatment allocation Unknown 4.5 5 5.5 Grand Total Control 3 5 1 8 IVP-A 2 2 4 1 9 IVP-B 4 4 8 IVP-C 3 5 8 Grand Total 2 12 18 2 33

[0301] Based on NZ body condition score as described in Roche et al., 2004.

[0302] BCS was not included in the final multivariate model as it had no effect on our analysis (p > 0.2). However, Vieira-Neto and colleagues (2021) found that cows with a BCS greater than 3.50 (equivalent to 4 BCS NZ) had more health benefits from calcitriol treatment than cows with BCS less than 3.50 (equivalent to 4 BCS NZ) at parturition. The fact that control and treatment cows had similar BCS may explain why we didn’t observe a larger difference in health benefits in some treated groups such as (IVP- A & B) vs control cows during the high risk period [3 days in milk (DIM)] (Table 14, Table 15 and Table 16).

[0303] In this study, the age of dairy cows ranged between three and seven years old (Table 11). The cows that were diagnosed with subclinical or clinical hypocalcaemia were those aged threeand seven. Goff, (2008) has shown that older cows are 50% more likely to experience hypocalcaemia than the younger cows (25%). The cows that were treated with IVP-C had an age range of 3-5 years. Cows in IVP-A had had an age range between 3-7 years while IVP-B group had cows in the age group 3 years and older (missing data). Cows treated with the IVP-C out performed those treated with IVP-A and B by age. There seems to have been no age difference in serum Ca in cows treated with IVP-C. However, cows aged 7 and treated with IVP-A had a higher serum Ca between 48-144 hr compared to the younger cows (3 or 4 year olds). These comparisons could not be made for IVP-B and the control cows because some records for age were missing. A study in New Zealand reported a 52% prevalence of hypocalcaemia in all age groups of dairy cows (Roberts & McDougall, 2019). These results suggest that when using this product, it would be ideal to select animals that are more at risk to hypocalcaemia than others.

[0304] In this study, we only had one cow that had tCa which was classified as clinical hypocalcaemia using the laboratory biochemistry standards (Goff, 2008). However, this was a retrospective, biochemical diagnosis as no clinical signs were observed by the farmer or the study team. A similar finding was reported in a larger scale study in the USA optimal (Vieira-Neto et al., 2021). The cow with biochemistry range for clinical hypocalcaemia was in the treatment group IVP-A which was detected on day 11 postpartum. This finding in this study was unusual because the incidence of clinical hypocalcaemia in New Zealand has been reported as (2-2.9%) within three days post calving (Roberts & McDougall, 2019; McDougall, 2001) and abroad (5-7%) (DeGaris & Lean, 2008; Goff, 2008; Mulligan & Doherty, 2008; Kelton et al., 1998). The results in this study on the incidence of clinical hypocalcaemia may have been under reported somewhat due to the nature of a small sample size.

[0305] There are significant challenges in establishing an optimal dosage of calcitriol and the frequency of administration in dairy cows. In this study, the optimal dosage for the immediate (initial burst) release calcitriol formulation was determined as single immediate-release doses of 108 µg / 600kg, 18 µg / 600kg, 18 µg / 600kg, and 12 µg / 600kg daily for day 1, 2, 3, and 4, respectively.The optimal dosage for the controlled sustained release (IVP-C) was 198 µg / 600 kg. However, as documented above, IVP-C was superior in enhancing tCa concentrations compared to IVP-A (Figure 1 and Figure 3, Table 16). Studies conducted in the USA found that a single dose of calcitriol (300 µg) was optimal (Vieira-Neto et al., 2017, 2021). However, repeated administration of calcitriol has been suggested to increase the risk of hypercalcemia after 1-2 DIM. The other risk of administering calcitriol in cows is that elevated calcitriol concentrations could lead to suppressing the body production of PTH. It is also likely that excessive doses or repeated administration of calcitriol could pose risk of toxicity and disruptions of the vitamin D endocrinesystem. Massive toxicity in cows that have been injected with cholecalciferol (375 mg, vitamin D3) about 32 d prepartum has been reported (Littledike & Host, 1982). The use of parental administration of vitamin D3 was found to have prolonged hypercalcemia, hyperphosphatemia, and also increases of vitamin D3 and its metabolites in serum of non-lactating non-gravid and gravid cows prepartum (Littledike & Host, 1982). However, in the present study, we did not observe any side effects associated with repeated treatment of calcitriol (IVP-A). This suggests that the dosage and frequency that were used of the IVP-A were safe and appropriate. Viera-Neto and colleagues (2017) have suggested combining the use of appropriate diet and calcitriol if administration was going to be 12-24 hr post-partum to overcome the decrease in serum calcium that occurs immediately before calving. In this study, the administration of IVP was carried out within 12 hr postpartum and the cows were normocalcemic at the start were less likely to experience post-partum decrease in serum Ca.

