AU2016100352B4

AU2016100352B4 - Reproductive dosage regime

Info

Publication number: AU2016100352B4
Application number: AU2016100352A
Authority: AU
Inventors: Ray Drew Castle; Forbes Richard Emslie
Original assignee: Elanco New Zeeland
Current assignee: Elanco New Zeeland
Priority date: 2015-05-13
Filing date: 2016-04-01
Publication date: 2017-03-16
Anticipated expiration: 2024-04-01
Also published as: NZ708025A; AU2016100352A4

Description

James & Wells ref: 300776AU/91 2016100352 01 Apr 2016

REPRODUCTIVE DOSAGE REGIME

TECHNICAL FIELD

This invention relates to a reproductive dosage regime. 5 Referenced throughout the specification will be made to the use of the present invention in relation to cattle, but it can be used in relation to other species for example horses, sheep, dogs and pigs.

BACKGROUND ART 0

It should be appreciated that the reproductive performance of cattle is one of the most important factors affecting the profitability of dairy and beef farms. Considerable research has been conducted to develop reproductive programs that ensure oestrus amongst cows within a herd are synchronized when required, and that reproductive rates are high. 5 There are numerous factors that have to be taken into account when farmers are choosing the optimum reproductive programs for their herd. These programs usually involve the introduction of hormones in various forms and in a staged manner.

These treatments can take considerable time and money to implement, particularly when multiplied over hundreds of cows that are found in typical large herds. >0 For example, a basic method of synchronizing oestrus is to administer prostaglandin by injection to each cow in a herd, which is expected to result in around 75% of cycling animals to display oestrus in the following 2 to 5 days. This method would not be successful for anoestrus cows, since a corpus luteum is not present on the ovary. A more sophisticated protocol involves multiple interactions. For example, a treatment may start 25 with the injection of an equivalent to endogenous gonadotropin releasing hormone (GnRFI), which stimulates the maturation of ovarian follicles, ovulation and development of the corpus luteum.

In one version of a typical reproductive program, the next stage is to then inject the cow seven days later with an analog of prostaglandin (PGF2-a). This causes luteolysis (degradation) of the 30 corpus luteum.

Next the, cows that appear to be in oestrus are mated. The cows that do not seem to be in oestrus are given another dose of GnRFI and then inseminated shortly afterwards. 1

James & Wells ref: 300776AU/91 2016100352 01 Apr 2016

While this program is fairly successful, reproductive rates can be increased by the addition of further treatments into the program. It should be noted that every failure to conceive (and carry progeny successfully) is highly expensive for the farmer.

In addition to the above program is the intravaginal introduction of a drug delivery device 5 carrying progesterone. Devices of this type usually have a spine into which progesterone is infused (such as the device sold under the CIDR™ brand), or to which are attached silicon gills (such as the device sold under the brand CUEMATE™).

These devices are introduced between the initial introduction of GnRH and the introduction of PGF2-a analog. The introduction of progesterone is to inhibit follicle maturation for around o seven days ensuring that the cows are synchronised for the dose of PGF2-a analog.

Again, this regime increases the rate of reproduction in cows.

However, a further treatment has been found that can increase even further the reproductive rate. This is the introduction of equine chorionic gonadotrophin (eCG) at the same time as the introduction of a PGF2-a analog. It has been found the eCG is highly effective in stimulating 5 follicular development thereby increasing ovulation rates.

Unfortunately, there are a number of problems associated with the use of eCG.

Firstly, GnRH and PGF2-a analog treatments are provided in ready-to-use liquid form enabling for them to be readily injected into the animal.

In contrast however, eCG is unstable in liquid form and therefore is sold separately in powdered !0 form with a diluent. This requires the user to mix the powder with a phosphate buffered saline diluent prior to injection and then separately inject the PGF2-a analog.

