AU2022204326A1 - Anti-infection formulation and methods of use - Google Patents

Abstract

According to first aspect of the present invention there is provided a formulation, for administration to the teat canal or/and lower portion of teat cistern of a mammary gland of an animal, wherein the formulation prior to delivery is in the form of a paste, the formulation including: an oil-based physical barrier material which is able to form a cohesive mass in the teat canal and/or the lower portion of the teat cistern, and at least one antiseptic compound mixed with the barrier material.

Description

ANTI-INFECTION FORMULATION AND METHODS OF USE TECHNICAL FIELD

This invention relates to an anti-infective formulation and methods of use.

More specifically, this invention relates to an anti-infective formulation and methods

of use to prevent or ameliorate mammary gland infections, including mastitis.

BACKGROUNDART

One of the major problems associated with dairy cows is the high occurrence of

bovine mastitis, especially new infections during the dry or drying off period, when

dairy cows are particularly susceptible to mastitis. This problem also applies to

other lactating animals, but for the purposes of this discussion reference is made

to bovine mastitis.

The dominant pathogens associated with dry period mastitis infections may

include: Staphylococcus aureus, Streptococcus uberis and Streptococcus

dysgalactiae .

A number of treatments have previously been developed to overcome the

occurrence of mastitis, these include DCT (dry cow therapy), external teat seals

and internal teat seals. However, as discussed in detail below, all of the products

currently available have significant disadvantages.

Bovine mastitis is usually treated or prevented with intramammary antibiotics.

One previously used method to prevent new outbreaks of mastitis was DCT. This is the treatment of all cows (healthy and mastitis positive) with antibiotic therapy by

an infusion of all udder quarters with long acting antibiotic at the start of the dry off period to cover the dry period.

However, this method is only specific against the particular pathogen species that

the antibiotic used is susceptible to and will not prevent infection by other pathogen

species. Another disadvantage is that the antibiotic may not remain present at

5 therapeutic levels for the entire dry period. This may lead to increased

susceptibility when the level drops, or the requirement for re-administration.

Although this method can in some instances be effective, the use of antibiotics is

highly undesirable. When cows are treated with antibiotics, antibiotic residues can

be absorbed by the animal, and can be excreted in the milk. As a result of this, the

milk needs to be withheld for a period of time following antibiotic administration.

The use of antibiotics in this manner can also lead to increased risk of the mastitis

causing micro-organisms developing resistance to the antibiotic.

As may be appreciated, the need to withhold milk is an inconvenience to the

farmer. In order to overcome this, the farmer has to either ensure that the milk is

diverted away from the holding tank through separate milk lines, or depending on

their systems keep the treated animal(s) separate, and milk these at a separate time.

The loss of milk, and the time required to implement withholding periods after

treatment with antibiotics can result in significant economic losses. Further, consumers want confirmation of the non-treatment of animals from which milk or

meat is produced with antibiotics.

To overcome the above problems, teat seals, both external and internal were

developed to provide a physical barrier to prevent micro-organisms from gaining

access into the teat or udder. Again, both these have significant disadvantages.

External teat seals provide an external physical barrier across the entrance to the

teat canal. The end of each teat is dipped in an acrylic, latex or polymer based

teat seal after milking has finished, and the dry period commences.

Once applied, the external teat seal dries to generate a latex, acrylic or other

polymer based film that prevents entry of pathogenic bacteria into the teat canal.

However, the application of external teat seals only once during the dry period, at

drying off does not provide sufficient protection for the entire dry period. One

reference showed that numerous trials undertaken to validate the persistency of

external teat seals had shown that the medium length of protection, or persistency

was less than six days.

It has therefore been recommended that external teat seals are applied at the start

of the dry off period, and again as needed starting approximately ten days prior to

calving.

This therefore increases the time required by the farmer to check and re-treat cows

multiple times during the dry period. It also increases the amount, and therefore

cost of the external teat seal required.

A further method of preventing mastitis infection during the dry period is the use of

internal teat seals.

Internal teat seals are a thick paste that is infused into each quarter of the cow's udder at drying off. The internal teat seal then forms a physical barrier, either in a paste form or via solidifying in the teat canal, thereby preventing micro-organisms

from accessing the teat canal.

Many currently available internal teat seals include a heavy metal salt such as

bismuth sub-nitrate mixed into a gel base. The gel base may by polyethylene gel, which solidifies after administration. The gel base may also include a vehicle such as liquid paraffin. For example see New Zealand Patent No. 336153. Like, external teat seals, internal teat seals if used alone do not include antibiotics, and therefore do not require a withholding of milk.

5 One significant disadvantage of using an internal teat seal alone is that it does not

prevent or act against micro-organisms which are already present in the teat, or

which may be introduced during administration of the teat seal.

The effectiveness of Teatseal, an internal teat seal has been analysed. It was

found that although cows treated with Teatseal showed a significant reduction in

10 the risk of developing new infections by the major pathogens, and environmental

streptococci pathogen groups. This treatment showed no effect on the treatment of

new infection caused by either contagious or gram negative pathogens.

Although, treatment with internal teat seals is only required once at drying off, there

are still inconsistencies in the success of using this method to combat new mastitis

infections, with new infections of bovine mastitis still being observed in both treated

quarters and animals.

In some instances, such as when an animal is known to be infected, antibiotic treatment might be applied along with, or prior to administration of the internal teat

sealant.

New Zealand patent number 258199 relates to a veterinary composition containing the antibiotic cloxacillin in an aqueous suspension including a suspension aid, a

buffer and a surfactant. The composition also includes a heavy metal salt and gel

base which acts as a physical teat seal. This patent discloses the use of two independent compositions that are administered at the same time. The

intramammary composition including cloxacillin is administered followed by the teat seal.

The product disclosed in New Zealand patent number 258199 has a number of significant disadvantages. The most significant disadvantage is that like other antibiotic treatments the aim was for the antibiotic to be absorbed by the animal.

5 The aim is not necessarily for absorption when udder infections are being treated.

The aim is to get the antibiotic to the infected sites in the udder. So, although the aim may not be absorption, absorption does occur and this leads to residues in

other tissues. For example, page 8 lines 9 to 13 reads as follows: 'Within 30 minutes the antibiotic administered also crosses the blood-milk barrier establishing 10 a drug reservoir which aids the maintenance of therapeutic levels of cloxacillin for

the 72 hour treatment period.' This product therefore still retains all the problems associated with antibiotic treatment discussed above.

To overcome the deficiencies of using internal teat seals alone, and to avoid the

requirement to withhold milk, internal teat seals including bacteriocins have been developed. Bacteriocins, such as nisin and lacticin have broad spectrum action against a number of micro-organisms. The use of bacteriocins provides a multiple

approach to prevention of mastitis, including both a physical, and a chemical

feature.

A drawback of a seal is that microbes which may already be present in the teat are sealed inside and without natural challenges may become infectious. In addition,

the natural sealing process may also be disrupted, slowed or otherwise altered,

which may cause harm to the animal.

It should therefore be appreciated that a product which prevented new infection by

pathogenic micro-organisms during the dry period and also avoids altering natural

teat sealing processes would be advantageous.

It is an object of the present invention to address the foregoing problems or at least

to provide the public with a useful choice.