[0306] The findings from this pilot study demonstrate that calcitriol treatment of cows 12 hr postpartum was safe and effective in increasing tCa in the first 7 days after treatment. There was no significant difference in tCa concentrations despite differences in the overall calcitriol administered (156 vs.198 µg) to treatment cows (p> 0.05, Figure 5). IVP-C treated cows achieved a considerable higher tCa by 12 hr (up to the end of study day 12) post treatment compared with IVP-A or IVP-B (Figure 1). The overall treatment effect on serum calcium concentration was significantly higher than control cows (p=0.000, Figure 3).

[0307] This study suggests that IVP-C formulation could provide a better treatment and preventative option for hypocalcemia in cows because no cow in this group experienced subclinical / clinical hypocalcaemia. Cows that are at risk of subclinical or clinical hypocalcaemia may benefit significantly from IVP-C treatment.

[0308] This study has some limitations; although the sample size for the pilot study was small, we were able to support findings from a large scale study conducted in North America. In this study, we did not examine the feed composition supplemented to all cows. This is important because feed or supplementation has been used to prevent subclinical hypocalcaemia in dairy cows. The inclusion criteria to select subclinical hypocalcaemic cows based on Ca laboratory analysis were strict in this study leading to a high exclusion rate 56.5% (n=43 / 76). We therefore cannot extrapolate the observed effect in those cows that were excluded. On the other hand, given that we used clinically healthy cows may suggest that the observed increase in total calcium could potentially be replicated in the at risk cows. Our findings provide important guidance for effective calcitriol dosage determination and the overall results can serve as a basis for further studies of the effect of calcitriol in pasture fed herds in New Zealand.

[0309] Conclusions

[0310] In this study in dairy cows, we demonstrated that calcitriol treatment of cows 12 hr postpartum was safe, and was effective in increasing tCa in the first 7 DIM. Although all the three calcitriol formulations were not significantly different from each other, IVP-C treated cows achieved the highest tCa by 12 hr (up to the end of study day 12) post treatment compared with IVP-A and B. This study suggests that IVP-C formulation could provide a better treatment and preventative option for hypocalcemia in cows because no cows in this group experienced subclinical / clinical hypocalcaemia. Our findings provide important guidance for effective calcitriol dosage determination and the overall results can serve as a basis for further studies.

[0311] Based on these studies, it is likely that a derivative, prodrug or analogue of calcitriol may also work as well, but the Vitamin D3 active concentration may need to be determined theoretically or empirically.

[0312] Based on these studies, it is likely that a concentration of about 45-80% w / v SAIB formulation may work as well, but the controlled-release carrier may need optimising in some instances. It is likely that various solvent / s may work just as well. Also, Tween 80 or other surfactant may need to be added, especially for a SAIB concentration greater than about 70% w / v.

[0313] In the present specification, the word ‘comprising’ and its derivatives including ‘comprises’ and ‘comprise’ include each of the stated integers but does not exclude the inclusion of one or more further integers.

[0314] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0315] The terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±10 %, preferably ±5 %, more preferably ±2 %, and even more preferably ±1 %.

