LYSOBACTIN FOR USE IN THE TREATMENT OF BOVINE MASTITIS

Abstract

The present invention relates to lysobactin for use in the treatment of bovine mastitis.

Claims

1. A method of treating bovine mastitis caused by Streptococcus bacteria, comprising administering to a bovine in need thereof a therapeutically effective amount of lysobactin, wherein the lysobactin is administered intramammarily.

2. The method according to claim 1, wherein the bovine mastitis is a clinically manifest bovine mastitis.

3. The method according to claim 1, wherein the bovine mastitis is a subclinical bovine mastitis.

4. The method according to claim 1, wherein the lysobactin is provided at a dose of 25 to 1000 mg per udder quarter.

5. (canceled)

6. The method according to claim 1, wherein the bovine mastitis is caused by Streptococcus uberis, Streptococcus dysgalacticae and/or Streptococcus agalacticae.

7. The method according to claim 6, wherein the bovine mastitis is caused by Streptococcus uberis.

8. (canceled) .

9. A pharmaceutical composition formulated for intramammary administration into bovine mammaries, wherein said composition comprises lysobactin in a paraffin-based carrier.

10. The method according to claim 1, wherein the lysobactin is administered intracisternally in a paraffin-based formulation.

11. The method according to claim 1, wherein the lysobactin is administered two times with an interval of 24 hours.

12. A method of treating bovine mastitis caused by Truperella bacteria, comprising administering to a bovine in need thereof a therapeutically effective amount of lysobactin, wherein the lysobactin is administered intramammarily.

13. The method according to claim 12, wherein the bovine mastitis is a clinically manifest bovine mastitis.

14. The method according to claim 12, wherein the bovine mastitis is a subclinical bovine mastitis.

15. The method according to claim 12, wherein the lysobactin is provided at a dose of 25 to 1000 mg per udder quarter.

16. The method according to claim 12, wherein the bovine mastitis is caused by Trueperella pyogenes.

17. The method according to claim 12, wherein the lysobactin is administered intracisternally in a paraffin-based formulation.

18. The method according to claim 12, wherein the lysobactin is administered two times with an interval of 24 hours.

19. The method according to claim 1, wherein the Strepotococcus is a resistant strain to antibiotics other than lysobactin.

20. The method according to claim 12, wherein the Trueperella is a resistant strain to antibiotics other than lysobactin.

Description

EXAMPLES

[0039] The present invention will be further elucidated with reference to the following examples and figures without being limited to them. [0040] FIG. 1 shows the kill kinetics of lysobactin against Staphylococcus aureus and Streptococcus uberis (example 2) [0041] FIG. 2 shows the MIC changes of lysobactin against Staphylococcus aureus and Streptococcus uberis during serial passaging (example 4) [0042] FIG. 3 shows the efficacy of lysobactin in a Staphylococcus aureus acute mouse mastitis model (example 5) [0043] FIG. 4 shows the efficacy of lysobactin in a Streptococcus uberis challenge mouse mastitis model (example 6) [0044] FIG. 5 shows the concentration-time profile of lysobactin in milk after intramammary (IMAM) application to lactating Holstein cows (example 7)

Example 1: In Vitro Antibacterial Activity Against Mastitis Pathogens

[0045] The in vitro antibacterial activity of lysobactin against common mastitis pathogens such as Staphylococcus aureus , Coagulase-negative staphylococci, Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, Trueperella pyogenes, Escherichia coli, or Klebsiella pneumoniae was assessed by microbroth dilution MIC methodology as described by the Clinical and Laboratory Standards Institute (CLSI) in order to obtain the Minimal Inhibitory Concentration (MIC), which is defined as the lowest concentration of a substance that prevents visible growth of a bacterium. The results expressed as MIC.sub.90 are summarized in the following table, where the MIC.sub.90 is defined as the concentration at which the growth of at least 90% of the strains of a given species is inhibited.

TABLE-US-00002 Tested bacteria MIC.sub.90 [μg/ml] of lysobactin Staphylococcus aureus 0.5 Coagulase-negative staphylococci 0.5 Streptococcus uberis 0.125 Streptococcus dysgalactiae 0.25 Streptococcus agalactiae 0.25 Trueperella pyogenes 0.5 Escherichia coli >16 Klebsiella pneumoniae >16

Example 2: In Vitro Kill Kinetics for Mastitis Pathogens

[0046] In order to assess the ability of lysobactin to kill bacteria in milk, flasks containing different concentrations of lysobactin in store-bought full-fat milk were inoculated with 1-2×10.sup.6 colony forming units/nal of a representative strain of either Staphyloccus aureus or Streptococcus uberis. The flasks were incubated for 24-48 hours in a shaking water bath at 35+/−2° C., and viable bacterial counts in each flask were determined at several time-points by diluting and plating samples on agar plates. A reduction of the number of viable bacteria in the initial inoculum by at least 99.9% is defined as bactericidal activity.

