NO763773L - - Google Patents

Description

The invention relates to a method for preventing and regulating mammary gland infections in livestock, and more particularly the invention relates to a method for treating mastitis in cattle, caused by bacterial infection.

It has long been recognized that the occurrence of mastitis in lactating dairy cows has resulted in significant amounts of milk being spoiled. Examples of the organisms that usually cause bacterial mastitis are E. coli, Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae. In the past, the most effective methods for treating the clinical condition have involved the administration of antibiotic agents in the mammary gland, e.g. penicillins, streptomycin, cephalosporins and tetracyclines. However, it has been shown that some cows become immune to such therapy. In some cases, this is because the disease organism belongs to a strain that is resistant to antibiotics. In other cases, it turns out that even if the causal bacterium is not inherently resistant when isolated, it still presents in vivo such a disease state that even if it can be reduced to a subclinical level, it will constantly be found in the animal for a certain time after the treatment has stopped. It is believed that this type of condition is caused, at least in part, by the causative organism being deposited in the udder folds or in the cells and becoming physically inaccessible to antibiotics. If the condition is the result of an infection by a resistant organism, then even prolonged treatment with broad-spectrum antibiotics may not be effective.

If the infection is of the recurring type, then the condition can be regulated by normal therapy with antibiotics, but the cows can never be completely free of the disease. If a cow suffers from this condition, it is not only an unreliable milk producer, but also poses a constant threat of infection to other cows in the herd. This condition can never be resolved satisfactorily, and it ends with such a cow having to be slaughtered.

In the above cases, a persistent, high level of residual antibiotic, due to the dose required to treat even non-resistant micro-organisms, remains in the udder and is in turn transferred to the milk. Consequently, significant quantities of milk must be drained down the drain after treatment is completed, before the level of antibiotic in the milk is reduced to a level acceptable for human consumption. Therefore, milk spoils not only as a result of the disease, but also as a result of the treatment.

In order to minimize this lost milk production, it is very important to prevent or suppress such infections to subclinical levels during lactation. To reduce the general level of infection in a foal and to manage to remove all infection before calving, it is possible to apply preventive antibiotic therapy in the mammary gland during the animal's weaning period or "drying period". Such treatment only provides limited protection during lactation. As a further preventive measure, certain orally absorbed antibiotics are occasionally added to animal feed. However, on the basis of dairy animals' high body weight and their high food intake, relatively large amounts of antibiotic must be administered in order to be able to maintain effective levels of antibiotic in the animal. This method again only results in limited protection. Because of the aforementioned considerations, improvements and new methods are constantly sought in the treatment of mastitis.

It has now been discovered that certain significant improvements in the treatment of mastitis can be achieved by administration into the mammary gland of certain double-stranded polynucleotides so that it is now possible to treat immune mastitis conditions. It is believed that the polynucleotides exert their effect at least in part by their ability to stimulate the natural defense mechanism present in the udder.

According to the invention, a method for the prophylaxis and treatment of bacterial mastitis in dairy animals is provided, and the method comprises administering to such animals a veterinary preparation comprising a double-stranded ribonucleic acid (dsRNA) or a double-stranded derivative of a double-stranded ribonucleic acid (dsRNA) or a complex of a double-stranded ribonucleic acid with a polybase together with a veterinary acceptable base via the mammary gland.

The term "double stranded" is used herein as applied to ribonucleic acids to refer to the characteristic whereby two ribonucleic acid molecules are associated by hydrogen bonding between complementary bases in each molecule.

The dsRNA compounds that can be used in connection with the invention can be synthetic or of natural origin. Synthetic dsRNA compounds useful in the method are poly I:poly C; poly A:poly U; poly Grpoly C; in particular polyI:poly C. Complexes between synthetic double-stranded ribonucleic acids and polybases, which can be used in the method, are e.g. the complex between poly I:poly C or poly A:poly U and DEAE dextran, dimethrin bromide, positively charged proteins and polyamino acids, e.g. histone, polyarginine, poly-lysine and polyornatin which are described in US Patent Nos. 3,679,654 and 3,725,545.