[0316] Reference in this specification to any number range includes all possible numbers / numerical values falling within that range, and further includes all possible subranges falling within that range, context permitting.

[0317] Reference throughout this specification to ‘one embodiment’ or ‘an embodiment’ means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrases‘in one embodiment’ or ‘in an embodiment’ in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more combinations.

[0318] In compliance with the statute, the invention has been described in language more or less specific to structural or methodical features. It is to be understood that the invention is not limited to specific features shown or described since the means herein described comprises preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims appropriately interpreted by those skilled in the art.References 1. Altieri B, Cavalier E, Bhattoa HP et al.2020. Vitamin D testing: advantages and limits of current assays. Eur. J. Clin. Nutr.74, 231-247. 2. Barton BA.1978. Studies of vitamin D, calcium and phosphorus metabolism of the dairy cow. Master’s thesis dissertation. University of Wisconsin, Madison, WI, USA. 3. Bates D, Mächler M, Bolker B, Walker S. 2014. Fitting Linear Mixed-Effects Models using lme4. arXiv:1406.5823 [stat.CO]. 4. Blanc CD, Van der List M, Aly SS, Rossow HA, Silva-del-Räo N. 2014. Blood calcium dynamics after prophylactic treatment of subclinical hypocalcemia with oral or intravenous calcium. J. Dairy Sci.97, 6901–6906. 5. Beede DK, Pilbean TE, Puffenbarger SM, Tempelman RJ. 2001. Peripartum responses of Holstein cows and heifers fed graded concentrations of calcium (calcium carbonate) and anion (chloride) three weeks before calving. J. Dairy Sci.84, 83. 6. Chapinal N, Carson M, Duffield TF, Capel M, Godden S, Overton M, Santos JEP, LeBlanc SJ. 2011. The association of serum metabolites with clinical disease during the transition period. J. Dairy Sci.94, 4897–4903. 7. Cobas. 2011. Parathyroid hormone (parathorne, parathyrin)-PTH, intact. Elecsys and cobas e analyzers_Ref 11972103-122. http: / / webcache.googleusercontent.com / search?q=cache:E- 4OMtBwlY4J:labogids.sintmaria.be / sites / default / files / files / pth_2019- 06_v26.pdf+&cd=11&hl=en&ct=clnk&gl=nz 8. Craig AH & Stoll IV.1947. Milk fever (parturient paresis) as a manifestation of alkalosis. Am. J. Vet. Res.8, 168. 9. DeGraris DG & Lean JJ. 2008. Milk fever in dairy cows- a review of pathophysiology and control principles. Vet. J. this issue. 10. Dusso AS, Brown AJ, Slatopolsky E. 2005. Vitamin D. Am. J. Physiol. Renal Physiol. 289: 445-446. 11. Goff JP. 2008. The monitoring, prevention, and treatment of milk fever and subclinical hypocalcemia in dairy cows. Vet J.176(1):50-7. doi: 10.1016 / j.tvjl.2007.12.020. 12. Goff JP & Horst RL.1997. Effects of the additional potassium or sodium, but not calcium, to prepartum ratios on milk fever in the dairy cows. J. Dairy Sci.80, 176-186.13. Goff JP & Horst RL.1993. Oral administration of calcium salts for treatment of hypocalcemia in cattle. J. Dairy Sci.76, 01-8. 14. Hernández-Castellano LE, Hernandez LL, Bruckmaier RM.2020. Review: endocrine pathways to regulate calcium homeostasis around parturition and the prevention of hypocalcemia in periparturient dairy cows. Animal.14: 330–338. 15. Hendriks SJ, Phyn CVC, Turner SA, Mueller KM, Kuhn-Sherlock B, Donaghy DJ, Huzzey JM, Roche JR.2019. Lying behavior and activity during the transition period of clinically healthy grazing dairy cows. J. Dairy Sci.102, 7371-7384. 16. Horst RL, Goff JP, Reinhardt TA. 2005. Adapting to the transition between gestation and lactation: differences between rat, human and dairy cow. J. Mammary Gland Bio.10,141- 56. 17. Horst RL, Goff JP, Reinhardt TA.2003. Role of vitamin D in calcium homeostasis and its use in prevention of bovine periparturient paresis. Acta Vet Scand.97 (Suppl):35–50. 18. Kerslake JI, Amer P, O’Neill P, Wong S, Roche J, Phyn C.2018. Economic costs of recorded reasons for cow mortality and culling in a pasture-based dairy industry. J. Dairy Sci.101, 1795– 1803. 19. Hustmyer FG, Beitz DC, Goff JP, Nonnecke BJ, Horst RL, Reinhardt TA. 1995. In vivo parathyroid hormone stimulates in vitro bone resorption by bovine monocytes. J. Dairy Sci. 78, 2700-2708. 