All references, including any patents or patent applications cited in this

specification are hereby incorporated by reference. No admission is made that any

5 reference constitutes prior art. The discussion of the references states what their

authors assert, and the applicants reserve the right to challenge the accuracy and

pertinency of the cited documents. It will be clearly understood that, although a

number of prior art publications are referred to herein, this reference does not

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.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be

attributed with either an exclusive or an inclusive meaning. For the purpose of this

specification, and unless otherwise noted, the term 'comprise' shall have an

inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components

or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

Further aspects and advantages of the present invention will become apparent

from the ensuing description which is given by way of example only.

DISCLOSURE OF INVENTION

According to first aspect of the present invention there is provided a formulation,

for administration to the teat canal or/and lower portion of teat cistern of a

mammary gland of an animal, wherein the formulation prior to delivery is in the

form of a paste, the formulation including: an oil-based physical barrier material which is able to form a cohesive mass in the teat canal and/or the lower portion of the teat cistern, and at least one antiseptic compound mixed with the barrier material.

According to a further aspect of the present invention there is provided a use of a

formulation as substantially described above, in the manufacture of a medicament

to prevent or ameliorate mastitis.

According to a further aspect of the present invention there is provided a use of a

formulation as substantially described above, in the manufacture of a medicament

to prevent or ameliorate infection within the teat cistern of a mammary gland.

The term 'infection' or grammatical variations thereof refers to the invasion of an

animal's body by micro-organisms.

The term 'prevent' or grammatical variations thereof refers to keeping, averting or hindering an infection from occurring.

The term 'mammary gland' or grammatical variations thereof refers to the whole of

the udder of an animal, including the secreting tissue, connective tissue, glands

and teats.

The term 'teat canal' or grammatical variations thereof refers to the teat canal or

aperture from the teat cistern, where the milk is extracted from.

The term 'antiseptic' or grammatical variations thereof refers to any agent that kills

or prevents the spreading of infectious organisms in order to prevent the spread of

infections. Antiseptics are generally applied to the external surface tissue.

Whereas the term 'antibiotic' refers to a drug that treats infections internallywithin the body.

The term 'gelling compound' or grammatical variations thereof refers to

compounds that promote coagulation or thickening of one or more further

compounds together.

The term 'dry period' or grammatical variations thereof refers to the period where

milking for the lactation season has ceased as the cows have no milk to expel and

are therefore 'dry'. A person skilled in the art will understand that this time period

may commence is early to mid summer until spring, when the cows calve.

The term 'coherent' or grammatical variations thereof refers to something that is

sticky and binding together and to other material.

The term 'temporary' or grammatical variations thereof refers to the physical barrier

as not lasting or not being permanent.

Preferably, the formulation, prior to use, is a paste. Preferably, the paste is

administered into the teat canal, for example through a tube or syringe. Once

administered, the formulation solidifies in the teat canal to form a substantially solid

and coherent temporary physical barrier.

Preferably, the paste has a viscosity range between 800,000 - 3,000,000 cps @

20 0C using a Brookfield LVT on a Helipath stand, spindle TF at 0.3 RPM.

The inventors have found that this viscosity range is advantageous as it helps form

a solid product that effectively seals the teat, thereby helping to prevent microbes

from entering the canal to cause mastitis.

The inventors have found that if the paste's viscosity is lower than 800,000 cps, the

paste tends to be runny, and unlikely to be able to form such a seal.

Similarly, if the paste is too thick (e.g. above 3,000,000, the inventors have found

the paste often is not capable of moulding to the shape of the teat canal to form a plug after it has been administered. Further, a high viscosity paste can be difficult to administer to canal - requiring some force from the farmer.

Furthermore, this viscosity range discussed above may be advantageous as it

helps to provide the dissolution profile of the active, preferably chlorhexidine. If the

paste's viscosity was outside of the range discussed above, the active would likely

be released to quickly or to slowly for desired outcome.

Essentially an oil based product in the viscosity range described with infused

antiseptic forms a cohesive mass upon administration to the teat canal.

Preferably, the physical barrier may have sufficient coherence to allow it to flex with

movement of the sides of the teat canal or/and lower portion of teat cistern. It

would be appreciated that this increases the seal created by the formulation and/or

physical barrier and the side of the teat canal or/and lower portion of teat cistern, to

decrease the channel between the seal and the teat canal or/and lower portion of

teat cistern.

All previous formulations when used alone have acted in an entirely physical

manner and do not often provide a complete and consistent seal across the entire

teat canal. For example, with movement of the animal, or due to the inflexibility of

the formulation gaps or a channel are present between the formulation and the

side of the teat canal.

Throughout this specification, such a gap shall be referred to as a channel, this

should be taken to include, any gap, moisture film or passageway between the formulation and the side of the teat canal.

Any such channels provide a means by which micro-organisms can enter the teat canal and migrate into the teat cistern. Micro-organisms that enter the teat cistern

may then cause new infections, including mastitis.

This ineffectiveness of previous physical teat seals and formulations indicate that

sealing is not complete or consistent, and that gaps or a channel is present

between the formulation and the side of the teat canal. This defeats the purpose

of the seal to some extent, and limits its viability and effectiveness. The inventors

consider that there may be a narrow, moisture filled channel, or film between the

administered formulation and the side of the teat canal, which allows the migration

of micro-organisms from the exterior of the teat into the teat cistern. Therefore, the

present formulation has sufficient elasticity to increase the physical barrier within

the canal or/and lower portion of teat cistern, and therefore prevent the migration

of micro-organisms into the teat cistern.

It should be appreciated that even if the mechanism by which the present invention

work is found not to be as suspected by the inventors, the data shows that the

present invention is highly effective irregardless of the mechanism by which it

works. For example, the present invention may work by not necessarily stopping

migration of all microbes past the teat seal within the teat canal or/and lower

portion of teat cistern, but once microorganisms enter the teat cistern, the

antiseptic may then operate on them.

The formulation of the present invention may be used to prevent and/or ameliorate

mastitis. Preferably, the formulation may be used to prevent and/or ameliorate

mastitis in milking animals, preferably the formulation may be administered to

sheep and goats or cows, preferably dairy cows.

Preferably, the formulation of the present invention is administered at substantially

the start of a dry period, during the drying off period or prior to first calving in

primiparous heifers.

Preferably, the physical barrier includes barium based compound which may be a

barium salt. In one preferred embodiment, the barium salt may be barium sulphate. Barium sulphate is advantageous in that it is cheaper and more environmentally friendly compound than previously used compounds, such as bismuth sub-nitrate.

Preferably, the barium based compound is micronised.

In a preferred embodiment the barium sulphate is present in the formulation at a

concentration of approximately 40% to 85% w/w. More preferably, the barium

sulphate may be present in the formulation at a concentration of approximately

67% w/w.

It is envisaged that the barium sulphate particles settle into the teat canal or/and

lower portion of teat cistern, contribute to substantially sealing off the teat canal

or/and lower portion of teat cistern with the temporary physical barrier.

Preferably, the antiseptic may be substantially incapable of being absorbed into the

body/cells or metabolised in the teat sinus (or udder). An advantage with this

feature is that the antiseptic is readily removed so as not to contaminate the milk

supply. This is highly desirable, as the antiseptic remains in the localised area and

is virtually incapable of entering the cow's body physiology.