This separate step of mixing and injecting the eCG is highly time consuming particularly multiplied over the hundreds of cows in a large herd. Further, having to conduct two quite different procedures (eCG and PGF2-a) at the same time and deliver separately to each cow is 25 considered fussy and off-putting to the farmer. Although there are benefits to using eCG, uptake of this regime is not too high because of the additional hassle it entails

Further, the manufacturer is required to have significant packaging for the two separate products with bulky diluent being provided for the two separate products. This is another disincentive, adding extra cost to the product. 30 It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice. 2

James & Wells ref: 300776AU/91 2016100352 01 Apr 2016

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly 5 understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country.

Throughout this specification, the word "comprise", or variations thereof such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or o group of elements integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

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

5 DISCLOSURE OF THE INVENTION

According to one aspect of the present invention there is a provided a reproductive dosage regime for a non-human animal, the regime characterised by the steps of: >0 mixing a powdered equine chorionic gonadotrophin (eCG) or equivalent thereof with ProstoglandinF2 (PGF2-a) or equivalent thereof in an aqueous solution, and injecting the animal with the mixture arising from step a).

According to another aspect of the present invention there is provided a kit set including eCG or equivalent thereof in powdered form, and 25 an aqueous solution of PGF2-a, or equivalent thereof and instructions for the mixing of the eCG and PGF2-a, and instructions for the administration of the mixture from step c) to a non-human animal.

According to another aspect of the present invention there is provided a kit set when used in the control or synchronisation of oestrus in a non-human animal, the kit set including 30 eCG or equivalent thereof in powdered form, and an aqueous solution of PGF2-a or equivalent thereof and 3

James & Wells ref: 300776AU/91 2016100352 01 Apr 2016 instructions for the mixing of the eCG and PGF2-a to form a mixture, and instructions for the administration of the mixture to a non-human animal.

According to another aspect of the present invention there is provided a kit set including eCG or equivalent thereof in powdered form, and 5 an aqueous solution of PGF2-a or equivalent thereof and instructions for the mixing of the eCG and PGF2-a to form a mixture, and instructions for the administration of the mixture to a non-human animal including a vial containing an aqueous solution of PGF2-a, or equivalent thereof, and a cap attached to the vial, wherein the cap contains the eCG or equivalent thereof in o powdered form, wherein the cap and vial are configured so that the powdered eCG or equivalent thereof mixes with the aqueous solution before administration of the PGF2- a or equivalent thereof.

According to another aspect of the present invention there is provided a composition suitable for injection, comprising an aqueous solution of PGF2-a, or equivalent thereof, and eCG or 5 equivalent thereof.

Preferably the pH of the composition is from 6 to 8.

More preferably, the pH of the composition is from 6.3 to 7.5.

It is thought that the present invention could be used in many cases as part of a larger reproductive program to control or synchronise oestrus in animals. The present invention will 20 be discussed primarily for use with cattle, however it should be appreciated the present invention can also be used with numerous other species including the like of sheep, horses, goats, pigs and dogs.

The eCG acts to stimulate follicular development and to induce oestrus in anoestrus animals thereby increasing ovulation rates. Therefore, an equivalent to this product could be something 25 along the lines of hCG or pharmaceutical equivalents thereof which have a similar action on the animals into which the product is induced.

It should be noted that eCG is unstable when in aqueous solution, which is why as it is normally sold in freeze dried powder form, thereby requiring the supply of diluent so that the eCG can be reconstituted just prior administration to the animal. For example, the product Pregnecol™ 30 provides in a single box a vial containing the eCG powder along with a bottle of diluent of a 4

James & Wells ref: 300776AU/91 2016100352 01 Apr 2016 phosphate buffered saline solution. The freeze-dried powder form of eCG has a shelf-life of up to 3 years when refrigerated. When reconstituted with a diluent it has limited stability and is usually used immediately. Although, the efficacy of the reconstituted eCG can be maintained for a short while. For instance Novormon eCG by AgriHealth states on the label that the product 5 should be discarded 21 days after reconstitution with the diluent. PGF2-a can come in a number of forms and the form as it is widely used in the veterinary industry is Cloprostenol, which is a functional synthetic analogue of PGF2-a. Therefore an equivalent thereof would be expected to have similar effect to Cloprostenol in the animal’s system. o In the product Ovuprost™, Cloprostenol as the sodium salt is sold premixed with citrate buffered saline solution, at a concentration of 0.25 g/L of cloprostenol sodium.