20. Kelton DF, Lissemore KD, Martin RE.1998. Recommendations for recording and calculating the incidence of selected clinical diseases of dairy cattle. J Dairy Sci.81, 2502- 2509. 21. Kimura K, Reinhardt TA, Goff JP.2006. Parturition and hypocalcemia blunts calcium signals in immune cells of dairy cattle. J. Dairy Sci.89, 2588–2595. 22. Liang D, Arnold L, Stowe C, Harmon R, Bewley J.2017. Estimating US dairy clinical disease costs with a stochastic simulation model. J. Dairy Sci.100, 1472-1486. 23. Little ET & Horst RL.1982. Vitamin D3 toxicity in dairy cows. J. Dairy Sci.65, 749-750. 24. Machin D, Campbell MJ, Tan SB, Tan SH. 2009. Sample size tables for clinical studies.3rd ed. Chichester: Wiley-Blackwell.25. McArt and Neves, 2020. Association of transient, persistent, or delayed subclinical hypocalcemia with early lactation disease, removal, and milk yield in Holstein cows https: / / doi.org / 10.3168 / jds.2019-17191 26. McDougall S.2001. Effect of periparturient disease on the reproductive performance of New Zealand dairy cows. N Z Vet. J.49, 60–67. 27. Martinez, N., L. D. P. Sinedino, R. S. Bisinotto, R. Daetz, C. Lopera, C. A. Risco, K. N. Galvão, W. W. Thatcher, and J. E. P. Santos. 2016. Effects of oral calcium supplementation on mineral and acid-base status, energy metabolites and health of postpartum dairy cows. J. Dairy Sci.99:8397–8416. 28. Martinez N, Lima FS, Bisinotto RS, Greco LF, Ribeiro ES, Maunsell F, Galvao KN, Risco CA, Santos JEP. 2012. Evaluation of peripartal calcium status, energetic profile, and neutrophil function in dairy cows at low or high risk of developing uterine disease. J. Dairy Sci. 95, 7158- 7172. 29. Mulligan F & Doherty M. 2008. Production diseases: A major health, welfare and economic problem on dairy farms. Vet. J.176, 1-2. 30. Phillipo M, Reid GW, Nevison IM.1994. Parturient hypocalcaemia in dairy cows: effects of dietary acidity on plasma minerals and calciotrophic hormones. Res. Vet. Sci.56, 303-309. 31. Oetzel GR & Miller BE.2012. Effect of oral calcium bolus supplementation on early-lactation health and milk yield in commercial dairy herds. J Dairy Sci.1-16. 32. Oetzel GR. 1996. Effect of calcium chloride gel treatment in dairy cows on incidence of periparturient diseases. J. Am. Vet. Med. Assoc.209, 958-61. 33. R Core Team.2021. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Sourced March, 2021, from URL https: / / www.R- project.org / . 34. Roberts KI, Bennison J, & S McDougall. 2019. Effect of treatment with oral Ca boluses following calving on concentrations of Ca in serum in pasture-based dairy cows. N Z Vet. J.67, 20-26. 35. Roberts KI & McDougall S.2019. Risk factors for subclinical hypocalcaemia, and associations between subclinical hypocalcaemia and reproductive performance, in pasture-based dairy herds in New Zealand. N Z Vet. J.67, 12-19.36. Reinhardt TA, Lippolis JD, McCluskey BJ, Goff JP, Horst RL.2011. Prevalence of subclinical hypocalcemia in dairy herds. Vet J.188, 122-1244. 37. Roche JR, Dillon PG, Stockdale CR, Baumgard LH, Van Baale MJ. 2004. Relationships among international body condition scoring systems. J. Dairy Sci.87, 3076–9. 38. Salgado-Hernández EG, Bouda J, Villa-Godoy A, Romano Muñoz JL, Gutiérrez-Chávez JA, Velásquez-Forero FH. 2014. Metabolites of vitamin D and minerals in blood and colostrum of primiparous and multiparous dairy cows postpartum. Czech J. Animal Sci.59, 11–18. 39. Vieira-Neto A, Negro G, Zimpel R, Poindexter M, Lopes Jr. F, Thatcher WW, Nelson CD, Santos JEP. 2021. Effects of injectable calcitriol on mineral metabolism and postpartum health and performance in dairy cows. J. Dairy Sci.104, 683-701. 40. Vieira-Neto A, Lima IRP, Lopes Jr. F, Lopera C, Zimpel R, Sinedino LDP, Jeong KC, Galvão K, Thatcher WW, Nelson CD, Santos JEP.2017. Use of calcitriol to maintain postpartum blood calcium and improve immune function in dairy cows. J. Dairy Sci.100, 5805–5823. 41. Vieira-Neto A, Negro G, Zimpel R, Poindexter M, Lopes F Jr, Thatcher WW, Nelson CD, Santos JEP. 2020. Effects of injectable calcitriol on mineral metabolism and postpartum health and performance in dairy cows. J. Dairy Sci.104, 683-701.