This is in direct contrast with antibiotics which enters the cow physiology and

causes the farmer to withhold milk for a number of milkings, this costing the farmer

significantly. Further, a continued application of antibiotics can build up resistance in the microorganisms causing mastitis and it is desirable to be able to avoid using

antibiotics as much as possible. Finally, teat seals can only be used during the dry

period, whereas usually the farmer has to resort to antibiotics during the June

milking (in the Southern Hemisphere). Therefore, the inventor considers it

important to be able to use a product that is not containing antibiotics in the only

period that this is possible.

Ideally, the antiseptic is physiologically acceptable, and have a broad spectrum

activity. More preferably, the antiseptic may be at least active against the major

and minor pathogens associated with bovine mastitis. This allows the antiseptic to

be active against a wide range of micro-organisms, and overcome some of the

5 associated problems with the use of antibiotics - that these are targeted to, and

active against an individual, or small related group of micro-organism.

Preferably, the antiseptic may have one or more of the following properties:

a) the chemical has antimicrobial activity at physiological'pH, and the pH of

milk, and/or

b) addition of the antiseptic does not change the physical characteristics of the

formulation, and/or

c) the antiseptic has a release rate such that the concentration in the aqueous

channel is greater than the minimum inhibitory concentration (MIC) for at

least 2-4 weeks, and/or

d) the antiseptic is not readily absorbed by the cow's body, and/or

e) the antiseptic is reasonably stable, and/or

f) the antiseptic is non-irritant to the cow's body.

Preferably, the antiseptic is chlorhexidine. Preferably, the chlorhexidine may be in

the form of the base or HCI salt of chlorhexidine, or any other salt which is physiologically equivalent, or can provide the desired characteristics to the

formulation.

Using chlorhexidine provides a further advantage, in that this compound is

acceptable for veterinary use and has been used in dairy practices for sometime.

Chlorhexidine, or formulations containing the compound, for example, are used by

farmers to clean and sterilize milking machines, and is used as a teat spray after

milking. Teat spraying after milking helps to prevent the entry of micro-organisms

into the teat canal which remains open for a period of time after milking has

finished. As such, these compounds are well known and tested. It is well known

that they are acceptable for use around and alongside current diary practices. It is

also stable and non-toxic to the animal.

One further significant advantages of using chlorhexidine, is that according to

current literature, and based on its known chemical structure, it is not believed to

be absorbed through the teat wall into the body.

However, the use of chlorhexidine should not be seen as limiting, as any other anti

septic with the desired characteristics may be utilised with the present invention.

Antiseptic with poor absorption characteristics are preferred, these may include, for

example ionised anti-septic. For example, quaternary ammonium compounds may be suitable, including, but not limited to Cetrimide and BZK. Similarly povidone

iodine which is currently used in the dairy industry may be suitable.

Preferably, the antiseptic, once released or diffused from the formulation may be

localised into the area between the physical barrier and the side of the teat canal or/and lower portion of teat cistern.

Preferably, the antiseptic may be released from the formulation at such a

concentration that it provides a localised concentration of antiseptic in the area

between the formulation and the side of the teat canal, or/and lower portion of teat

cistern which is sufficient to prevent the passage of, kill, or deactivate any micro

organisms that are present in the teat canal or/and lower portion of teat cistern.

Release of the antiseptic from the formulation is by diffusion of the small amount of

chlorhexidine which is dissolved in the base compound of the formulation to the

surface of the formulation and then partitioning into the aqueous channel.

Equilibrium will be established between the concentration of chlorhexidine in the

aqueous channel and the concentration of the chlorhexidine in the base compound

near the surface of the formulation.

The fact that the antiseptic is not absorbed into the body (or very little is) results in

an equilibrium being formed between the concentration of antiseptic in the

formulation and the concentration of antiseptic in the channel between the

formulation and the side of the teat canal or/and lower portion of teat cistern.

This equilibrium, the small area between the formulation and the side of the teat

canal or/and lower portion of teat cistern, and the fact that no, or very little

antiseptic is lost from this area due to absorption means that only a small volume of the antiseptic needs to be released from the formulation at any given time. This

increases the stability of the formulation, and decreases the amount of compounds

required. For example, this overcomes the necessity when bacteriocins are used

for the addition of Tween 80, to enhance the release of the bacteriocin from the

formulation. As previously discussed, this leads to a decreased stability and longevity of the formulation, which is highly undesirable.

Given the small area between the formulation and the side of the teat canal or/and

lower portion of teat cistern a small volume of antiseptic released from the

formulation will result in a high concentration of antiseptic between the formulation and the side of the teat canal or/and lower portion of teat cistern.

In some embodiments, the formulation may include the use of the more insoluble salts of chlorhexidine. The use of these salts may be utilised to reduce the release rate of chlorhexidine from the formulation.

In another embodiment, the formulation may also include a complexing agent. It will be appreciated that the complexing agent may assist with stabilising the

antiseptic within the formulation.

In a preferred embodiment the antiseptic may be retained in the formulation, at

least in part due to suspension in the viscous formulation.

Complexion of chlorhexidine may reduce the free concentration of chlorhexidine in

the base compound which would slow release and also possibly reduce the

equilibrium concentration in the aqueous channel.

Chlorhexidine is cationic, therefore it may be possible to utilise this organic ion to

form an ion pair within the oil or formulation which may lead to a greater retention

of antiseptic in the teat.

The inventors also anticipate that increasing the barium based concentration may

be used to slow the release rate of the chlorhexidine.

The formulation may include a carrier. The carrier may be a combination of both a

gel and oil, the gel may act to increase the viscosity of the oil, on administration

through the teat canal .

Preferably, the gelling compound or compounds are present at a concentration

within the range of approximately 1 to 12 % w/w of the oil component. More preferably, the gel compound or compounds may be present at a concentration of

approximately 5.5 to 6.0 %w/w of the oil compound.

Preferably, the gelling compound is aluminium stearate. One skilled in the art

would be aware, that any other suitable gelling compounds could also be used in

conjunction with the present invention. Preferably, the aluminium stearate may be present at a concentration of approximately 1.8 % w/w of the final product.

In embodiments where the carrier includes at least one oil, the oil is preferably

selected as being an oil that may be difficult for the animal to metabolise. More

preferably, the oil may be non-absorbable. More preferably, the oil may act as a

vehicle.

Preferably, the oil may be present at a concentration of approximately 30 % w/w. It

will be appreciated that the concentration may vary depending on the other

components used, and may be varied to provide the desired characteristics of the

formulation. Therefore, more preferably the oil may be at a concentration

determined by the calculation of: 100% minus the other components to be

included.

Preferably, the oil may be liquid paraffin. However, this should not be seen as

limiting, as one skilled in the art would be aware that a range of different oils may

be utilised, for example in some instances vegetable or mineral oil may be utilised.

The relationship between the ratio of oil to barium sulphate, and the particle size

and shape of the barium sulphate are factors considered to be important to the

effectiveness of the formulation of the present invention, as they affect how the

formulation packs into a cohesive mass in the teat canal or/and lower portion of

teat cistern, and irritability of the formulation.

It will be appreciated that the preferred physical characteristics of the formulation of

the present invention may be provided by the barium sulphate, along with at least

one oil and a gelling agent.

