IE80454B1 - Veterinary compositions for treating mastitis - Google Patents

Description

Veterinary Compositions for treating Mastitis The invention relates to a veterinary composition, particularly for the prophylaxis and treatment of mastitis in cows.

Bacterial infection via the teats of a cow is the most common cause of mastitis.

It is known to treat teats of a cow with a long acting antibiotic with effective cover only being provided whilst minimum inhibitory concentration (MIC) levels of the antibiotic are maintained. This period of cover can vary from 4 to 10 weeks.

It is also known to provide a physical barrier in the teat canal to try to prevent the ingress of pathogens. One such system is described in UK 1,441,747 (Lazonby).

One commercially available barrier system comprises a twin injector pack, one injector containing an antibiotic formulation and a second injector containing a barrier or seal formulation. The antibiotic formulation comprises penicillin salts and dihydrostreptomycin which is infused into the udder following the last lactation and before the cow is dried off. The seal formulation comprises a gel of aluminium stearate and liquid paraffin containing approximately 35% by weight of bismuth subnitrate. This is infused into the udder after the antibiotic formulation to seal the teat canal.

B0454 This invention is therefore directed towards providing an improved veterinary composition for the prophylaxis and treatment of mastitis in dry cows.

According to one aspect of the invention provides a veterinary composition for intramammary use in non-human animals comprising an antibacterial formulation, and a seal formulation, the seal formulation comprising a gel base and a non-toxic heavy metal salt in the base, wherein the heavy metal salt is present in an amount of at least 40% by weight of the base.

Preferably, the heavy metal salt is present in an amount of between 50% and 75% by weight, most preferably approximately 65% by weight.

In a preferred embodiment of the invention the heavy metal salt is bismuth sub-nitrate.

In one embodiment of the invention the base is a gel based on aluminium stearate. Preferably in this case the gel includes a vehicle such as liquid paraffin.

In one embodiment of the invention the antibacterial agent comprises an antibiotic in the form of a substantially insoluble salt in an aqueous suspension. Preferably, the antibiotic agent is a substantially insoluble salt of a synthetic penicillin such as cloxacillin.

Most preferably the antibiotic comprises cloxacillin benzathine.

Preferably the antibiotic is in a micronised form having an average dimension of less than 25μ. Preferably a substantial proportion of the antibiotic has an average dimension of less than 10μ. - 3 Preferably the composition is a unit dose. Typically the composition contains 600 mg of cioxaciliin as cioxaciliin benzathine.

In one embodiment of the invention antibacterial agent is present in the gel base.

Alternatively the antibacterial formulation and the seal formulation are separate.

In another embodiment of the invention the gel comprises a polyethylene gel. The gel may be based on low density polyethylene or on high density polyethylene.

The invention also provides a veterinary composition for use in the prophylaxis as treatment of mammary disorders in non-human animals during an animals' dry period.

Detailed Description of the Invention The invention will be more clearly understood from the following description thereof given by way of example only.

EXAMPLE A A veterinary composition comprising a first antibiotic - containing injector (1A) and a second seal-containing injector (2A) were prepared as follows: INJECTOR ΙΑ Component g/Kg Function Cloxacillin Benzathine 212.6 Antibiotic PVP 0.59 Suspension Aid Sodium Citrate 7.87 Buffer Tween 80 (Trade Mark) 0.983 Surfactant EDTA (disodium) 0.0787 Cation Scavenger Antifoam M30 0.0157 Production Aid Water for Injection QS Aqueous Vehicle * (i) will be adjusted depending on potency (ii) the cloxacillin benzathine was in a micronised form having an average dimension less than 25μ with approximately 75% less than 15μ and 50% less than 10μ and 85% was greater than 2μ. (1) Place most of the water for injection in a production vessel. (2) Add and dissolve separately, sodium citrate, E.D.T.A., P.V.P. and Tween 80. Mix well. (3) Add antifoam and mix well, the solution will have a slight haze. (4) Add and suspend Cloxacillin Benzathine and homogenise for 15 minutes. (5) Bring to final weight with addition of further water for injection. (6) Fill 3.6g into intramammary injectors. - 5 This formulation is stable when subjected to extended storage periods in its proposed marketing container.

We have surprisingly found that cloxacillin benzathine in an aqueous base gives rapid absorption in a very short time period.

A number of studies have been conducted in dry cows to establish the following : Study 1: Stuffy 2: Study 3: Bioavailability of cloxacillin in bovine colostrum following intramammary infusion of Injector 1A.

Residues of cloxacillin in bovine colostrum following intramammary infusion of the Injector LA.

