WO2000023085A1

WO2000023085A1 - Mastitis treatment with particulate glucan

Info

Publication number: WO2000023085A1
Application number: PCT/AU1999/000910
Authority: WO
Inventors: Graham Edmund Kelly; Alan James Husband
Original assignee: Novogen Research Pty. Ltd.
Priority date: 1998-10-19
Filing date: 1999-10-19
Publication date: 2000-04-27
Also published as: AUPP659198A0

Abstract

A method for the treatment and prevention of mastitis is described which comprises administering to a mammary site a composition including particulate glucan in association with a physiologically acceptable carrier and optionally an excipient. Also described are compositions for the treatment of mastitis.

Description

MASTITIS TREATMENT WITH PARTICULATE GLUCAN

The present invention relates to processes for the treatment of mastitis and compositions for effecting the same in the treatment of mastitis. 5

BACKGROUND OF THE INVENTION

Mastitis is a disease condition which effects the preponderance of lactating mammals. Mastitis presents serious economic consequences in commercial milk producing 10 operations, such as those involving cows, sheep and goats, which may collectively be regarded as the "dairy industry".

Bovine mastitis (subclinical and clinical) is a disease complex that results from the interaction of the cow, the environment (including the milking machine), and

15 microorganisms. It is a major cause of loss in milk production to the dairy industry. Many of the costs of mastitis are unrecognised, but a single infected gland may result in a loss of 770 kg of milk per year from an animal. Major losses from mastitis include decreased milk production, increase in replacement of heifers, loss of antibiotic-contaminated milk, drug and veterinary costs, extra labour and loss of genetic material from the herd (Blosser,

20 1979). One of the dominant etiological pathogens contributing to economic loss is Staphylococcus aureus (McDonald, 1977). Although antibiotic therapy can be effective in the control of mastitis from a variety of organisms, it is only moderately efficacious for S. aureus infections and requires a 3 to 5 days milk withdrawal (Aungier and Austin, 1987). Improved mastitis therapeutics remain a major unfilled need of the dairy industry.

25 Treatments that do not introduce antibiotics into the food chain offer great advantage.

S. aureus mastitis infection is characterised by the presence of viable bacteria in the milk and elevated numbers of somatic cells in the mammary gland and milk (Anderson, 1982; Craven and Williams, 1981; Daley et al. 1991; Fox and McDonald, 1988). In an infected 30 gland, these host cells are >95% polymorphonuclear leukocytes (PMN). The development of chronic S. aureus mastitis depends on the interaction between invading bacteria and these cells of the defence system (Derbyshire and Berman, 1968; Paape and Wergin, 1977). Although the defence mechanisms of mammary gland can be effective in eliminating the infection from mammary gland as much as 3 to 4 logs of bacteria. It has been suggested that less activated PMN may contribute to the relapse of an existing infection and the chronic cyclic nature of S. aureus mastitis (Daley et al. 1991). Furthermore, once organisms have been phagocytosed, these may be protected from the effects of antibiotic therapy (Craven and Williams 1981; Madgwick et al 9189; Russell and Reiter. 1975).

The treatment of mastitis in other dairy animals presents essentially the same problems as those set out above.

A clear need exists for processes and compositions for the treatment of mastitis.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the invention there is provided a process for the treatment of mastitis which comprises administering to an affected mammary site of a lactating mammal a composition including paniculate glucan in association with a physiologically acceptable carrier and optionally an excipient.

In another aspect of the invention there is provided a composition for the treatment of mastitis which includes particulate glucan in association with a physiologically acceptable carrier and optionally an excipient.

The composition may be presented in a manner specifically indicated for the treatment of mastitis such as by labelling of a container which contains the composition.

In another aspect of the invention there is provided a method for the prevention of mastitis in a dairy animal which comprises administering to a teat of the animal a composition as herein described. In another aspect the invention relates to a composition which includes paniculate glucan, an antibiotic, and a physiologically acceptable carrier and/or excipient. The antibiotic is preferably present at a low dose.

DETAILED DESCRIPTION OF THE INVENTION

Glucan is a generic term referring to an oligo- or polysaccharide composed predominantly or wholly of the monosaccharide D-glucose. Glucans are widely distributed in nature with many thousands of forms possible as a result of the highly variable manner in which the individual glucose units can be joined (glucosidic linkages) as well as the overall steric shape of the parent molecule.