[0047] The kill kinetics of lysobactin against Staphylococcus aureus ATCC 29740 and Streptococcus uberis ATCC 27958 in milk were determined for concentrations of lysobactin of 4, 8, 16, 32 and 64 μg/mL.

[0048] Results are depicted in FIG. 1 (CFU: colony forming units). A cidality at 24 h and 48 h, respectively, can be postulated for S. aureus and S. uberis.

Example 3: In Vitro Assessment of Spontaneous Resistance Development:

[0049] The frequency of spontaneous resistance development was assessed by plating at least 1×10.sup.9 colony forming units of the respective bacterial strain on agar plates containing lysobactin at either 4x or 8x the MIC and incubating the plates at 35+/−2° C. After 48 h, the number of colonies that had grown on the plates at lysobactin concentration above the MIC was divided by the number of bacteria that was initially plated. The resulting number is defined as the spontaneous resistance frequency, and is an indication for the likelihood of resistant isolates to appear during an infection.

[0050] As a conclusion, lysobactin at 4- and 8-fold MIC displays a very good resistance profile: no resistant isolates were detectable. The results are summarized in the following tables:

[0051] Staphylococcus aureus ATCC 29740:

TABLE-US-00003 24 h 48 h Lysobactin plated obtained frequency of obtained frequency of concentration CFU colonies resistance colonies resistance 2 μg/ml (4x MIC) 3.52 × 10.sup.9 0 <2.84 × 10.sup.−10 0 <2.84 × 10.sup.−10 4 μg/ml (8x MIC) 3.52 × 10.sup.9 0 <2.84 × 10.sup.−10 0 <2.84 × 10.sup.−10

[0052] Streptococcus uberis ATCC 27958:

TABLE-US-00004 24 h 48 h Lysobactin plated obtained frequency of obtained frequency of concentration CFU colonies resistance colonies resistance 2 μg/ml (4x MIC) 2.4 × 10.sup.10 0 <4.17 × 10.sup.−11 0 <4.17 × 10.sup.−11  4 μg/ml(8x MIC) 2.4 × 10.sup.10 0 <4.17 × 10.sup.−11 0 <4.17 × 10.sup.−11

Example 4: In Vitro Assessment of Resistance Development During Serial Passaging

[0053] The appearance of MIC changes during constant exposure of bacteria to sub-MIC concentrations of lysobactin was assessed by serial passaging experiments. On the first day the MICs of S. aureus and S. uberis were assessed by microbroth dilution MIC methodology as described by the Clinical and Laboratory Standards Institute (CLSI). Each day for the following 33 consecutive days, bacteria from the well containing the highest concentrations allowing full growth were collected and used to inoculate new 96-well plates and to assess the MIC during constant exposure to lysobactin. The MIC obtained on each day was plotted over time. The results are shown in FIG. 2. Rifampicin, which is known to result in quick changes of the MIC, was used as positive control.

[0054] The results indicate a very good profile of lysobactin for resistance development during constant exposure, as the MICs for S. aureus and S. uberis remain constant over the 33 days period.

Example 5: Efficacy in an Acute Mouse Mastitis Model with S. aureus

[0055] The efficacy of lysobactin (formulated in a hydrogel at pH 4.7) was tested in a Staphylococcus aureus acute mouse mastitis model established at the University of Sherbrooke, Canada (Broui-llette et al, Vet. Microbiol. (2004) 4:253-262), that is hereby incorporated by reference. Both the abdominal mammary glands (L4 and R4) of lactating CD-1 mice were intramammarily infected with 100 CFU (colony forming units) of S. aureus. The mice were treated intramammarily with lysobactin four hours after infection. Each treatment group contained at least 3 mice (6 quarters) 14 hours later (18 hours after inoculation) mice were sacrificed, mammary glands were harvested and the CFU content evaluated by plating 10-fold serial dilutions of mammary gland homogenates. The CFU content was expressed as log.sub.10 count. The detection limit was 200 CFU/g of gland. Glands with less than 200 CFU/g were regarded as cleared. The results are shown in FIG. 3.