Claims

Claims 1. A controlled-release Vitamin D3 formulation comprising an active form of Vitamin D3 (‘Vitamin D3 active’) and a carrier, said carrier providing both an initial burst release as well as sustained release of the Vitamin D3 active.

2. The formulation of claim 1, wherein the formulation is in an injectable form, preferably for subcutaneous or intramuscular injection.

3. The formulation of claim 1 or claim 2, wherein the Vitamin D3 active is present in the range of approximately 0.0001% w / v to 10% w / v, preferably approximately 0.001% to 1% w / v, more preferably approximately 0.005% to 0.05% w / v, and even more preferably approximately 0.005 % to 0.01% w / v.

4. The formulation of any one of the preceding claims, wherein the Vitamin D3 active is calcitriol, or a derivative, prodrug or analogue thereof.

5. The formulation of claim 4, wherein the Vitamin D3 active is calcitriol.

6. The formulation of any one of the preceding claims, wherein the formulation is a solution, suspension, dispersion, emulsion or low viscosity gel, suitable for injection.

7. The formulation of claim 6, wherein the formulation is in the form of a low viscosity, injectable gel which gels rapidly at an injection site.

8. The formulation of claim 7, wherein the carrier comprises one or more gelling agents.

9. The formulation of claim 8, wherein the one or more gelling agents comprises sucrose acetate isobutyrate (SAIB).

10. The formulation of claim 9, wherein the formulation comprises SAIB in the following approximate ranges: 30% to 90%, 35% to 90%, 40% to 90%, 45% to 90%, 50% to 90%, 30% to 85%, 35% to 85%, 40% to 85%, 45% to 85%, 50% to 85%, 30% to 80%, 35% to 80%, 40% to 80%, 45% to 80%, 50% to 80%, 30% to 75%, 35% to 75%, 40% to 75%, 45% to 75%, 50% to 75%, 30% to 70%, 35% to 70%, 40% to 70%, 45% to 70%, or 50% to 70% w / v.