Glucan is a well described molecule and is found primarily in the cell wall of most fungi such as yeasts, moulds and bacteria.

Paniculate glucan which may be used in this invention includes glucan having a molecular weight from about 500,00 to about 3,000,000 daltons or more, preferably about 1,000,000 to about 3,000,000 daltons. Paniculate glucan generally has a particle size from about 0.1 μm to about 20 μm, preferably about 5 μm to about 10 μm and may be referred to as microparticulate glucan.

Preferably the glucan is isolated from the yeast Saccharomyces cerevisiae which has been shown by many groups to be acceptable in terms of efficacy and safety as an immune stimulant in animals and humans. Glucan prepared from Saccharomyces cerevisiae is referred to hereafter as ("Sc")-glucan. Predominantly or wholly -1,3 glucans from other fungi, bacteria or plants may also be used.

Various processes have been described for the production of microparticulate glucan. See for example Hassid et al (1941), Manners (1973), DiLuzio (1979) and US Patents Nos. 4,810,694 and 2,992,540, which are incorporated herein by reference. The extraction of microparticulate glucan from whole yeast cells depends on the fact that the bulk of the cell wall glucan is insoluble in water, strong alkali, acid and organic solvents whereas all other cell wall components are suspended in one or more of these solutions.

The essential principles of microparticulate glucan extraction of Sc-glucan are (i) lysis of the yeast cell to allow the intact cell walls to be separated from the less dense cytoplasmic contents, and (ii) subsequent or concomitant dissolution of unwanted wall components such as other carbohydrates (glycogen, mannan, glucosamine), lipids and proteins using various combinations of water, alkali, acid and organic solvents. It is preferred in such processes that the three-dimensional matrix structure of the cell wall remains unaltered and intact as a cell wall skeleton (also known as a "cell sac"), comprised predominantly of -(l,3)(l,6)-glucan. The cell wall skeletons characteristically are spherical, hollow structures of approximately 4 to 20 μm diameter and with a molecular weight of between approximately 1,000,000 to 3,000,000 daltons and they are insoluble in water.

A number of methods of extraction of microparticulate Sc-glucan are known as mentioned above, although most are essentially variations of a common method. The described methods entail the following steps.

1. Contact of whole yeast cells with strong alkali solution (pH 12 to 14). This effects lysis of the cells and dissolution of most of the non-glucan components except lipids. This step is uniformly rigorous in all described processes. The contact usually is repeated two to three times using fresh batches of alkali and heat also usually is applied to speed the reaction time.

2. The cells then are exposed to acid (pH 1 to 5) with heat to effect dissolution of certain residual non-glucan components and to effect some hydrolysis of the -1,6 side-branches. The rigour of this step varies considerably between the known processes of relatively mild acid treatment where the conformational changes are minimal and many of the side-branches are retained, through too extensive acid treatment where little or no side-branches remain. 3. Contact of the cell residue with alcohol with heat with or without additional subsequent exposure to solvents, particularly ether or petroleum ether to effect removal of lipids.

The manner in which microparticulate glucan is formed is not significant to this invention. The inventors have found that glucan having a molecular weight of about 500,000 to 3,000,000 daltons or more, which is insoluble in water, and which forms a microparticulate suspension in water is useful in the invention. Large glucan particles which may be produced by extraction processes can be easily ground to a relatively fine particle size.

The microparticulate glucan is combined with a physiologically acceptable carrier and optionally an excipient in a form suitable for administration to an effected mammary area. Examples of caniers include: water physiologically saline, isotonic solutions containing dextrose, glycerol or other agents conferring isotonicity, lower alcohols, vegetable oils, polyethylene glycol, glycerol triacetate and other fatty acid glycerides. The glucan is present suspended or admixed in the corners. Examples of other caniers which may be used include cream forming agents, gel forming agents and the like. Excipients include buffers, stabilisers, emulsion forming agents, colouring compounds, salts, amino acids, antibiotics and other anti-bacterial compounds chelating agents and the like. More than one excipient and canier may be used.

It is believed that antibiotics interact synergistically with the paniculate glucan in the treatment and prevention of mastitis. As a result lower amounts of antibiotics than are currently used can be employed, for example preferably 50% or less of the standard antibiotic dose cunently employed in the art for the treatment of mastitis. This is advantageous from a perspective of cost, and from a perspective of reduced levels of exposure to antibiotics.