In some further preferred embodiments, the formulation may also include other

additives to provide the required consistency, physical properties or behaviour. For example, the formulation may include a thickening agent, such as Aerosil 200, and/or preservatives, such as methylparaben (as free acid) and/or propylparaben

(as free acid).

The applicants consider the rheology of the formulation is important, this includes

such features as the ability to flow under high shear forces, being sufficiently fluid

that once administered it spreads to form a reasonable seal with the side of the

teat canal or/and lower portion of teat cistern and at least some elastic properties

to allow the formulation to be able to move and flex with movement of the teat

canal or/and lower portion of teat cistern.

The present invention overcomes the inconsistent effectiveness of previous

formulations and formulations used in combination with bacteriocins.

Another of the major advantages of the present invention is that the formulation

provides a physically stable product. This is important, as it ensures the physical

barrier to be effective over a set period of time. Physically stable should be taken

to include the characteristics of minimal dispersion of the formulation.

This is a significant advantage over previous attempts, such as the use of

bacteriocins. Products containing bacteriocins are not physically stable, which

leads to a lack of persistence within the teat canal of the teat. This may be due to

the formulation being used in conjunction with bacteriocins being an emulsion

which therefore has low elasticity, resulting in easier dispersion. In fact, it has

been found that formulations containing bacteriocins totally disperse or break down

with approximately eight days of administration. Further, no long-term studies or

farm studies have been reported for either lacticin or other bacteriocins.

It is expected that when either antibiotics or bacteriocins -are used at a high

concentrations (or designed to provide a high local anti-bacterial concentration) with an internal physical formulation further problems and disadvantages may be experienced. Bacteriocins may not be localised within the area of the formulation, and indeed all previous work has been directed towards increasing the release of the bacteriocin from the formulation. The applicant believes that the bacteriocin peptides may be metabolised or degraded by the animal, and the products of same may be absorbed into the animal's body.

Therefore, while bacteriocins may be present within any gaps or channels between

the formulation and the side of the teat canal, they are not specifically targeted to

this area, nor are they limited to this area. Because of the more general release of

bacteriocins, they probably only last a short time in the channel before being

metabolised/degraded, it has been reported that there is no recovery of

bacteriocins from formulations eight days after administration.

The present invention overcomes problems with existing formulation/chemical

preparations by providing an antiseptic which is targeted to and limited to the area

between the formulation and the side of the teat canal or/and lower portion of teat cistern. This is advantageous as it provides a consistent and effective barrier, both

physical and chemical across the entire teat canal or/and lower portion of teat cistern, either preventing the passage of micro-organisms into the teat cistern or

treating those that are present in the cistern. Limiting the antiseptic to this small area decreases the amount of antiseptic required to provide an effective concentration. This lowers the cost of the formulation, and as the antiseptic is

limited to one area of relatively high concentration, the possibility of the micro organisms becoming resistant to the antiseptic is significantly reduced.

The present invention therefore provides a number of advantages over existing

formulations and methods of preventing bovine mastitis during the dry period

currently available, these include the following:

• Acts in both a physical and chemical manner,

* Acts to specifically target and prevent the migration of micro-organisms

through a channel or moisture film present between the formulation and the

side of the teat canal or/and lower portion of teat cistern, or treats those

micro-organisms that enter the cistern

• Makes use of an antiseptic which is not absorbed by the body cells, this

decreases the concentrations required, ensures specific localization and

targeting and prevents the antiseptic getting into the milk flow,

* Provides more consistent and effective prevention of mastitis during a dry

period and the drying off period.

• Decreases the possibility of further micro-organisms being introduced

during administration because if a few are introduced they will be killed by

the antiseptic.

• It can kill micro-organisms which are:

- existing in teat canal (fissures) or/and lower portion of teat cistern

- introduced during infusion

- ascending during dry period

* The applicant believes that the finished product may not need sterilization;

BRIEF DESCRIPTION OF DRAWINGS

Further aspects of the present invention will become apparent from the following

description which is given by way of example only and with reference to the

accompanying drawings in which:

Figure 1 shows how the zone of inhibition was measured;

Figure 2 shows graphs comparing of the drug (antiseptic) release rate from

different formulation matrix (F16 and F17) indicated by the

antibacterial activity F16-2 and F17-2 contain 0.5% CH HCI and F16

4 and F17-4 0.5% CHX base;

Figure 2a shows graphs relating to testing of the formulation against S.

aureus;

Figure 2b shows graphs relating to testing of the formulation against S.

pyogenes;

Figure 3 shows graphs comparing of the drug (antiseptic) release rate from

formulations (F16 and F17) indicated by the antibacterial activity.

F16-1 and F16-2 contain 0.15% and 0.5% CHX HCI respectively and

F16-3 and F16-4 contain 0.15% and 0.5% CHX base respectively;

Figure 3a shows graphs relating to testing of the formulation against S.

aureus;

Figure 3b shows graphs relating to testing of the formulation against S.

pyogenes;

Figure 4 shows graphs comparing the antibacterial activity against S. aureus (left) and S. pyogenes (right) of CHX HCI salt (0.15% in F16/F17

land 0.5% in F16/F17-2) and CHX base (0.15% in F16/F17-3and

0.5% in F16/F17-4);

Figure 5 shows a graph illustrating the cumulative release profiles of chlorhexidine from F 17-4 and F 16-4;

Figure 6 shows a graph illustrating the release rate profiles of chlorhexidine from F 17-4 and F 16-4;

Figure 7 shows a graph illustrating the post challenge combined udder mean

scores by day;

Figure 8 shows a graph illustrating the incidence of clinical mastitis per group;

Figure 9 shows a graph illustrating the group average somatic cell counts

data post calving;

Figure 10 shows average clincal scores per group

BEST MODES FOR CARRYING OUT THE INVENTION

Examples are now provided, describing the invention. It should be appreciated

that the following best modes are given by way of example only.

EXAMPLE 1: FORMULATION AND MANUFACTURE - NO THICKENING

AGENTS

Table 1 below provides a summary of embodiments of the formulation.

Table 1 - Formulation Summary

Ingredient Invention Trial Trial Ingredient Formulations Formulation I Formulation 2 Function

(% w/w) (% w/w) (% w/w)

Chlorhexidine 0.15-0.5 0.25 0.5 Active base or HCI ingredient/ salt (can be antiseptic other salt types)

Barium 67 67 67 Barium Sulphate base compound

Liquid paraffin Approximately Approximately Approximately Vehicle 30 30 30

Aluminium 5.5 - 6 % of oil Approximately Approximately Gelling stearate 1.8 % of final 1.8 % of final agent Approximately product product 1.8 %of final product

As noted above in Table 1, the key difference between Formulation 1 and 2 is the

amount of Chlorhexidine (CHX) added.

A manufacturing process to produce the above formulations is now described.

1. Weigh out the heavy liquid paraffin and aluminium stearate,

2. Add aluminium stearate to the liquid paraffin, and stir till a smooth

suspension with no lumps forms,

3. Heat the mixture from step 2 and allow the temperature to rise to 1500C,

4. Hold the temperature at approximately 1500C for Ihour (to sterilise),

5. Cool the mixture from step 4 to below 40°C, stir and homogenize until a

smooth base is formed

6. Weigh out, and add chlorhexidine, mix by stirring and then homogenize to

ensure the chlorhexidine is homogenously dispersed throughout the base,

7. Weigh out, and add barium sulphate to the gel base, stir to mix,

8. Keep stirring till a smooth paste is formed. Homogenize if necessary.

In vitro studies using the above formulations have shown that limited release of

chlorhexidine occurs during time. This is desired in the present invention where

only a small volume of chlorhexidine is required to provide an antiseptic

concentration of chlorhexidine in the required area.