However, it is preferred to use a double-stranded ribonucleic acid of natural origin, or a double-stranded derivative thereof, or a complex thereof with a polybase.

The term "double-stranded ribonucleic acid of natural origin" shall be used herein to mean any double-stranded ribonucleic acid that can be isolated from a naturally occurring source, and excludes synthetic double-stranded ribonucleic acids such as those specified above. Among the specific, naturally occurring sources of dsRNA are, for example, virus particles found in virally infected fungi, e.g. Penicillia, e.g. P. Chrysogenium, see British Patent No. 1,170,929;

P. Stoloniferum, cf. Bankes et al. in Nature 223 155 1968; and Aspergilli, e.g. A. niger and A. foetidus; reovirus 3 viron and the replicative form of MS 2 coliphage and MU G mutant coliphage.

The term "double-stranded derivative of a double-stranded ribonucleic acid of natural origin" means any double-stranded ribonucleic acid of natural origin which has been subjected to chemical or biochemical (e.g. enzymatic) transformation which changes the primary and/or secondary and/or tertiary structure, e.g. .ex. the N-oxides described in British Patent No. 1284150 or the alkali-modified dsRNA compounds according to British Patent No. 1356263, or the formaldehyde-modified dsRNA compounds described in British Patent No. 1,411,748, provided that the resulting derivative preserves a substantial degree of base-pairing between complementary strands.

The complexes between dsRNA of natural origin and polybases suitable for use in the method are, for example, those between quaternary amino polymers described in British Patent No. 1410013 of which the most suitable are the "water-soluble" complexes described therein, in particular the complex which is defined there as C3.

The other complexes between dsRNA compounds of natural origin and polybases are those disclosed in Belgian Patent No. 834859 between dsRNA and polyarginine. Of these, one that is particularly suitable is the complex described there with a molecular weight for polyarginine of essentially 6,500.

Another class of complex which is suitable for use in the method according to the invention is described in Belgian Patent Document No. 824947.

If a complex is used, it is preferred to use the complex between dsRNA derived from P. chrysogenum and polyarginine.

However, it is preferred in this method to use a dsRNA of natural origin, especially that obtained from P. chrysogenum.

This method can be used for the treatment of cattle-threatening mastitis either during lactation or in the dry period. Alternatively, the treatment can be given to the apparently uninfected

cow in the dry period. The medication is administered directly to the udder via the strek channel. When the cow is lactating, the drug is administered after milking.

For the treatment of the clinical condition, a therapeutic dose, in accordance with the method according to the invention, essentially consists of the daily administration of from 0.1 to 50 mg of dsRNA or a derivative or complex thereof,

per day to the infected teat or to each of the infected teats in the udder. By 50 mg of a complex of a dsRNA is meant the amount of the complex containing 50 mg of dsRNA.

The daily dose of dsRNA is preferably from 5 to 30 mg per teat, but is preferably 20 mg per teat.

The exact amount of dsRNA, its derivative or complex, to be administered to the animal, as well as the treatment time, depends on the degree of infection and inflammation in the udder. Administration of the above polynucleotides and their derivatives and complexes causes some local inflammation, at a dose approaching 50 mg per teat. This inflammation is visible to a person who administers the substance. It would therefore be preferable to administer smaller doses over a longer period of time than a large dose over a short period of time, so that one would not unnecessarily worsen any inflammation caused by the infection. Treatment can continue until the udder produces normal, uninfected milk.

In the case of clinical mastitis, it is often not necessary to maintain administration for the entire period in which the disease symptoms are present. In general, administration of dsRNA for a period of 3 days will be sufficient to cause the disease to subside.

For the purpose of prophylaxis or treatment of the subclinical condition, a dose of between 0.1 and 50 mg, but more suitably 0.1-20 mg, is administered daily to each teat in the udder for at least part of, but more suitably the whole dry period. Preferably, this administration is achieved by using a preparation with a slow release, which will be discussed below.