Antibiotics which may be used include tetracyclines, penicillin-streptomycin combination, penicillin G-neomycin combination, penicillin-nitrofurazone combination, penicillin-tylosin combination, novobiocin, sodium cloxacillin, ampicillin, furaltadone, erythromycin, spiramycin, cephalosporins (e.g. cefoperazone), and combinations of said one or more antibiotics (see Radostits, D.C., et al, Veterinary Medicine, 8th Edition, 1994, Bailliere Tindall).

One or more antibiotics may be used at a "low dose", that is at a dose of 50% or less than that currently in use for the treatment of mastitis, such as from 5 to 50% , 5 to 40%, 5 to 30% or 5 to 20% of the conventional daily dose. For example, a standard daily dose of a tetracycline is 400 mg per day per treatment. A low dose may be from 20 to 200 mg.

Therefore, in another aspect of the invention there is provided a composition which includes paniculate glucan, an antibiotic, and a physiologically acceptable carrier and/or excipient. The antibiotic is preferably present at a low dose.

The above composition may be used in dry cow therapy, that is, during the non-milking period. In this regard, it is standard practice to dose the teats of dairy animals with antibiotics during the "dry" period to prevent mastitis. The composition of the invention either obviates the need for antibiotics, or uses low doses of antibiotics in conjunction with glucan. Such composition may be routinely used during the "dry" period, for example as a once-treatment preventative, or as a therapeutic for mastitis treatment.

Compositions according to the invention are prepared by simple admixture of microparticulate glucan particles with carriers and/or excipients.

The composition of this invention may be in various forms, such as a solution with particles suspended therein, a cream, a gel or ointment or like forms for infusion into an infected mammary gland or topical application to a mastitis affected mammary area.

In the process of the invention, microparticulate glucan solutions, creams, gels or the like may be infused into an infected mammary gland by way of a syringe, a needle or other tubular projection for introducing the composition into the mammary gland and being of a sufficient diameter to allow glucan particles to pass into the mammary gland. Creams, gels, foams, ointments and the like may be topically applied to a mastitis infected mammary area. Topical application has been found to be effective.

A one off treatment, such as a one off or single infusion of a microparticulate glucan suspension, has been found to be effective in the treatment of mastitis. A microparticulate glucan containing suspension may be infused or injected into a mastitis affected teat, and a cream containing particulate glucan and optionally an antibiotic may be topically applied to the effected area. Rapid treatment ensues within about one to five days. Prior art treatment regimens include by way of contrast a three day or weekly treatment such as once per day.

This invention may be used in the treatment of any lactating mammal such as cows, sheep, goats, buffalo, horses, dogs, cats, and human females.

Mastitis may be prevented by treatment during the non-milking period. Therefore according to an aspect of this invention there is provided a method for the prevention of mastitis in a dairy animal which comprises administering to a mammary area, such as a teat and/or udder or breast area of an animal, a composition as described herein.

This invention will now be described with reference to the following non-limiting examples:

Example 1

Preparation of glucan

Microparticulate glucan is prepared as follows:

A 400 g sample of commercial Saccharomyces cerevisiae in dry form is added to four litres of 4% w/v sodium hydroxide and heated to 100°C for one hour with vigorous stirring. The suspension is allowed to cool to between 45°C and 50°C before the lysed yeast cells are separated from the alkaline hydrolysate by centrifugation at 800 g for ten minutes. The lysed yeast cells are resuspended in a fresh batch of three litres of 3% w/v sodium hydroxide and boiled for 15 minutes. Following separation by centrifugation, the lysed yeast cells are resuspended in a fresh batch of two litres of 3% w/v sodium hydroxide and boiled for 15 minutes followed by standing at 70 °C for 16 hours. Following separation by centrifugation, the lysed yeast cells are resuspended in water and boiled for 10 minutes. The latter step is repeated once. Following centrifugation, the lysed yeast cells are resuspended in a fresh aliquot of 2 L water, the pH adjusted to 4.5 by the addition of phosphoric acid and the suspension then boiled for thirty minutes. Five hundred mL of chloroform then is added and the suspension subjected to vigorous agitation for ten minutes, following which the suspension is allowed to settle for 10 minutes in a separating funnel. The lower chloroform phase plus a greyish intermediate phase are discarded, leaving an aqueous phase which is collected and exposed as before to a fresh batch of 500 mL of chloroform. The final aqueous phase is collected and boiled for 10 minutes to remove any residual chloroform and then dried using a spray-drier. The product may be used as is for formulation, or ground to a desired particle size.