The inventors have ascertained that the diluent used for PGF2-a is also effective when used as the diluent for eCG.

The inventors have also found that despite having never been mixed together before, the 5 combination of eCG and PGF2-a in a single dose to be injected caused no problems.

Stability of this product is important and it has been a surprising result that this new combination of the two actives with a single diluent gives unexpected stability.

Indeed, the inventors postulate that a combination of the compositions, or a composition containing the two actives was not considered as suitable due to stability concerns, other than !0 when the combination would be used immediately.

The applicants have tested for efficacy and stability the combination of Pregnecol™ and Ovuprost™, in which the freeze-dried eCG was reconstituted using the cloprostenol solution as the diluent. The combination was found to be stable and maintain efficacy of both actives 21 days after reconstitution. 25

Patent literature discusses the stability of protein formulations. For instance WO2012108828 includes the following statement: “The stability of protein drugs in vivo and in vitro is a complex matter where 30 multiple degradation reactions occur in parallel, such as oxidation, deamidation, aggregation etc. One major reaction occurring is formation of aggregates. Protein aggregates can form via covalent or non-covalent pathways, and can be of soluble or insoluble nature. The presence of protein aggregates is a major concern from safety perspective as it may impact the secondary and tertiary 5

James & Wells ref: 300776AU/91 2016100352 01 Apr 2016 structure of the protein. Presence of specific non-native protein structures has been associated with increased immunogenicity of proteins potentially causing reduced efficacy or even in vivo immunological reactions to native proteins, with life threatening conditions as result.” 5

The 1938 journal article by F. Bischoff (Chemical Studies on the Gonadotropic Hormone of Pregnant Mare Serum J. Biol. Chem. 1938, 125:697-702) discusses chemical studies of gonadotrophic hormone of pregnant mare serum (eCG). In the preparation stage the author precipitated the eCG from solution using pH 6.0. This indicates that the eCG is more soluble at o the pH conditions of blood at around 7.4, and is prone to precipitation at slightly acidic pH.

In the experiments testing the effect of various reagents on the biological effect of eCG, the author found that the pH was a critical factor on whether the protein was denatured by the reagent. The results showed that some reagents had no effect at slightly acidic pH, but when used in a slightly alkaline solution the outcome was a significant or total degradation in the 5 biological effect of the eCG. A typical diluent for eCG, is that used by Pregnecol™, which is a saline solution that uses a phosphate buffer, with a pH of approximately 7.4. This is a common form of diluent used for injectable products, particularly intravenous injectables.

The use of pH 7.4 is clearly suitable for the stability of eCG when it is the sole active, since it !0 avoids the possible precipitation caused by acidic pH, and there is no issue with the degradation at slightly alkaline pH because there is no other active in the reconstituted composition.

The results of the experiments in the Bischoff article indicate that there would be considerable issues to be expected when mixing eCG with another active agent such as cloprostenol. In particular, if a skilled person were to consider mixing eCG with cloprostenol, selecting an 25 appropriate pH would present a critical dilemma. A slightly acidic formulation would not be seen as favourable since it presents conditions that lead to precipitation of the eCG. A slightly alkaline formulation has been demonstrated as being unsuitable for eCG when combined with other chemical entities that are capable of interacting and causing degradation of the biological acitivity of the eCG. An interaction between 30 cloprostenol and eCG would appear likely at slightly alkaline conditions due to the presence of reactive moieties, such as the carboxyclic acid functional group on cloprostenol.