11. The formulation of claim 10, wherein the formulation comprises about 50% w / v SAIB.

12. The formulation of claim 10, wherein the formulation comprises about 70% w / v SAIB.

13. The formulation of any one of claims 8 to 12, wherein the carrier comprises one or more solvents or a solvent system.

14. The formulation of claim 13, wherein the one or more solvents is present in an amount of approximately 10% to 50% w / v, preferably approximately 15 to 30% w / v.

15. The formulation of claim 14, wherein the one or more solvents comprises ethanol, diethylene glycol monoethyl ether (DEGEE), N-methylpyrrolidone (NMP), triacetin, benzyl benzoate, miglyol, propylene carbonate, benzyl alcohol, ethyl lactate, glycofurol, 2-pyrrolidone, propyleneglycol, acetone, methyl acetate, ethyl acetate, methyl ethyl ketone, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, caprolactam, decylmethylsulfoxide, oleic acid, or 1- dodecyazacycloheptan-2-one, or any mixture of these.

16. The formulation of any one of the preceding claims, wherein the carrier comprises a surfactant, detergent or emulsifier.

17. The formulation of claim 16, wherein the surfactant, detergent or emulsifier is present in an amount of between approximately 0.1% to 10% w / v, preferably approximately 0.4% to 10% w / v, and more preferably approximately 0.5% to 5% w / v.

18. The formulation of claim 17, wherein the surfactant, detergent or emulsifier is present in an amount of about 2% w / v.

19. The formulation of any one of claims 16 to 18, wherein the surfactant, detergent or emulsifier is at least one type of polysorbate, preferably Tween 80.

20. The formulation of any one of claims 1 to 19, wherein the carrier comprises at least one type of preservative, preferably at least one type of antioxidant.

21. The formulation of claim 20, wherein the, or each type of, preservative or antioxidant is present in an amount of at least approximately 0.001% w / v, more preferably at least approximately 0.01% w / v.

22. The formulation of claim 21, wherein the formulation comprises approximately 0.1% to 1% w / v preservative or antioxidant, such as a phenolic antioxidant and / or ester of 4-hydroxybenzoic acid.

23. The formulation of any one of claims 20 to 22, wherein the at least one preservative or antioxidant comprises one or more of butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), methyl paraben and propyl paraben.

24. The formulation of claim 1, wherein the formulation comprises (Formulation 1): Vitamin D3 active, preferably calcitriol: approximately 0.005% to 0.1% w / v, preferably approximately 0.005% to 0.01% w / v; SAIB: approximately 45% to 80% w / v, preferably approximately 50% to 70% w / v; Optionally, preservative / s such as antioxidant / s with a quantity to suit, preferably one or more of BHT, methyl paraben and propyl paraben; Solvent / s, preferably a mixture of ethanol, triacetin and diethylene glycol monoethyl ether (DEGEE): quantity to suit; and Optionally, surfactant / s or detergent / s for any SAIB concentration above about 70% w / v, preferably Tween 80: approximately 0.5% to 5% w / v, preferably approximately 2% w / v.

25. The formulation of claim 1, wherein the formulation comprises (Formulation 2):Vitamin D3 active, preferably calcitriol: approximately 0.005% to 0.01% w / v; SAIB: approximately 50 % w / v; Optionally, preservative / s such as antioxidant / s, preferably one or more of BHT, methyl paraben and propyl paraben; and Solvent / s: approximately 9.8% w / v ethanol, approximately 8.6% w / v triacetin, and quantity to suit diethylene glycol monoethyl ether (DEGEE).

26. The formulation of claim 1, wherein the formulation comprises (Formulation 3): Vitamin D3 active, preferably calcitriol: approximately 0.005% to 0.01% w / v; SAIB: approximately 70% w / v; Optionally, preservative / s such as antioxidant / s, preferably one or more of BHT, methyl paraben and propyl paraben; Solvent / s: approximately 9.8% w / v ethanol, approximately 8.6% w / v triacetin, and quantity to suit diethylene glycol monoethyl ether (DEGEE); and Surfactant / s or detergent / s, preferably Tween-80: approximately 2% w / v.