Irritancy of aqueous cloxacillin in the bovine mammary gland.

The results can be summarised as follows.

R IA - Stutfy-1 A total of 8 cows were infused with Injector LA and drug LeveLs were measured in colostrum for a period of 144 hours following infusion. A peak of 5233.4 ug/ml was seen one hour post infusion. This declined exponentially and no drug was detectable at 6 days post infusion. The results are presented graphically in Fig. 1. These results are precisely in line with the concept of a teat seal as described herein with a high initial peak effectively sterilizing the udder, with the second Injector providing a barrier to ingress of pathogens thereafter. This provides the advantages of reduced risk - 6 of drug residues both to the producer and consumer based upon a unique dry cow antibiotic profile.

Fig. 1 is a graphic representation of the results of this study.

Fig. 2 is a log transformation of the results of Fig. 1.

These graphs illustrate a linear decline in drug levels over time which is an ideal pharmacokinetic profile.

INJECTOR 1A -Study 2 Study 2 was conducted to specifically determine the end point for milk withholding. Animals were infused in each of four quarters with the first Injector. Samples were taken every 24 hours and analysed for cloxacillin levels. Eight days after administration of Injector 1A the levels of drug were below the acceptable maximum residue level for cloxacillin.

INJECTOR 1A - Study 3 This study involved an assessment of the irritancy of the aqueous cloxacillin formulation in the bovine udder. It was found that cloxacillin is mildly irritant in the bovine udder but has no effect on milk yield and the somatic cell count returns to normal 72 hours after infusion.

From these studies it can be concluded that cloxacillin in an aqueous suspension offers a safe and effective means of controlling mastitis in the dry cow and offers significant advantages over existing preparations especially with regard to consumer health and animal welfare.

INJECTOR TYPE 2A Various gels based on liquid paraffin with aluminium stearate were prepared.

Formulation Mass Constituents YV/(Na2) 2A1 3.5g 14% AS-LP gel + 37%BSN + 0. l%Ac 110.3 2A2 7.0g 14% AS-LP gel + 37%BSN + 0.1%AC 110.3 2A3 3.5g 14% AS-LP gel + 37%BSN + 0.1%Ac 215.5 LP BSN Ac AS YV Liquid Paraffin Bismuth Subnitrate Acriflavin Aluminium Monostearate Yield value - (A measure of the relative fluidity of the gel. Low yield values indicate a more liquid gel).

Products 2A1 to 2A3 were considered appropriate candidates for use as test seals.

An ideal teat seal should have the following characteristics : 1. it should be non-irritant to the bovine udder; 2. Persistence - the seal should remain in situ for the duration of the dry cow period; 3. Consistency - the seal should not break up within the udder; 4. Compatibility - the seal should be compatible with the antibiotic formulation used in association with it, either aqueous or oily; . Ease of Removal - at the end of the dry period, the seal should be readily removable for the udder and not give rise to persistent residues of either the seal or antibiotic.

Irritancy of the seals was the first characteristic to be assessed as any product which was irritant would have to be rejected irrespective of its performance against the other criteria. Irritancy was measured by conducting somatic cell counts in treated and untreated quarters of lactating cows and comparing these results by measuring area under the curve ratios using the following formula: AUC Ratio = AUC of treated Quarter AUC of untreated quarter This allows for a relative assessment of the various formulae. seal Formulation AUC Ratio Peak (cells/ml) Condition of milk Duration before return to pre-dose level (hours) 2A1 125 9.0 z 10s Normal 160 2A2 133 1.0 x 10* Normal 144 2A3 128 83 x 105 Normal 160 Formulations 2A1 and 2A3 were considered appropriate for further investigation. It was concluded that AS-LP gels are viable candidates for sealing teats.

In vitro studies have shown that whilst AS-LP gels have relatively high yield values, their tensile strength is not as great as other possible seal formulations. The relative merits of these two properties were studied by Xray analysis of various formulations in dry cows. - 9 A series of studies were undertaken to optimise these parameters : INJECTOR TYPE 2A - Study 1 Formulation Gel former % LP °c AC % BSN % Mass(g)P 8 cm3 YV Nm* 2A1 83 AS 54.1 0.1 37 7.0 132 110.0 AS = Aluminium Stearate LP - Liquid Paraffin AC = Acriflavin BSN = Bismuth Subnitrate p = Density YV = Yield Value The test formulation was infused into quarters of dry cows. The effectiveness of sealing was measured by x-ray analysis. In addition the mass of seal recovered, % BSN recovered and the effective seal duration [ESD] were estimated.