EXAMPLE 2: FORMULATION AND MANUFACTURE - INCLUDING

THICKENING AND PRESERVATIVE AGENTS

Table 2 below outlines the preferred formulations, with the inclusion of thickening

and preserving agents.

Table 2 - Formulations with Thickening and Preserving Agents

Ingredient Concentration Function (g/kg)

Chlorhexidine (as base) 5.0 Active ingredient/ antiseptic

Barium Sulphate 670.0 Barium base compound

Heavy liquid paraffin 300.4 Vehicle

Aluminium stearate 15.4 Gelling agent

Aerosil 200 8.6 Thickening agent

Methylparaben (as free acid) 0.4 Preservative

Propylparaben (as free acid) 0.2 Preservative

A manufacturing process to produce the formulations above is now described.

1. Weigh out heavy liquid paraffin, barium sulphate, aluminium stearate, Aerosol 200, methylparaben, and propylparaben;

2. Heat up liquid paraffin to 80°C. While holding the temperature, add barium sulphate, aluminium stearate, Aerosol 200, methylparaben, and

propylparaben, and into liquid paraffin with agitation; continue stirring until a smooth suspension is formed;

3. Heat the mixture from step 2 and allow the temperature to rise to 150°C;

4. Hold the temperature for 2 hours with containers lidded (sterilisation). No

stirring is required;

5. Cool the mixture to around 400C, stir the gel making it smooth;

6. Weigh out and add chlorhexidine (sterile) to the gel from step 5, mix

thoroughly by stirring, then homogenize to ensure the active is well dispersed

and the gel is smooth with no visible particles in the product;

7. Test syringibility on the second day of completion of step 6. Adjust viscosity

if required;

8. Fill the teat seal into syringes.

This formulation is tested in the in vivo studies described in Examples 5 to 7 below.

EXAMPLE 3: BACTERIAL CHALLENGE TESTING RESULTS OF THE BARIUM

TEAT SEALANTS

The aim of this study was to predict the optimal antiseptic loading concentration in

the formulation that can produce a chemical barrier to the bacteria ascending through the teat canal.

Another aim was to specifically look at factors affecting antiseptic release rate, including drug concentration, chemical form (salt or base) and formulation.

Chlorhexidine at higher concentrations (>100pg/ml) acts as a bactericide to most ofthebacteria. At lower concentrations chorhexidine (1-100pg/ml) acts as a

bacteriostatic agent (European Medicines Agency: EMEA).

Ideally, the formulation should contain such amount of chlorhexidine that after administration into the teat canal or/and lower portion of teat cistern, it will have a bactericidal concentration. An excessive higher concentration should be avoided as it will cause tissue irritation along with adding to the cost of the formulation.

The formulations tested in this study are shown in Table 3 below. These

formulations and method of manufacture are also described in Example 1 above.

Table 3: Formulations of the barium teat seal tested

F16-1 F16-2 F16-3 F16-4 F17-2 F17-4

Chlorhexidine

form HCI salt HCI salt Base Base HCI salt Base

Concentration

(%, w/w) 0.15 0.5 0.15 0.5 0.5 0.5

Paraffin gel

matrix with

BaSO4 F16 F16 F16 F16 F17 F17

Note that F16 and F17 contain 6 and 5.5 % aluminium stearate respectively (in

relation to the paraffin oil).

Challenge testing of the six teat seal formulations was carried out using

Staphylococcus aureus (ATCC-6538) and Streptococcus pyogenes (ATCC-12344),

as these organisms are related to mastitis, and therefore provide realistic

challenge organisms. Tryptic Soya Agar was used in both the disc assay and the

direct inoculation testing.

Aqar diffusion test

First an agar diffusion test was conducted. Here, a 13mm disc was cut from the

agar (Tryptic Soya Agar) and 0.5g of sample was dispensed into the well. The diameter of the zone of inhibition was measured with callipers to the first colonies observed around the disc.

Measurement of the zone of inhibition was measured as shown in Figure 1.

The results from this test showed the zone of inhibition against Staphylococcus

aureus was only observed in test samples: F16-4 (6.5 mm) and F17-4 (5.4mm).

Therefore showing these test samples are more effective at killing the bacteria.

Direct inoculation

In this test, 2g of the formulations were used in the direct inoculation of 0.02ml of

bacterial suspension (5.8 *105 cfu/ml), then mixed with 2.7ml Tryptic Soya

Broth(lecithin (0.5 %) Tween 20 (4%) and peptone (0.1%)) by vortexing for 30

seconds.

The mixture was dispensed in a plate and cultured in Tryptic Soya Agar (volume to

Tryptic Soya Broth was 20 to 15).

For each formulation, 6 samples were prepared and the recovery level of bacteria

(cfu/ml) at 0, 1, 3, 6, 24 and 48 hours were measured respectively.

The results of the direct inoculation tests are presented in Tables 4 and 5 below,

along with Figures 2, 3 and 4.

Table 4: Microbiological challenge test for antimicrobial properties by direct

inoculation of Staphylococcus aureus (ATCC-6538). The inoculum level was

approximately 5.8 x10 5cfu/ml.

Time (hr) F16-1 F16-2 F16-3 F16-4 F17-2 F17-4

0 1.1 0.9 1.3 1.7 0.9 1.3

1 2.3 2.7 2 3.3 2.9 3

3 2.5 2.5 3.1 4.3 3.5 5.8

6 3.1 3.2 2.8 4.8 3.5 5.8

24 3.8 3.1 5.8 5.8 4.8 5.8

48 3.2 2.9 5.8 5.8 3.6 5.8

Data are expressed as Log Reduction: Log Reduction = log (inoculum

level/recovery level) assumed 0 =1).

Table 5: Microbiological challenge tests for antimicrobial properties by direct

inoculation of Streptococcus pyogenes (ATCC-12344). The inoculum level was

approximately 6.6 x105 cfu/ml.

Time (hr) F16-1 F16-2 F16-3 F16-4 F17-2 F17-4

0 1 0.8 1.3 1.7 0.9 1.8

1 2 3.1 2.4 2.8 2.9 3.7

3 3.1 3.9 3.1 5.8 3.5 5.8

6 3.8 4.1 3 5.8 3.4 5.8

24 5.8 4.5 4.3 5.8 5.8 5.8

48 3.4 5.8 5.8 5.8 5.8 5.8

Data are expressed as Log Reduction: Log Reduction =log (noculum

level/recovery level, assumed 0 =1).

These results show the different formulation matrix's (F 16 and F17) had a different

drug release rates. This can be observed in Figure 2. As indicated, the same

drug, at the same concentration (see samples F17 (F17-2 and F17-4)) produced

faster release profiles than F16 (F16-2 and F16-4). The difference can be

observed in the results of the challenge test against S. aureus (Figure 1A).

The results also showed that the log reduction over 48 hours increased with the

increase of concentration of chlorhexidine base (0.15% vs. 0.5%, P=0.056).

However, there was very little difference between the two loading levels of

chlorhexidine salt (by Staph test).