For the purposes of such administration, dsRNA is presented as an aqueous solution or suspension for infusion, or as a liquid or semi-liquid ointment for extrusion into the udder. For infusion purposes, the preparation is prepared in sterile pyrogen-free water, pyrogen-free salt water or a pyrogen-free puff that is acceptable for administration into the mammary gland. For extrusion purposes, the preparation is prepared in an oil or an oily ester which is acceptable for administration into the mammary gland, e.g. a di- or triglyceride of a Cg - C1Q fatty acid, or alternatively an anhydrous medium, e.g. propylene glycol, which is acceptable for such a mode of administration, is also used. If desired, the aqueous or oily preparation can also contain solubilizing, suspending, dispersing, preserving, stabilizing, emulsifying or buffering agents.

For prophylaxis purposes, dsRNA, its derivative or complex is suitably prepared in a viscous ointment or a slow-release granule. Suitable ointments for administration into the mammary gland during the cows' dry period are known in antibiotic therapy

in the dry period. Such ointments are formulated so as to allow slow release of drug.

When formulated in a slow-release granule, the dsRNA is dispersed in a parenterally acceptable non-irritating wax substance, e.g. carnauba wax, montan wax, beeswax and glycerides, e.g. hydrogenated castor oil or hydrogenated lard.

These granules can be produced by known methods.

In a suitable granulation technique (according to Lachman et al.,

"The Theory and Practice of Industrial Pharmacy", Lea&Febieger, 1970, Ch. 12, p.322) a binder, as discussed above, and the drug are mixed in a planetary mixer or Z-mixer, and a suitable organic solvent is added to produce a mixture of granulation consistency, which is then mashed through a rotary granulator. The resulting granules are oven-dried and hammer-milled to the desired size. Other methods for producing granules are spray drying (see Lachman et al., "Theory and Practice of Industrial Pharmacy", Lea&Febiger 1970, p.403-5), spray evaporation, low-temperature spray drying, or coaservation (see Lachman et al., "The Theory and Practice of Industrial Pharmacy", 1970, p.393; and Ranney M.W., Microencapsulation Technology, 1969, Noyes Data Corporation,

New Jersey, U.S.A. The granules can be coated with a conventional coating agent and preferably administered in an oily suspension or emulsion.

All these preparations are advantageously filled in tubes or syringe packs of a conventional type for administration into the mammary gland, i.e. they are equipped with a needle holder for infusion directly into the mammary gland via the strek channel.

Alternatively, dsRNA or double-stranded derivatives or complex with a polybase will be supplied for use as a freeze-dried powder suitable for dissolution or suspension in an aqueous or other acceptable parenteral vehicle, especially pyrogen-free saline. In such cases, the user will be provided with a two-part pack or syringe or a container with two packs, where one part or pack contains a measured amount of dsRNA, a derivative or complex thereof and the other contains a measured amount of aqueous carrier. The first part or package will suitably contain a unit dose of drug as previously mentioned, and the second part or package will contain an amount of aqueous carrier. An appropriate amount is sufficient so that when mixed, the concentration of the dsRNA, derivative or complex thereof will be from 0.1 to 10 mg ml

The general method according to the invention is also useful for improving the overall effectiveness of normal antibiotic therapy. It has been shown that it is particularly effective to combine polynucleotide treatment with antibiotic treatment rather than using the treatments separately.

For the purpose of such a combined treatment, the polynucleotide can be administered in an independent or a combined preparation. During the dry period, the double-stranded ribonucleic acid or the double-stranded derivative thereof or complex thereof with a polybase and the antibiotic can be administered simultaneously. During the lactation period, each drug can be administered either alternatively or simultaneously after milking. The dose of antibiotic that is appropriate is that which is normally administered when it comes to clinical mastitis.

Primarily, the antibiotics used in combination with the polynucleotides are penicillin G and salts thereof, especially the procaine salt; semi-synthetic penicillins, e.g. ampicillin, amoxycillin, carbenicillin, cloxacillin, flucloxacillin, ticarcillin, penicillin, talampicillin, nafcillin, dicloxacillin, oxacillin, methicillin, carfecillin; and mixtures of such penicillins, e.g. ampicillin/cloxacillin, amoxycillin/cloxacillin or tiacarcillin/flucloxacillin, and their veterinary acceptable salts and esters; cephalosporins, e.g. cephalonium and cefoxazole, their veterinary acceptable salts and esters.