Formulation ESD (days) Mass of seal recovered (t) BSN recovered 2A 28.5 ± 13.1 8.19 (117.1) 64.1 The mass of seal recovered was in excess of that applied probably due to the presence of aqueous material.

INJECTOR TYPE 2A - Study 2 The effect of the product volume and density was examined in another series of X-ray studies.

Formulation Gel former % LP % AC % BSN % Mass ( P g cn YV P NmJ 2A4 3.1 AS 313 0.1 65 5.0 1.70 161.7 2A5 3.1 AS 31.8 0.1 65 10.0 1.70 161.7 The results of these studies > are shown in the table below: Formulation Mass(g)* recovered (t) % BSN *♦ recovered 2A4 2.47 (49.4) 2A5 4.52 (45.2) * Adjusted for water content 89.8 100.2 ** % BSN were higher than in the previous study indicating a greater integrity of seal. As this trial was of a shorter duration than study 1, this may have been a contributing factor. However, increasing the density and reducing the volume had a clearly beneficial effect on the product performance.

INJECTOR TYPE 2A - Study 3 As it is intended to use the teat seal in conjunction with an antibiotic preparation this study looked at the effect of an aqueous and oily based antibiotic suspension on seal integrity over time. Additionally, the mass of seal was reduced to 3.5g to see if this could further enhance the seals effectiveness.

Products used are given i in the table below • • Formulation Gel LP Ac. BSN Mass P YV Anti* former % % % % 1 g/cm’ NM* biotic 2A6 3.1% AS 313 0.1 65 33 1.70 161.7 Oily A6’ 3.1% AS 313 0.1 65 33 1.70 16L7 Aq.

The products were then examined for their effective seal duration [ESD].

Formulation ESD (days) Recovery of material (g) BSN Recovery % BSN recovery io fluid portion ppm 2A6 2A6' 473 * 13.5 0 633 * 5.4 106 752 2191 117 A combination of a seal and an aqueous based antibiotic offers a superior combination to a seal and an oily based antibiotic. This can be deduced from the fact that the ESD's for the seal/aqueous combinations were significantly better than those for oily combination. This is further evidenced by the high BSN recoveries (>75%) for the aqueous products, indicating retention of seal in situ. In contrast, the amount of bismuth found in the fluid portion at parturition was far lower for the aqueous than the oily combination. This shows that there was a larger degree of dispersal of the seal into the udder with the oily combinations.

The amount of BSN lost in absolute terms can be calculated as follows : A χ Β x M - Loss of BSN (g) A = % BSN lost.

B = % of BSN in product M = Mass of Product Using the formula, product 2A1 lost 1.30 g. 2A61 lost 0.57g. Considering that product 2A61 was in situ for 70 days as compared to 49 days for 2A1, this demonstrates at least a 2-3 fold reduction in BSN loss and a major improvement in product design.

PREFERRED METHOD OF MANUFACTURE FOR INJECTOR TYPE 2A Aluminium Stearate (Alugel 30 D.F.) Heavy Liquid Paraffin Bismuth Subnitrate Acriflavin (Pigment) Qm/Kg 48.9 300.4 650.00 0-994 Each injector contains 3.5g.

The aluminium stearate used is a distearate compound having a melting point in the region of 150°C to 160°C.

Bismuth subnitrate (6Bi2O3.5N2O3.9H2O) is a white, slightly hydroscopic powder. (1) Place heavy liquid paraffin in reactor vessel, heat to 160°C for one hour. Cool to 40sC. (2) Start emulsifiers and mixers and add the aluminium stearate. Heat to 145°C ± 5°C and maintain for one hour. Cool to 40°C. (3) Add and blend the Bismuth Subnitrate and Acriflavin. (4) Fill 3.5 g into intramammary injector.

EXAMPLE B A veterinary composition was prepared comprising a first antibiotic-containing injector having the same formulation as Injector 1A described above and a second seal injector 2B as described below.

INJECTOR TYPE 2B Various gel based on liquid paraffin with polyethylene were prepared. Two grades of polyethylene were used in manufacturing the gels: low density (LDPE) and high density (HDPE). They differed in the degree of side chain branching but produced similar gels.