The difference in effect between the two chemical forms of chlorhexidine (base and

salt) is also shown in the results. The formulations with chlorhexidine base

produced an increasing log reduction-time profile over the 48 hours against S.

aureus and S. pyogenes as shown in Figure 4. The chlorhexidine base at 0.15

0.5% in both formulation matrix acted as a bactericide as there was no bacterial

recovered (log reduction 5.8) at 3- 24 hours after inoculation of the bacteria. F17-4

produced fasted bactericidal activity with no bacterial recovery at 3 hours. The

base formulations produced a higher antibacterial activity compared with the

corresponding salt formulations. Only F16-4 and F17-4 which containing 0.5%

chlorhexidine base produced a zone of inhibition against the bacteria.

In comparison, formulations that contained chlorhexidine salt, except for F16-2 and

F17-2 against the bacteria which containing higher concentration of drug, showed

an increase in the log reduction from 0 to 24 hours, with a decline of log reduction

at 48 hours, indicating a bacteriostatic rather than a bactericidal effect had taken

place. This is due to the slow release of chlorhexidine salt and the local drug

concentration was low and could only produce a bacteriostatic action.

The results in most of the cases showed that S. pyogenes was more sensitive to

chlorhexidine than S. aureus. In summary, the chlorhexidine base appeared to have the advantage over the corresponding HCI salt, as it was easier to release

and to reach the bactericidal concentration desired. The F17 formulations showed

a quicker release of the active integrant than the F16 formulations.

EXAMPLE 4: IN VITRO RELEASE STUDY BY HPLC ASSAY

To test the release of formulations F17-4 and F16-4, the method below was

followed: 1. Place 2g formulation in the cap (C=2cm)

2. 2g formulation forming a slab (3.14cm2 x 3.4mm)

3. Release medium: 10 ml water

4. Reverse the vials and place the vials vertically in a 350 C water shaking

bath

5. HPLC assay for the release medium by an ion-paired HPLC method

with UV detector (254nm).

The results of these tests are shown in Figures 5 and 6. As shown, a burst of

release of the formulation (in both F17-4 and F16-4) was observed in the first 1

hour, with a gradual decline thereafter. The release rate after the first 24 hours

was minor.

EXAMPLE 5: CHALLENGE STUDY FOR THE ASSESSMENT OF MASTITIS

PREVENTION

This study is a 'challenge study'to assess the biocompatibility (chronic irritability

and acceptability) and efficacy against experimental microbial challenge of the

formulation of the present invention (ATS-Barium) (see Example 2 for composition

and manufacture) in comparison with the existing internal teat sealant product

30 `4

Teatseal TM, marketed by Pfizer Animal Health (Teatseal), in 'drying off' dairy cattle.

Cattle Selection

None of the animals in this test had received any antibiotic preparations within 30

days of study initiation (as confirmed by inhibitory screening test on day -6). No

animals were found to be debilitated, suffering from disease or injury, fractious or otherwise unsuitable for inclusion in the study, in the opinion of the Investigator on

day -6.

The health status of the mammary gland was checked by palpation and RMT on

day -6. Animals with palpable changes in any of the quarters on day -6 were

excluded from the study. Some animals had a very mild precipitation on RMT Test

but were included if they had an acceptable somatic cell count (SCC) from a

sample collected on the same day.

A herd test including a SCC was conducted on day -12. Animals with counts

>200,000 cells/mL for cows were not assigned to the study. A follow-up SCC was

conducted on day -6 to ensure counts remained under 300,000 cells/mL

Eligible cows with the lowest SCC and with four functional quarters, free of clinical

signs of mastitis or teat abnormalities were assigned to study.

The animals were ranked according to ascending somatic cell count data (Day -6

sample). Every seventh animal (n=6) was assigned as an untreated control. The

remaining 42 animals were randomly assigned to treatment groups, as follows:

Group 1 (n=14), were treated with ATS-Barium; Group 2 (n=14), were treated with

Teatseal.

Procedure

Table 6 below outlines the timeline of events during the present investigation.

Table 6 - Timeline of events Study Day Event -10 Acclimation, Start daily health observations -6 Screening for study assignment: Milk collection for inhibitory substance and SCC, clinical observations, RMT, udder palpation -1 Allocation -4 &0 Sterile milk sampling for pre challenge microbiological assessment. o Groups 1 &2 treatment with teat sealant formulations followed by teat disinfection &control teat disinfection only 2 &4 Challenge with S. uberis broth 2, 4 to 34 All animals'daily clinical observation of quarters for mastitis. All animals with clinical mastitis sterile milk samples for microbiological analysis and treatment Study end Collection of the remaining teat seal by stripping off the teats individually. (at calving) Sterile milk sample for microbiological assessment and milk sample Day 0 SCC test. calving Day 4 Sterile milk sample for microbiological assessment and milk samples for SCC and SAITL inhibitory substances test Day 0 to 10 Recording if any flecks present on the filter socks

Pre-treatment milk sampling

Four days prior to and on the day of drying-off (Day 0), single-quarter milk samples

were collected 'aseptically' into sterile P35 plastic containers. Before sampling, the

teats were wiped with proprietary teat wipes (teat wipes containing ethanol 70%,

chlorhexidine 0.1%, and cetrimide 0.16%). Front teats were cleaned first. Once the

teats were clean, care was taken to avoid contact with the tail, feet and legs. The

teats were allowed to dry for several seconds before sampling. Disposable latex

gloves were worn throughout the milk sampling procedure and changed between

cows. To minimise cross-contamination, milk samples were taken first from the

rear teats and then from the front teats. The first two to three squirts of milk were

discarded and samples taken into sterile plastic containers P35 with watertight lids. The samples were frozen for later microbiological examination.

32 O'

Treatment Administration

The ATS-Barium formulation tested in this Example is described above, under

heading Example 2.

Prior to application of treatments and starting with teats on the most distant part of

the udder, teats were wiped, using the method as discusses above.

For each animal assigned to groups 1, 2 and 3 all four teats were treated with one

of the test formulations. The control group was untreated animals. Table 7

outlines the treatment group, drug and number of animals tested.

Table 7 - Overview of the test groups Treatment Drug Route No. of Animals/ Groups Treatment Group 1 ATS Barium One tube/ syringe per quarter at drying IMM 14 off. 2 Teatseal "vPfizer Animal Health One tube/ syringe per quarter at drying IMM 14 off. 3 Untreated control N/A 6

Overall Total 34

After application of treatments, all teats including those of control group cows were sprayed with diluted iodine teat spray (Teatguard plus - Ecolab Ltd, an iodophore

based teat sanitizer with 23 g/L active iodine).

Challenge

The challenge was undertaken as below and described previously by Fernandez

(Fernandez, C. Masters degree thesis Massey University 2007 - in preparation).

All cows were exposed to the challenge broth, containing S. uberis, strains

previously isolated-in New Zealand on two occasions, two and four days after dry

off, by dipping of each teat, entirely, for 1-2 seconds.

The microbial suspension contained 1.031 x 108on the day 2 challenge and 1.27 x

108on day 4 challenge.

Administration of S. uberis

To reduce the milk letdown response and consequent opening of the teat

sphincter, the experimental challenge was performed in separate facilities from the

normal milking shed. Each cow was identified by ear tag and checked for the

presence of mastitis. Each of the four teats was dipped in microbial suspension

broth for 1-2 seconds and the cows released.