27. A controlled-release Vitamin D3 formulation for: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, said controlled- release Vitamin D3 formulation comprising the formulation of any one of claims 1 to 26.

28. A controlled-release Vitamin D3 formulation for use or when used for: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, said controlled-release Vitamin D3 formulation comprising the formulation of any one of claim 1 to 26.

29. Use of a Vitamin D3 active and a carrier in the manufacture of a medicament for: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, said medicament being formulated to provide to the cow both an initial burst release as well as sustained release of the Vitamin D3 active, wherein said Vitamin D3 active is as described in any one of claims 1 to 26, and said carrier is as described in any one of claims 1 to .

30. A method of: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, said method comprising the step of administering to the cow the controlled-release Vitamin D3 formulation of any one of claims 1 to 26.

31. Use of a controlled-release Vitamin D3 formulation comprising a Vitamin D3 active and a carrier for: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, wherein said Vitamin D3 active is as described in any one of claims 1 to 26, and said carrier is as described in any one of claims 1 to 26.

32. An injector containing a controlled-release Vitamin D3 formulation comprising a Vitamin D3 active and a carrier for: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, wherein said Vitamin D3 active is as described in any one of claims 1 to 26, and said carrier is as described in any one of claims 1 to 26.

33. A kitset for use or when used in a method of: preventing or treating hypocalcaemia in a cow; maintaining normal and / or healthy blood calcium levels in a cow; normalizing blood calcium levels in a cow; increasing blood calcium levels in a cow; avoiding calcium deficiencies in the blood of a cow; or, avoiding insufficient calcium levels in the blood of a cow, wherein the kitset comprises: an injector capable of administering the controlled-release Vitamin D3 formulation as defined in any one of claims 1 to 26.

34. The controlled-release Vitamin D3 formulation of claim 27 or claim 28, the use of claim 29 or claim 31, the method of claim 30, the injector of claim 32, or the kitset of claim 33, wherein approximately 0.01 μg to 10 μg per kg bodyweight of the cow, preferably approximately 0.03 μg to 3.3 μg per kg bodyweight of the cow, more preferably approximately 0.25 μg to 0.5 μg per kg bodyweight of the cow, and even more preferably approximately 0.33 μg per kg bodyweight of the cow is injected.

35. The controlled-release Vitamin D3 formulation of claim 27, 28 or 34, the use of claim 29, 31 or 34, the method of claim 30 or claim 34, the injector of claim 32 or claim 34, or the kitset of claim 33 or claim 34, wherein approximately 0.05 to approximately 10 ml of formulation is injected.

36. The controlled-release Vitamin D3 formulation of claim 27, 28, 34 or 35, the use of claim 29, 31, 34 or 35, the method of claim 30, 34 or 35, the injector of claim 32, 34 or 35, or the kitset of claim 33, 34 or 35, wherein the formulation is used for the prevention or treatment of sub-clinical hypocalcaemia, or the prevention or treatment of clinical hypocalcaemia.

37. The controlled-release Vitamin D3 formulation of claim 27, 28, 34, 35 or 36, the use of claim 29, 31, 34, 35 or 36, the method of claim 30, 34, 35 or 36, the injector of claim 32, 34, 35 or 36, or the kitset of claim 33, 34, 35 or 36, wherein: the formulation is injected before calving; the formulation is injected at the time of calving; the formulation is injected within approximately 12 hours after calving (postpartum); the formulation is administered to a periparturient dairy cow; approximately 198 µg of calcitriol / 600 kg body weight is injected into the cow; the formulation is effective in increasing serum tCa (total sero-calcium) in approximately the first 7 days postpartum or after injection; the formulation is effective in achieving a serum calcium level of > / = 2.0 mM within approximately 24 hours of injection; the formulation is effective in increasing serum calcitriol beginning approximately an hour after injection; or the formulation is effective in maintaining an elevated serum calcitriol concentration until approximately 48 hours after injection.

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