Formulation 2B1 Mass 3-5g Constituents 37% BSN + 0.1%Ac YV (NM*2) 40.9 3% HDPE - LP gel + 2B2 7.0g 3% HDPE - LP gel + 35% BSN + 0.1%Ac 40.9 2B3 70g 5% HDPE - LP gel + 37% BSN + 0.1%Ac 110.0 2B4 7Dg 5% HDPE - LP gel + 37% BSN + 0.1%Ac 220J 2B5 3-5g 3% HDPE - LP gel + 37% BSN + 0.1%Ac 653 2B6 7.0g 3% HDPE - LP gel + 37% BSN* + 0.1%Ac 36.6 2B7 7.0g 3% LDPE - LP gel + 37% BSN* + 0.1%Ac 54.1 LP = Liquid Paraffin BSN= Bismuth Subnitrate Ac = Acriflavin YV = Yield value - (A measure of the relative fluidity of the gel. Low yield values indicate a more liquid gel).

* - BSN was in micronised form.

Products 2B1 and 2B7 were considered appropriate candidates for use as test seals.

Irritancy of the seals was the first characteristic to be assessed as any product which was irritant would have to be rejected irrespective of its performance against the other criteria. Irritancy was measured by conducting somatic cell counts in treated and untreated quarters of lactating cows and comparing these results by measuring area under the curve ratios using the following formula: AUC Ratio = AUC of treated quarter AUC of untreated quarter This allows for a relative assessment of the various seal formulae.

Formulation AUC PEAK (CELLS/mL) CONDITION DURATION BEFORE RATIO OF MILK RETURN TO PREDOSE LEVEL (HOURS) 2B1 17.0 5.3 X 106 N 160 2B2 4.8 2.4 X 106 N 160 2B3 16.4 >3 X 106 N 184 2B4 3.7 1.7 X 106 N 136 2B5 10.8 1.0 X 106 N 112 2B6 13.0 7.0 X 106 N 120 2B7 51.8 >1.0 X 107 C 112 N = Normal C = Clotted Formulations 2B1 and 2B5 were considered appropriate for further investigation. 2B6 and 2B7 (micronised Bismuth Subnitrate) were excluded on the basis of there being more irritant that the other formulas tested and having no significant advantages. Thus, it is concluded that PE-LP gels are viable candidates for sealing teats.

In vitro studies have shown that PE-LP gels have high tensile strengths for relatively low yield values. The merits of these two properties were studied by X-ray analysis of various PE-LP formulations in dry cows.

A series of studies were undertaken to optimise these parameters: INJECTOR TYPE 2B - Study 1 Formulation Gel former % LP % Ac. % BSN % Mass 8 P gem3 YV NM* 2B3 3.1% PE 59.8 0.1 37 7.0 132 136.6 2B2 1.9% PE 61.0 0.1 37 7.0 132 40.0 2B3' 3.1% PE 59.8 0.1 37 7.0 132 2203 PE = Polyethylene LP = Liquid Paraffin AC = Acriflavin BSN= Bismuth Subnitrate P = Density YV = Yield Value 2B31= Formulation is identical to 2B3. Gel was formed using different temperature profile, leading to different yield values.

Each of the test formulations was infused into quarters of dry cows. The effectiveness of sealing was measured by Xray analysis. In addition to mass of seal recovered, % BSN recovered and the effective seal duration [ESD] were estimated.

Formulation ESD (days) BSN recovered Mass of Seal recovered (%) 2B3 47.3 ♦ 16.0 0.62 (8.81) 40.8 2B2 54.1 ♦ 10.4 3.00 (43.0) 40.0 2B3; 49.0 t 12.9 1.38 (19.7) 44.6 There was a direct correlation between the ESD and the mass of seal recovered for these products.

INJECTOR TTPE 2B--.-Sty<3Y 2 product volume and density was examined The effect of the in another series of X-ray Formulation Gel LP former % * 2B8 1.7% PE 332 2B9 1.7% PE 332 studies.

Ac. % BSN % Mass 8 P g/cm3 YV NM* 0.1 65 5.0 1.70 2163 0.1 65 10.0 1.70 2163 The results of these studies are shown in table below: Formulation Mass (g) * % BSN ** recovered (%) recovered 2B8 3.78 (75.6) 85.6 2B9 3.92 (39.2) 92.2 * Adjusted for water content ** % BSN were greater than in the previous study indicating a greater integrity of seal. As this trial was of a shorter duration than study 1, this may have been a contributing factor. However, increasing the density and reducing the volume had a clearly beneficial effect on the product performance.

INJECTOR TYPE 2B - Study 3 As it is intended to use the teat seal in conjunction with an antibiotic preparation this study looked at the effect of an aqueous and oily based antibiotic suspension on seal integrity over time. Additionally, the mass of seal was reduced to 3.5g to see if this could further enhance the seals effectiveness.