To minimise cross-contamination, the challenge was started from the distant teats

and then the nearest teats. One container was used for all four teats in each cow.

Post-challenge Clinical Assessments

All quarters were examined daily, with exception of days 1 and 3 post-treatment, by

an experienced dairy technician for the presence of mastitis starting on the day of

the first microbial challenge and continuing until the last observation on day 34.

Individual quarters were observed and palpated for the clinical signs associated

with mastitis, i.e. heat, swelling, redness, swollen lymph nodes, painful quarter/s

and if required, by an examination for the presence of clots and flakes in the

secretion.

Each quarter was subjectively judged as mastitic or non-mastitic according to the

criteria below in Table 8. The personnel making assessments of clinical mastitis

was blinded to the treatment of each quarter.

Examinations were also performed observing the teat for evidence of irritation.

Mastitic quarters were sampled for microbiological culture before treatment was

initiated. After sampling each affected quarter was treated as for lactating cow

clinical mastitis with penicillin based antibiotics.

Both udder and teat examinations were scored using the following parameters

outlined in Table 8:

Table 8 - Ratings for Pain or Distress

Pain or distress:

0 = No or virtually no stress, irritation, pain, erythema or oedema

1 = Stress, irritation or pain of a minor intensity or slight erythema or oedema

2 = Stress, irritation or pain of a moderate intensity or moderate erythema or oedema

3 = Stress, irritation or pain of a severe intensity or severe erythema (beet redness to slight eschar formation) or severe oedema (raised more than 1mm and extending beyond the area of exposure)

4 = Severe eschar and/or corrosion

A score of 3 was determined to be the score for calling an udder as mastitic.

Allowance was made by the experienced person during examinations for any udder

enlargement taking place as part of the normal drying-off process.

Any affected quarter that was found with a score of 3 or over was determined to be

clinical mastitis and subsequently treated.

Prior to treatment, all clinically mastitic quarters were sampled for bacteriological culture in same way as described for pre-treatment sampling. The quarters were

then treated under supervision of a veterinarian with an intramammary product

(Ubro yellow, Boehringer Inge/heim) once daily after complete milking out of the

affected quarter. If all four quarters were affected in the same cow then injectable

product was used (Mamyzin, Boehringer /nge/heim,) once a day in following dose

rate: first day 10g and two consecutive days 5 g a day intramuscularly. Quarters were continued to be observed daily and treated as appropriate but any subsequent mastitis was not included in statistical measurements.

Post Calving Study-end procedures

For the first ten days post calving, filter socks were checked for presence of flecks.

For the first 21 days, observations reported were minor non-specific flecks. There

were no further comments from observations.

Aseptically taken milk samples were collected by quarter for microbiological

analysis on days 0 and 4 after calving. Samples were as per collection procedures

described above. Samples were taken on a quarter level for somatic cell counts on

days 0 and 4 and inhibitory substances on day 4 following the micro sampling.

Samples were taken by filling a P35 sample pottle by manual stripping of each

quarter.

Results

The test items and positive control were able to significantly prevent the

development of S. uberis mastitis subsequent to challenge.

Infection rates (No. with positive ('+ve') S. uberis infection in dry period/no. of

quarters) in the treatment groups were ATS-Barium 1/56 and Teatseal was 0/56

quarters.

The infection rate in the control group was 11/24 quarters.

The test items did not show any evidence of irritability as measured by daily teat

examinations up to day 34 post-treatment, and somatic cell counts after calving.

The average somatic cell counts were lower for ATS-Barium on both days 0 and 4

comparable to the negative and positive control groups.

Figure 7 shows examination scores for clinical Mastitis on days post treatment (i.e.

during the dry period). Tables 9 to 14 below outline various data from the

assessments made. Table 9 shows a summary of the number of Microbiological

cultures (clinical cases) prior to calving and daily udder check scores (>3). Table

10 shows the microbiological culture results from samples of cows with udder

exam scores >3 during Days 2-34. Table 11 shows a summary of microbiological

culture results of all quarters including those previously treated, post-calving.

Table 12 shows a summary of microbiological culture results, all quarters including

those previously treated post-calving. Table 13 shows an average daily udder

lo exam scores by group by day. Table 14 shows an average Somatic Cell Counts, all

quarters including those previously treated at calving.

Table 9 - Summary of Microbiological cultures (clinical cases) prior to calving and daily udder check scores (>3)

No./qtrs with positive No./qtrs with scores 3 Group Treatment No. of quarters strep uberis or greater

ATS 1 Barium 56 1 1

3 Teatseal 56 0 2

4 Control 24 11 14

Table 10 - Microbiological culture results from samples of cows with udder exam scores >3 Days 2-34

Cow number Quarter Prel. bacteriol result Group Treatment

29 BR S.uberis 4 Control

74 FL S.uberis 4 Control

160 FR S.uberis 4 Control

160 FL NEG 4 Control

164 BR S.uberis 4 Control

2 BR S.uberis 4 Control

89 BL S.uberis 4 Control

2 BL NEG 4 Control

29 FL S.uberis 4 Control

29 FR S.uberis 4 Control

89 BR S.uberis 4 Control

89 FL S.uberis 4 Control

89 FR S.uberis 4 Control

124 BL S.uberis 1 ATS-Barium

29 LH Strep not uberis 4 Control

152 LH Strep not uberis 3 Teatseal

Table 11 - Summary of microbiological culture results post-calving (all quarters including those previously treated)

Day 0

Negative S. uberis Bacillus Staph Contam Coryne

Control 17 1 2 0 4 0

Teatseal 32 0 7 5 8 0

ATS-Barium 35 0 8 3 6 0

Table 12 - Summary of microbiological culture results post-calving (all quarters including those previously treated)

Day 4

Negative S. uberis Bacillus Staph Contam

Control 16 1 2 1 4

Teatseal 18 0 9 0 20

ATS-Barium 28 0 9 2 8

Table 13 - Average daily udder exam scores by group by day

Group/Day 2 4 5 6 7 8 9 10 11 12 13

ATS Barium 0.00 0.16 0.32 0.20 0.14 0.13 0.16 0.14 0.11 0.20 0.18

Teatseal 0.00 0.16 0.30 0.34 0.23 0.45 0.29 0.29 0.40 0.46 0.33

Control 0.00 0.04 0.42 0.63 0.58 0.54 1.00 0.67 1.42 1.33 0.88

Group/Day 14 15 16 17 18 19 20 21 22 23 24

ATS Barium 0.07 0.11 0.04 0.11 0.04 0.04 0.02 0.04 0.04 0.07 0.02

Teatseal 0.38 0.25 0.13 0.17 0.21 0.15 0.23 0.21 0.15 0.13 0.15

Control 0.67 0.42 0.54 0.38 0.38 0.29 0.33 0.17 0.25 0.21 0.17

Group/Day 25 26 27 28 29 30 31 32 33 34

ATS Barium 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02

Teatseal 0.17 0.13 0.15 0.15 0.12 0.15 0.13 0.08 0.13 0.12

Control 0.38 0.21 0.29 0.21 0.10 0.17 0.17 0.13 0.04 0.08

Table 14 - Average Somatic Cell Counts at calving (all quarters including those previously treated)

Average No.of No.of Group Treatment quarters Day 0 quarters Average Day 4

1 ATS-Barium 51 1085 52 234

3 Teatseal 49 2657 52 310

4 Control 24 4240 20 584

EXAMPLE 6: EFFICACY STUDY BETWEEN ATS-BARIUM AND TEATSEALTM

This study assessed the efficacy of the treatment, during normal conditions where

the cows are kept. This study involved treating the cows with TeatsealT,

marketed by Pfizer Animal Health (Teatseal) or the formulation of the present

invention (ATS-Barium), at drying off and then leaving the cows in standard

grazing conditions.