[0056] Intramammary instillation of 50 μg lysobactin reduces the median CFU content by ca. 4 log.sub.10, 400 μg lysobactin eliminates the infection from all infected glands.

Example 6: Efficacy in an Acute Mouse Mastitis Model with S. uberis

[0057] The efficacy of lysobactin (formulated in a hydrogel at pH 4.7) was tested in a Streptococcus uberis challenge mouse mastitis model. The results are shown in FIG. 4.

[0058] Twenty lactating mice were experimentally infected on the fourth pair of mammary glands with S. uberis around 12-15 days after birth of the offspring. Four hours after inoculation, groups of four mice were treated on the same glands with lysobactin at 100, 200, 400, or 800 μg/ gland formulated as hydrogel. The fifth group was treated solely with the hydrogel vehicle as negative control. Eighteen hours after infection the animals were euthanized, the glands were removed, homogenized, and bacterial colony forming units (CFUs) were determined by established microbiological methods. Subsequently CFU/mL homogenate as well as CFU/g of gland were calculated. The detection limit was approximately 100 CFU/g of gland. Antimicrobial activity of lysobactin against Strep uberis was determined by comparison of the mean CFUs/gland of the different dosing groups and the negative control group.

[0059] Glands of all animals of the control group showed an optimum infection rate (>10.sup.7 CFU/g of gland) 18 hours after infection. With one exception in the highest dosing group all glands in the lysobactin treated groups had bacterial counts below the limit of detection (10.sup.2 CFU/g). It can be concluded that lysobactin formulated as hydrogel has outstanding antibacterial efficacy against S. uberis at intramammary doses between 100 and 800 μg/gland.

Example 7: Lysobactin In Vivo Cattle Data—Milk Pharmacokinetic Study in Lactating Holstein Cows

[0060] The study was designed as a non-pivotal study suitable to investigate the milk pharmacokinetics of the active substance lysobactin after single intracisternal application to lactating dairy cows.

[0061] The active substance was provided in a paraffine based service formulation suitable for intracisternal application containing 150 mg lysobactin B in 10 g oily suspension.

[0062] The test item was administered as single intracisternal treatment at a dose rate of 150 mg lysobactin to a single hind quarter of four lactating dairy cows each.

[0063] The dairy cows on study (Holstein cows) represented the target population in age, lactation number, lactation stage, milk yield and breed. The animals were stalled in a tie-barn and were fed with standard feed for dairy cows consisting of corn and gras silage and milk performance feed Milking was twice daily at a 12 hour interval using a bucket-type milking device.

[0064] Frequent milk sampling was performed from the treated and respective control quarters prior to (0 h) and over a period of 168 h after treatment (0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, 84, 96, 120, 144, and 168 h) by manual stripping of the respective udder quarters. Milk samples at routine milking times were gathered prior to milking

[0065] Concentrations of the active substance lysobactin in milk were analyzed by HPLC with detection by tandem mass spectrometry. The limit of quantitation was 0.05 mg/L.

[0066] Pharmacokinetic evaluation of milk concentration data was based on non-compartmental methods and comprised PK-parameters to adequately describe the absorption, distribution and elimination profile of the active substance in milk

[0067] Summarized results derived from the treated udder quarters are presented in the following table. No lysobactin was detected in the control samples (untreated udder quarters).

[0068] Mean Milk Pharmacokinetic Results of Lysobactin after Single Treatment

TABLE-US-00005 Matrix C.sub.max.sup.1 T.sub.max.sup.2 t.sub.1/2.sup.1 AUC.sub.inf.sup.1 AUC.sub.0-12 h.sup.1 AUC.sub.0-24 h.sup.1 mg/L h h mg*h/L mg*h/L mg*h/L Milk 342 4 11.6 2321 1870 2213 Dose applied to 1 quarter per cow was 150 mg lysobactin; Means are given as .sup.1geometric mean, .sup.2median

[0069] The concentration time curve of lysobactin is depicted in FIG. 5.