Suitable salts of the above penicillins and cephalosporins are their sodium, potassium, benzathine and procaine salts.

Particularly useful penicillins are normally ampicillin, cloxacillin, ampicillin/cloxacillin, amoxycillin/cloxacillin, and sodium and potassium salts thereof.

A particularly useful cephalosporin is cefoxazole.

For the purposes of such treatment, it is highly advantageous to present the drugs in a combined preparation.

For the purposes of administration into the mammary gland, the combination of polynucleotide and antibiotic is prepared in standard-. preparations for use in the mammary gland, as previously discussed. The same considerations regarding appropriate and preferred dsRNA, double-stranded derivatives and complexes with polybases, as well as antibiotics apply to these preparations as well as to the method for treating mastitis with these components individually.

Among the possible combinations of antibiotics and polynucleotides, it has been shown that those containing a semi-synthetic penicillin, particularly cloxacillin, or a semi-synthetic cephalosporin, particularly cefoxazole, and dsRNA are those originating from P. chrysogenum as referred to above.

Therefore, in a further aspect, the invention includes within its scope veterinary preparations suitable for administration into the mammary gland, comprising a semi-synthetic penicillin or semi-synthetic cephalosporin and the dsRNA derived from P.chrysogenum according to British Patent No. 1,170,929 as referred to herein, in combination with a suitable veterinary acceptable carrier.

The following examples illustrate the invention.

In order to aid the understanding of the following examples, it should be pointed out that the milk produced by animals contains a number of white cells similar to those found in the blood. The presence of local irritation or infection in the udder causes an increase in the number of white cells produced in the milk, giving a measure of the level of infection present. In the case of mastitis caused by micro-organisms, the number of neutrophils increases in particular, but the number of other cell types (eg macrophages) also increases; this may form part of the host's defense mechanism.

Examples 1-6 illustrate the treatment of mastitis with a purified double-stranded ribonucleic acid extracted from Penicillium chrysogenum ATCC 10002 containing a large amount of virus particles, as described in British Patent No. 1,170,929.

Example 1

A Jersey cow had symptoms of mastitis in her left hind teat (L.H.). The teat was swollen and warm to the touch. The milk produced contained blood clots. Bacterial examination of the milk showed that the infection was caused by the bacterium Streptococcus uberis. 20 mg of dsRNA was infused into the infected teat in 5 ml of pyrogen-free saline daily for 3 consecutive days. The bacteriology results are shown in Table 1.

It can be seen that the above treatment caused a reduction of the infection and a resolution of the clinical condition. On the fourth day after the third infusion, the milk samples became sterile and normal in appearance.

Example 2

A Jersey cow that had recently calved showed, 4 days after calving, a non-hemolytic coliform infection in the right foreleg (R.F.). The symptoms were the presence of a few small lumps in the milk and signs of Escher. coli by smear.

The R.F. teat was infused via the strek canal with 20 mg dsRNA in pyrogen-free saline (C.T.3243) daily for 3 consecutive days after the evening milking. The cell count and bacteriology are shown in table 2. After the first infusion, the lumps disappeared from the milk. The high number of white cells, 40,000,000, which was found in the milk before the infusion, immediately decreased to the normal value of

day 13. The level of E. coli infection decreased until it completely disappeared by the sixth milking after the last infusion (day 5) when the milk became sterile.

Example 3

A cow had symptoms of mastitis in her right hind teat caused by a hemolytic Coagulase +ve Staphylococcus infection and was infused with three daily doses of dsRNA in pyrogen-free saline via the streak canal. The cell count and bacteriology are as shown in Table 3.

Example 4

One cow had symptoms of mastitis in her left foreskin caused by a hemolytic E. coli infection and was infused with doses of dsRNA in pyrogen-free saline via the streak canal. The results are shown in table 4.