Method

The ATS-Barium formulation tested in this example is described above under

Example 2 heading.

Prior to application of treatments and starting with teats on the most distant part of

the udder, teats were wiped, using the previously discussed method.

The test articles were formulations manufactured commercially or in a commercial

manufacturing facility.

Confirmation of administration was recorded against the unique animal

identification and included in the raw data.

For each animal assigned to groups 1, 2 and 3 all four teats were treated with one

of the teat seal products. The control group was untreated animals. The numbers

of cows per group and in total are presented in Table 15.

Table 15 - Group and total number of cows calved up to day 84 post treatment

Group Number of cows ATS-Barium 107 Teatseal 116 Control 89 Total 312

Results - Incidence of clinical mastitis

The incidence of clinical mastitis per group and in total up to 30 days post calving

is shown in Table 16 and Figure 8. The number of cows with clinical mastitis

between ATS-Barium and Teatseal did not differ significantly (p=0.176), while there

is a statistical difference between the ATS-Barium and non-treated control group

(p=0.044). The data of clinical mastitis is based on cases occurring in the early

period post-calving. There were no cases of clinical mastitis during the dry period

in any of the groups.

Table 16 - Incidence of clinical mastitis per group up to 30 days post calving

Group Clinical mastitis Cows per group Percent ATS-Barium 8 107 7.5 Teatseal 7 116 6 Control 16 89 18 Total 23 312 9.9

Results - Somatic cell counts data

Average somatic cell counts per group at three time points post calving (Day 3,

Day 7 and Day 10) are presented in Table 17 and Figure 9.

Table 17- Raw somatic cell counts data per group and in total Day3 Day7 Day10

Group Cows Cows Cows SCC tested SCC tested SCC tested ATS Barium 566.570 105 485.617 115 342.531 96

Teatseal 590.113 115 357.040 125 238.481 108 Control 1.054.437 88 371.135 89 446.072 83

Total 737.040 308 404.597 336 342.362 297

Somatic cells are affected by a few factors, such as stage of lactation, age of the

cow, previous year somatic cell counts, dry cow treatment, stress, and finally

mastitis. In the present study, cows were blocked by results of previous year age

and somatic cell counts, trying to avoid confounding of these factors. As expected,

there is exponential decrease of somatic cells post calving in all three groups, with

ATS-Barium and Teatseal groups demonstrating similar results, while untreated

animals (control group) have higher average somatic cell counts.

All quarters were examined daily, with exception of days 1 and 3 post-treatment, by

an experienced dairy technician for the presence of signs of irritation or mastitis

from the time of treatment until 34 days later. Individual quarters were observed

and palpated for the clinical signs associated with mastitis, i.e. heat, swelling,

redness, painful quarter/s and if required, by an examination of the secretion.

Each quarter was subjectively judged as mastitic or non-mastitic according to

described criteria. Mastitic quarters were sampled for microbiological culture before treatment was initiated. After sampling each affected quarter was treated

as for lactating cow clinical mastitis with penicillin based antibiotics. Cows treated

with the test items (barium and chlorhexidine; and bismuth and chlorhexidine) or

Teatseal demonstrated significantly lower scores on clinical examination compared

to the cows in the negative control (untreated) subsequent to challenge.

This demonstrated that barium plus chlorhexidine product provides effective sealant in early dry period with no irritability recorded.

The results are illustrated in Figure 10.

The viscosity of the present invention was tested against that of other teat seals. It is important to ensure that a viscosity is obtained that allows ready application of the paste to the teat canal, yet forms a cohesive mass therein.

Results - Viscosity range measurement of the A TS-Barium paste

The viscosity range of the ATS-Barium was measured using a Brookfield LVT viscometer on Helipath stand, Spindle T-F, at 20°C. The ATS-Barium tests were compared to the Pfizer Teatseal in the table below. The preferred viscosity of the ATS-Barium ranges from 800,000 to 3,000,000 cps at 0.3 rpm.

Test sample RPM 1 st 2 Average Factor Viscosity reading reading reading (cps)

ATS-Barium 1 0.6 100+ 100+ 100+ 15600 0.3 94 93 93.5 31200 2917200 0.6 56 55 54 15600 842400 ATS-Barium 2 0.3 36 36 36 31200 1123200 0.6 48 46 47 15600 733200 ATS-Barium 3 0.3 31 31 31 31200 967200 Teatseal (Pfizer) 1 0.6 100+ 100+ 100+ 15600 0.3 77 78 77.5 31200 2418000 0.6 53 53 53 15600 826800 Teatseal (Pfizer) 2 0.3 31 31 31 31200 967200 0.6 100+ 100+ 100+ 15600 -- Teatseal (Pfizer) 3 0.3 74 76 75 31200 2340000

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.

Claims (16)

WHAT WE CLAIM IS:

1. A formulation, for administration to the teat canal or/and lower portion of

teat cistern of a mammary gland of an animal, wherein the formulation prior

to delivery is in the form of a paste, the formulation including:

an oil-based physical barrier material which is able to form a cohesive mass

in the teat canal and/or the lower portion of the teat cistern, and

at least one antiseptic compound mixed with the barrier material.

2. A formulation as claimed in claim 1 wherein the physical barrier material

includes barium.

3. A formulation as claimed in claim 2 wherein the barium is in the form of a

barium salt.

4. A formulation as claimed in claim 3 wherein the barium is in the form of

barium sulphate.

5. A formulation as claimed in either claim 3 or claim 4 wherein the barium is

micronised.

6. A formulation as claimed in any one of claims 1 to 5 wherein the antiseptic

is in the form of chlorhexidine or salt thereof.

7. A formulation as claimed in any one of claims 1 to 6 which includes a

carrier.

8. A formulation as claimed in claim 7 wherein the carrier is in the form of oil.

9. A formulation as claimed in claim 8 wherein oil is paraffin oil.

10. A formulation as claimed in claim 7 wherein the carrier is a gelling

compound.

11. A formulation as claimed in claim 10 wherein the gelling compound is

aluminium stearate.

12. The use of formulation as claimed in any one of claims 1 to 11 in the

manufacture of a medicament to prevent or ameliorate mastitis.

13. A method of treating or preventing infection within the teat cistern of a

mammary gland characterised by the step of administering a formulation as claimed in any one of claims 1 to 11 into the teat canal or/and lower portion

of teat cistern of the mammary gland of a non-human animal.

14. The formulation substantially as herein described with reference to Tables 1

- 3 of the "Best Modes" section.

15. The use of a formulation substantially as herein described with reference to

and as illustrated by Examples 3 - 6 of the "Best Modes" section.

16. A method substantially as herein described with reference to and as

illustrated by Examples 1 and 2 of the "Best Modes" section.