In addition to ensuring stability of the eCG, the stability of the cloprostenol must also be maintained. 6

James & Wells ref: 300776AU/91 2016100352 01 Apr 2016

From an article discussing prostaglandins (which includes cloprostenol), which are based on the eicosanoid structure: 5 o “Most eicosanoids have a very low chemical and metabolic stability (metabolic instability means that the biological degradation is high). For instance, PGE2 is only stable at pH 7; below pH 7 it is converted to PGA2, above pH 7 it is converted to PGB2. Pure PGI2 has a half-life of a few minutes and is hydrolysed to 6-oxo-PGF1a. The sodium salt of PGI2 is, however, sufficiently stable for transportation at low temperatures." http:/7onl

lol/l 0.1002/14356007.822 26f.DUb2/fuH

Given the disparity between the pH requirements for both the actives, it was surprising that the inventors were able to combine cloprostenol and eCG into a single composition that was stable for more than 24 hours, and is even stable for 21 days or more. 5 The expectation of a complete lack of stability is why a combined composition has not been marketed before, since if it were expected that it would have to be used immediately or discarded, it would not be a commercially viable product.

Significant advantages arise out of using this invention.

First, at this stage of the synchrony program, the farmer need only take the steps he/her »0 required for administering eCG to the cow. There is no additional requirement to separately inject the Cloprostenol, this reducing dramatically the amount of labor required.

Also reduced dramatically is the amount of diluent required as both the eCG and the Cloprostenol dosages (25mg in 100ml water 0.025%w/v) are capable of being dissolved in the same volume of diluent used for cloprostenol- namely 2mls 25 This ease of use and the fact that a preferred embodiment of the invention has the products together in one packages makes it easier for the farmer to choose to use the introduction of eCG in addition to Cloprostenol. This leads to greater chance of pregnancy occurring.

For the manufacturer, costs are also reduced due to their being less volume required of diluent, less vials required to provide a potent dosage with both actives and less packaging required to 30 house both actives compared to when they were sold independently.

Further the combination is unexpectedly stable, leading to greater shelf-life than related products. This leads to greater flexibility of use.

In a preferred embodiment of the present invention, the kit set is in a form of a box with similar dimensions to that sold under the Ovuprost™ brand. The main difference will be the inclusion 7

James & Wells ref: 300776AU/91 2016100352 01 Apr 2016 of an additional vial containing the eCG powder. No additional diluent is required as that is provided with the Ovuprost 20ml vials or 100ml flexi packs. In some embodiment of the present invention, the eCG may be held within a cap on the cloprostenol diluent container. Using existing technology, the membrane in the cap may be broken allowing the eCG to fall into the 5 Cloprostenol and diluent making it easy to mix the two actives together.

The present invention provides a one step process that encourages farmers to use methods that relate to greater reproductive rates. The present invention also has the advantages of providing less packing and ensuring proper ratios of eCG to PGF2-a are maintained.

0 BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present invention will become apparent from the ensuing description which is given by way of example only and with reference to the accompanying drawings in which: 5 Figure 1 Illustrates a possible oestrus synchrony program in accordance with one embodiment of the present invention.

BEST MODES FOR CARRYING OUT THE INVENTION »0 One embodiment of the present invention uses as the diluent, the cloprostenol-containing formulation as below.

Ingredient Name (Common or Chemical) CAS Number Quantity (g/L) Function Cloprostenol Sodium 55028-72-3 0.268* Active Ingredient Methyl Paraben 99-76-3 1.80 Preservative Propyl Paraben 94-13-3 0.20 Preservative Sodium Citrate 6132-04-3 5.03 Buffer Citric Acid Anhydrous 77-92-9 0.66 Buffer Sodium Chloride 7647-14-5 6.76 Isotonic agent/ Ionic Adjuster Citric Acid or Sodium Flydroxide Solution - qs to phi 6.3 - 6.9 pH Adjuster 8

James & Wells ref: 300776AU/91 2016100352 01 Apr 2016

Solvent

Qs 7732-18-5

Water for Injection A preferred eCG source is freeze-dried eCG powder. The powder includes sugars as cryo-protectants, which reduce degradation of the eCG during the freeze-drying process.