Example 2

Microparticulate Sc-glucan is prepared as follows:

A 400 g sample of commercial Saccharomyces cerevisiae in dry form is added to four litres of 4% w/v sodium hydroxide and heated to 100°C for one hour with vigorous stirring. The suspension is allowed to cool to between 45 °C and 50°C before the lysed yeast cells are separated from the alkaline hydrolysate by centrifugation at 800 g for ten minutes. The lysed yeast cells are resuspended in a fresh batch of three litres of 3% w/v sodium hydroxide and boiled for 15 minutes. Following separation by centrifugation, the lysed yeast cells are resuspended in a fresh batch of two litres of 3% w/v sodium hydroxide and boiled for 15 minutes followed by standing at 70 °C for 16 hours.

Following separation by centrifugation, the lysed yeast cells are resuspended in water and boiled for 10 minutes. The latter step is repeated once. Following centrifugation, the lysed yeast cells are resuspended in a fresh aliquot of 2 L water, the pH adjusted to 4.5 by the addition of hydrochloric acid and the suspension then boiled for ten minutes. Five hundred mL of chloroform then is added and the suspension subjected to vigorous agitation for ten minutes, following which the suspension is allowed to settle for 10 minutes in a separating funnel. The lower chloroform phase plus a greyish intermediate phase are discarded, leaving an aqueous phase which is collected and exposed as before to a fresh batch of 500 ml of chloroform. The final aqueous phase is collected and boiled for 10 minutes to remove any residual chloroform and then dried using a spray-drier.

Example 3

200mg of glucan (5mg/ml) and 40 ml of pyrogen free saline (0.9%) were infused into the mammary gland in infected and healthy cows to determine the effect of glucan therapy on the course of chronic Staphylococcal infection in the mammary gland. Milk samples were collected from the cows before, and 2,5 and 10 days after glucan treatment. Blood was also taken to examine the changes in total and differential cells in the blood. Body temperature was monitored during the course of the study.

There was a significant reduction in the staphylococcal infection in infected animals, as determined by measuring the amount of shedding and the production of antibodies. Blood neutrophils and lymphocytes levels were elevated in the mammary gland and milk of infected animals but dropped within two days to within 20% or normal, followed by a 100% normal distribution by 10 days. There was no evidence of a rise in body temperature after infusion of glucan in all cows. Cows showed no clinical symptoms of mastitis after about 2 to 5 days. A single treatment was effective.

Compositions containing 5 mg/ml paniculate glucan in 0.9% saline are prepared, which additionally contained antibiotics. One such composition contains 100 mg of tetracyline per dose. Such compositions were particularly effective in the treatment or prevention of mastitis.

Topical compositions containing 10 mg/ml paniculate glucan in a cream base are prepared. One such composition contains 150 mg antibiotic (streptomycin-penicillin combination). Such compositions were effective in the treatment and prevention of mastitis. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims

1. A method for the treatment of mastitis which comprises administering to the affected mammary site of a lactating mammal a composition including particulate glucan in association with a physiologically acceptable carrier and optionally an excipient.

2. A method according to claim 1 wherein the particulate glucan has a molecular weight from about 500,000 daltons to about 3,000,000 daltons or more and a particle size from about 0.1 μm to about 20 m.

3. A method according to claim 1 wherein the composition is infused into an infected teat or gland, or topically applied to a mastitis affected mammary site.

4. A method according to claim 1 wherein the lactating mammal is selected from cows, sheep, goats, buffalo, deer, dogs, cats, horses, and human females.

5. A composition for the treatment of mastitis which includes particulate glucan in association with a physiologically acceptable carrier and optionally an excipient.

6. A composition according to claim 5 wherein the glucan has a molecular weight from about 500,000 daltons to about 3,000,000 daltons or more and a particle size from about 0.1 m to about 20 m.

7. A composition according to claim 5 indicated for the treatment of mastitis.

8. A composition according to claim 5 which includes particulate glucan, an antibiotic and a physiologically acceptable carrier.

9. A composition according to claim 8 wherein the antibiotic is present at a low dose.

10. A method for the prevention of mastitis in a dairy animal which comprises administering to a mammary area of the animal a composition according to any of claims 5 to 9.

11. A method according to claim 10 for use during the non-milking period.