Example 8: Lysobactin In Vivo Cattle Data—Dairy Cow Udder Infection Model with S. Aureus

[0070] Fifteen healthy lactating dairy cows were experimentally infected with the mastitis pathogen Staphylococcus aureus on all four udder quarters. As soon as an udder quarter showed clinical symptoms of mastitis, such as swelling, pain, abnormal milk consistency, it was treated with either Lysobactin paraffine based ointment at two different concentrations, or Ubrolexin® (Cephalexin+Kanamycin, Boehringer Ingelheim), or saline solution as negative control. Treatments were randomly assigned to 42 udder quarters in total, either 50 mg lysobactin per quarter (in 11 quarters), or 150 mg Lysobactin (in 11 quarters), or Ubrolexin® (in 9 quarters), or saline solution (in 11 quarters). Only udder quarters that were bacteriologically positive for the challenge organism immediately prior to treatment (n=42) were eligible for assessment of microbiological and clinical cure. The diseased quarters were treated with these intramammary formulations two times with an interval of 24 hours in between. Udders were clinically examined and milk samples were taken before treatment and several times after that until three weeks after the second administration. Milk was inspected visually for deviation in its consistency and samples were evaluated for presence of the challenge organism and for somatic cell count to confirm the diagnosis mastitis. Diseased udder quarters were considered microbiologically cured when Staphylococcus aureus found in milk samples shortly before treatment could not be isolated from any milk sample taken within the time period between the third and the twenty-first day after the second treatment. Clinical cure was achieved when the local symptoms of mastitis had completely disappeared, and recovery of somatic cell counts (SCC) as parameter of udder inflammation was attained when all counts remained below 500.000 cells per mL of milk in the period mentioned above.

[0071] The microbiological cure rate on Day 3 and Day 21 after second treatment was 73% and 27% for Lysobactin 50 mg, 73% and 45% for Lysobactin 150 mg, 78% and 44% for Ubrolexin®, and 0% and 0% for saline solution, respectively. The local clinical cure rate on Day 3 and Day 21 after second treatment was 45% and 73% for Lysobactin 50 mg, 37% and 82% for Lysobactin 150 mg, 33% and 67% for Ubrolexin®, and 55% and 73% for saline solution, respectively. The SCC recovery rate on Day 3 and Day 21 after second treatment was 36% and 45% for Lysobactin 50 mg, 36% and 91% for Lysobactin 150 mg, 56% and 67% for Ubrolexin®, and 36% and 27% for saline solution, respectively.

[0072] It can be concluded that Lysobactin 150 mg shows similar or even better efficacy in comparison to the positive control product Ubrolexin® and superiority to Lysobactin 50 mg and Saline solution in the parameters microbiological and clinical cure, and SCC recovery.

Example 9: Lysobactin In Vivo Cattle Data—Dairy Cow Udder Infection Model with S. Uberis

[0073] Seventy-two healthy lactating dairy cows were experimentally infected with the mastitis pathogen Streptococcus uberis on two udder quarters per cow. As soon as an udder quarter showed clinical symptoms of mastitis (e.g. heat, swelling, redness, pain, abnormal milk consistency), it was treated with either lysobactin paraffine based ointment at two different concentrations, or Ubrolexin® (cephalexin+kanamycin, Boehringer Ingelheim).

[0074] Treatments were randomly assigned to 41 (clinical) or 37 (microbiological) udder quarters in total, either 50 mg lysobactin per quarter (in 15/13 quarters), or 150 mg lysobactin (in 15/14 quarters), or Ubrolexin® (in 11/10 quarters) as positive control. The diseased quarters were treated with these intramammary formulations two times with an interval of 24 hours in between. Udders were clinically examined and milk samples were taken before treatment and several times after that until three weeks after the second administration. Milk was inspected visually for deviation in its consistency and samples were evaluated for presence of the challenge organism to confirm the diagnosis mastitis. Diseased udder quarters were considered microbiologically cured when Streptococcus uberis found in milk samples shortly before treatment could not be isolated from any milk sample taken within the time period between the seventh and the twenty-first day after the second treatment. Clinical cure was achieved when the local symptoms of mastitis completely disappeared or were at the most of very slight nature.

[0075] The microbiological cure rate on Day 7 and Day 21 after second treatment was 84.6% and 92.31% for lysobactin 50 mg, 71.4% and 85.71% for lysobactin 150 mg, and 30% and 30% for Ubrolexin®, respectively. The local clinical cure rate on Day 7 after second treatment was 86.7% both for lysobactin 50 mg and lysobactin 150 mg, and 36.4% for Ubrolexin®, respectively.

[0076] It can be concluded that lysobactin 50 and 150 mg were clearly more efficacious in comparison to the positive control product Ubrolexin® in the pivotal parameters microbiological and clinical cure.