Example 5

The following preparations:-

a) dsRNA (20 mg) and cefoxazole (250 mg)

b) water-soluble complex according to British Patent No. 1,410,013 (200 mg) and

cloxacillin (200 mg)

c) poly I:poly C (20 mg) and cloxacillin (200 mg) were infused in 5 ml pyrogen-free saline, one more

each teat in the udder of an uninfected cow.

The number of white cells was measured before and after the infusions and is reproduced in table 5.

This was a normal cow without mastitis, and it will be seen from the white cell counts that all three treatments produced a remarkable response in the body's defense mechanism by virtue of the large increase in the number of white cells available to fight the infection.

Example 6

Three cows that had chronic mastitis caused by hemolytic coagulase-positive staphylococcus were selected. Each of these cows was known to be immune to combined ampicillin/cloxacillin therapy.

Bacterial examination showed that the first cow had staphylococcal infections in the right fore teat (R.F.) and right hind teat (R.H.) and left hind teat (L.H.) in the udder. The other cow had the same infection in the R.H. teat and the L.H. teat in the udder. The third cow had an infection only in the L.H. teat. A dose of 20 mg dsRNA in sterile water and 200 mg cloxacillin sodium in sterile water (1 ml) was administered sequentially to each infected udder over a period of three consecutive days. The infections subsided,

and ten days after the infusion, bacterial examination showed that the R.H. and L.H. teats of the first cow had a low level of infection. All remaining teats were clear.

Example 7

An oil-based preparation of dsRNA was made in the following way: - A portion of dsRNA equivalent to 300 mg of active material was dispersed into 45 g of oil base. The oil base consisted of: 3 g portions of the preparation were filled into disposable syringes, each containing 20 mg of active material.

Toxicity

Adult baboons were administered a dose of

20 mg/kg/day of dsRNA for 28 days in a first test and 10 mg/kg/day for 28 days in a second test, and no deaths occurred. In a third test, baboons were administered 0.2 mg/kg/day without any apparent ill effect.

Cows with a body weight of 200 kg have been administered with up to 50 mg/kg dsRNA intraperitoneally. Minimal side effects were observed and no deaths occurred.

Claims (12)

1. Method for improving the effectiveness of antibiotic preparations suitable for administration into the mammary gland of cattle for the prophylaxis and treatment of bacterial mastitis, characterized by mixing a double-stranded ribonucleic acid or a double-stranded derivative thereof or a complex thereof with a polybase and such known antibiotic preparations for use in the mammary gland. 2. Method as stated in claim 1, characterized in that the preparation comprises the double-stranded ribonucleic acid poly I: poly C. 3. Method as stated in claim 1, characterized in that the preparation comprises a double-stranded ribonucleic acid of natural origin or a complex between a polybase and a double-stranded ribonucleic acid of natural origin or a double-stranded derivative of a double-stranded ribonucleic acid of natural origin. 4. Method as stated in claim 3, characterized in that the double-stranded ribonucleic acid originates from P. chrysogenum. 5. Method as stated in claim 3, characterized in that the complex is complex C3 as previously defined. 6. Method as stated in claim 3, characterized in that the complex is between a polyarginine which has a molecular weight essentially of 6,500, and the double-stranded ribonucleic acid which originates from P. chrysogenum. 7. Method as stated in claims 1-6, characterized in that the antibiotic is a semi-synthetic penicillin or semi-synthetic cephalosporin. 8. Method as stated in claim 7, characterized in that the antibiotic is cloxacillin. 9. Method as stated in claim 8, characterized in that the antibiotic is cefoxazole. 10. Method as stated in claims 1 to 9, characterized in that the mixture is carried out by sequential administration of antibiotic and double-stranded ribonucleic acid or double-stranded derivative thereof or a complex thereof with a polybase for the udder. 11. Syringe/needle combination for use in the method according to claim 1, characterized in that the needle is of the type suitable for administration into the mammary gland and where the syringe contains a unit dose of a preparation prepared according to claims 1 to 9. 12. Syringe/cannula combination as stated in claim 11, characterized in that the cannula is of the type suitable for administration into the mammary gland, and that the syringe contains a unit dose of a preparation comprising a double-stranded ribonucleic acid or a double-stranded derivative thereof or a complex thereof with a polybase.