Products used are given in the table below • • Formulatioo Gel LP Ac. BSN Mass P YV Anti- former % % % % 8 g/cm3 NOT® biotic 2B8 L’% PE 332 0.1 65 33 1.70 2163 Oily 2B9 1.7% PE 332 0.1 65 33 1.70 2163 Aq. The products were then examined for their effective seal duration [ESD]. Formulation ESD (days) Recovery of BSN BSN Recovery material (gi Recovery in fluid % proportion ppm B8 563 - 13.9 0 0 . 1200 B9 603 ± 82 0.793 77.6 147 A combination of seal and a non aqueous based antibiotic offers a superior combination to seal and an oily based antibiotic. This can be deduced from the fact that the ESD's for the seal/aqueous combinations were significantly better than those for oily combinations. This is further evidenced by the high BSN recoveries (>75%) for the aqueous products, indicating retention of seal in situ. In contrast the amount of bismuth found in the fluid portion at parturition was far lower for the aqueous than the oily combination. This shows that there was a larger degree of dispersal of the seal into the udder with the oily combinations.

In conclusion, the use of polyethylene as a gelling agent in combination with heavy metal salts in a teat seal product in combination with an aqueous based antibiotic system provides a product with the desired properties as earlier outlined which should be efficacious in the treatment and prophylaxis of dry cow mastitis.

PREFERRED METHOD OF MANUFACTURE FOR INJECTOR TYPE 2B Gm/Kg H.D.P.E. 17.00 Heavy Liquid Paraffin 332.00 Bismuth Subnitrate 650.00 Acriflavin (Pigment) 0.994 Each injector contains 3.5g.

Bismuth subnitrate (6Bi203.5N20s.9H20) is a white, slightly hydroscopic powder (1) Place heavy liquid paraffin in reactor vessel. Heat to 160°C for one hour. Cool to 40°C. (2) Start emulsifiers and mixers and add the H.D.P.E. Heat to 145°C ± 5°C and maintain for one hour. Cool to 40°C. (3) Add and blend the Bismuth Subnitrate and Acriflavin. (4) Fill 3.5g into intramammary injector.

In vivo studies have suprisingly shown that levels of 5 greater than 40% Bismuth Subnitrate produces a seal product that remains in situ and shows less break-up than seals with lower levels of Bismuth Subnitrate. In addition, because the product is more dense a lesser quantity is required to achieve an effective seal.

Claims (15)

1. A veterinary composition for intramammary use in non-human animals comprising an antibacterial formulation, and a seal formulation, the seal formulation comprising a gel base and a non-toxic heavy metal salt in the base, wherein the heavy metal salt is present in an amount of at least 40% by weight of the base.

2. A veterinary composition as claimed in claim 1 wherein the heavy metal salt is present in an amount of between 50% and 75% by weight.

3. A veterinary composition as claimed in claim 1 or 2 wherein the heavy metal salt is present in an amount of approximately 65% by weight of the base.

4. A veterinary composition as claimed in any of claims 1 to 3 wherein the salt is bismuth nitrate. sub- 5. A veterinary composition as claimed in any preceding claim wherein the base is a gel based on aluminium stearate. 6. A veterinary composition as claimed in any preceding claim wherein the gel includes liquid paraffin as a vehicle.

5. 7. A veterinary composition as claimed in any of claims to 1 to 6 wherein the antibacterial agent comprises an antibiotic in the form of a substantially insoluble salt in an aqueous suspension.

6. 8. A veterinary composition as claimed in claim 7 wherein the antibiotic comprises a substantially insoluble salt of a synthetic penicillin.

7. 9. A veterinary composition as claimed in claim 8 wherein the synthetic penicillin is cloxacillin.

8. 10. A veterinary composition as claimed in claim 9 wherein the antibiotic comprises cloxacillin benzathine.

9. 11. A veterinary composition as claimed in any of claims 7 to 10 wherein the antibiotic is in a micronised form having an average dimension of less than 25μ.

10. 12. A veterinary composition as claimed in claim 11 wherein a substantial proportion of the antibiotic has an average dimension of less than 10μ.

11. 13. A veterinary composition as claimed in any of claims 7 to 12 wherein the antibiotic is a unit dose.

12. 14. A veterinary composition as claimed in claim 13 wherein the composition contains 600 mg of cloxacillin as cloxacillin benzathine.

13. 15. A veterinary composition as claimed in any preceding claim wherein the antibacterial agent is present in the gel base.

14. 16. A veterinary composition as claimed in claim 1 to 14 wherein the antibacterial formulation and the seal formulation are separate.

15. 17. A veterinary composition as claimed in any preceding claim for use in the prophylaxis or treatment of mammary disorders in non-human animals during an animals' dry period.