In one embodiment of the present invention, the animal treatment may be prepared accordingly. 5 Use a sterile syringe to withdraw 3 to 5 mL of the aqueous solution containing cloprostenol from a 30 mL pack of diluent.

Add the contents of the syringe to the vial containing 6000 i.u. of freeze-dried eCG.

Rock the contents of the eCG vial gently then withdraw the total contents and add to the diluent pack of cloprostenol supplied. o Repeat the procedure to ensure complete recovery of vial contents.

In another embodiment, the reproductive dosage regime of the invention is used in a treatment program to treat anoestrus and/or improve conception rates in cattle, as illustrated in Figure 1. The treatment program includes administration of actives according to a schedule.

On Day 0, administering a dose of Gonadotrophin Releasing Hormone (GnRH) to an animal. 5 The preferred GnRH is gonadorelin as the acetate in the form of an injectable solution at a concentration of lOOpg/mL. The typical dose for cattle is 100 pg of gonadorelin as an intramuscular injection.

Also on Day 0, a progesterone releasing device is inserted intravaginally. A preferred device contains approximately 1.56 g of progesterone in a controlled release silicone substrate. 20 On Day 7, the progesterone releasing device is removed from the animal, and a combination injection of eCG and PGF2-a is administered. The preferred PGF2-a is the prostaglandin analog cloprostenol. The combination injection is prepared by reconstituting eCG freeze-dried powder with an aqueous solution of cloprostensol. The preferred doses are 500pg of cloprostenol and 400-800 i.u. of eCG. 25 In the time between days 7 to 9, if the animal is seen to be in oestrus, then it is inseminated.

If oestrus is not seen, on Day 9, a second dose of 100 pg of gonadorelin is administered as an intramuscular injection.

On Day 10 (more specifically 16-20 hours after the second administration of gonadorelin) the animal is inseminated. 9

James & Wells ref: 300776AU/91 2016100352 01 Apr 2016

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

Claims

WHAT WE CLAIM IS:

1. A reproductive dosage regime for a non-human animal, the regime characterised by the steps of: a) reconstituting a powdered equine chorionic gonadotrophin (eCG) or equivalent thereof with an aqueous solution of ProstoglandinF2 (PGF2-a) or equivalent thereof, wherein the aqueous solution has a pH of 6.3 to 7.5, and b) injecting the animal with the mixture arising from step a).

2. A reproductive dosage regime as claimed in claim 1 wherein the dosage of eCG is the range of 0.5 to 20 i.u. per kg of animal and the PGF2-a dosage is in the range of 0.5 to 2 pg per kg of the animal.

3. A kit set when used in the control or synchronisation of oestrus in a non-human animal, the kit set including eCG or equivalent thereof in powdered form, and an aqueous solution of PGF2-a or equivalent thereof and instructions for the mixing of the eCG and PGF2-a to form a mixture, and instructions for the administration of the mixture to a non-human animal.

4. A kit set including eCG or equivalent thereof in powdered form, and an aqueous solution of PGF2-a or equivalent thereof and instructions for the mixing of the eCG and PGF2-a to form a mixture, and instructions for the administration of the mixture to a non-human animal including a vial containing an aqueous solution of PGF2-a, or equivalent thereof, and a cap attached to the vial, wherein the cap contains the eCG or equivalent thereof in powdered form, wherein the cap and vial are configured so that the powdered eCG or equivalent thereof mixes with the aqueous solution before administration of the PGF2- a or equivalent thereof.

5. A composition suitable for injection, comprising an aqueous solution of PGF2-a, or equivalent thereof, eCG or equivalent thereof, and a pH buffer wherein the pH of the composition is from 6.3 to 7.5.