KR100516418B1 - Veterinary Uses of Fluromutillin Derivatives - Google Patents

Abstract

It relates to the use of a compound of formula (I) in the treatment of livestock diseases in which expression is increased with increasing breeding density.

Formula I

Description

Veterinary Uses of Fluromutyline Derivatives

The present invention relates to fluromutillin derivatives. The present invention provides 14-O- [1- [R] -2 in the form of a compound of formula (I), ie, a free base or a veterinary acceptable salt, for the treatment of disease in livestock, the expression of which increases with increasing breeding density. -Amino-3-methylbutyrylamino] -2-methylpropan-2-ylthioacetyl] mutylin, hereinafter referred to simply as "use of the invention".

The compound of formula (I) (Econor®) in the form of a free base or veterinary acceptable salt will be referred to simply as "the formulation of the invention". The compounds of the invention are preferably in the form of salts, in particular in the form of hydrochlorides. The generic name of the compound having this form is known as balnemulin hydrochloride.

As used herein, the term "treatment" applies to therapeutic as well as prophylactic treatment.

Compounds of formula I in the form of free bases or in the form of chemotherapeutic or veterinary acceptable salts are for example EP 153 277 and their equivalents, for example USP 4 675 330, in particular Example 12 It is described in

From this, the following are known.

In vitro inhibitory activity against various bacteria at a concentration of 0.008-25 μg / ml.

In vitro inhibitory activity against mycoplasma and chlamydia in general at concentrations from 0.008 to 0.5 μg / ml.

In vivo inhibitory activity with various bacterial strains in rats and mycoplasma strains in hens when the dose is 12-50 mg / kg body weight.

In vivo antiparasitic activity against coccidia in chickens when the dose is 20-150 mg / kg feed.

In vivo growth promoting activity in hens and pigs when administered 10-50 mg / kg feed.

Therefore, the compounds of the present invention are generally useful as antibiotics and veterinary preparations with bacterial inhibitory activity, and in particular as chemotherapy treatments of coccidial disease in chickens as well as growth promoters in hens and pigs.

The inventors have found that the formulation of the invention is endemic pneumonia, Serpulina (formerly Treponema) hyodisenteriae in pigs caused by Mycoplasma hyopneumoniae infection. Swine colitis associated with swine colitis caused by infection, swollen colitis associated with serulina pilosicoli infection (inflammation of the colon), and ileitis of pig associated with Lausonia intracellularis infection Proliferative intestinal disease; swine intestinal adenomatosis), chronic respiratory disease and arthritis in poultry following mycoplasma gallisepticum infection, Pasteurella multocida, Actinobacillus (Hemophilus) (Actinobacillus Haemophilus) Secondary pneumonia, Pasteurella haemolitica in pigs following infection with Pleuroneumoniae and / or Haemophilus parasuis Breeding densities such as lamb, sheep and cattle pneumonia (shipping fever, transient fever, calf respiratory complications, bovine pneumonia Pasteurella infection), and polyarthritis in pigs following mycoplasma hyosynoviae infection It has been found to be particularly effective in the treatment of animal diseases in which expression is increased with increasing.

In addition, more surprisingly, the inventors found that the induction of resistance to the agent was extremely low.

Accordingly, the present invention relates to veterinary use as defined above.

The invention also relates to the use of the formulations of the invention for the preparation of a medicament for use in the treatment of livestock diseases in which expression is increased with increasing breeding density.

The present invention also relates to a method of treating livestock disease in which expression is increased with increasing breeding density, comprising administering a therapeutically effective amount of a formulation of the invention to an animal in need thereof.

In addition, the present invention includes a compound of formula (I) in the form of a free base or in the form of a veterinary acceptable salt and one or more veterinary acceptable carriers or diluents, the livestock whose expression is increased by increasing the breeding density It relates to a veterinary agent for treating a disease.

The invention also relates to a process for the preparation of a medicament for use as defined above, which comprises mixing a formulation of the invention with one or more veterinary acceptable carriers or diluents.

An animal suffering from a disease whose expression increases with increasing breeding density may not have been previously treated antimicrobially with a medicament according to the invention, or a medicament of the invention for promoting growth.

A) Mycoplasma hyopneumoniae infection:

Mycoplasma hyopneumoniae infections are described, for example, in Taylor, DJ, in Pig Diseases , 6th Ed. (1995), Publ. Diagnosis is done in a conventional manner as described in veterinary manuals such as DJ Taylor, Glasgow, UK on pages 164-165. The beneficial activity of the medicament of the present invention as used herein is measured as follows:

1. In vitro activity:

Ten field isolates and strain “J” types obtained from endemic pneumonia generation of various livestock herds are included in the test. Isolates are cloned by filtration, identified using disc growth inhibition test, and used in 7th to 10th passages. The minimum inhibitory concentration (MIC) test is carried out in a liquid medium in a 1.8 ml tube containing twice the concentration of Valnemulin and Thiamulin hydrogen fumarate. Friis, NF et al., Acta Vet. Scand . 32 [1991] 425-429. Mycoplasma is inoculated at a 10-fold dilution, and the culture is visually read for growth using a tube inoculated with 10 ² to 10 ⁴ color forming units. When the color shift no longer proceeds, the initial reading is done after 2-4 days and the final reading is after 10-14 days. MIC is determined as the lowest concentration of test compound that exhibits growth inhibition compared to the control test compound. Friis, NF and Szancer, J., Acta Vet. Scand. 35 [1994] 389-394.

The results are shown in Table 1:

TABLE 1

All mycoplasma hyopneumoniae strains tested were highly affected by balnemulin when the MIC value was 10-40 times smaller than thiamulin in both the initial and final readings. Thus, the compounds of the present invention are particularly useful for the clinical treatment of infected herds.

2. In vitro activity

Tissue samples are obtained from the lungs of pigs in a herd of livestock with various endemic pneumonia (EPP). The sample is delivered to dry ice and incubated. Mycoplasma Experimental Mycoplasma hyopneumoniae is recovered from freshly cut lung tissue by direct culturing on agar. This technique allows cloning from other faster growing mycoplasma species, such as Mycoplasma hyorinis, which are often present in EPP lung samples, to identify and easily isolate Mycoplasma hyopneumoniae's unique colonies. Isolates are passaged and identified as disc growth inhibition using specific rabbit antiserum derived against Mycoplasma hyopneumoniae NCTC 10110.

Cultures for antibiotic challenge are prepared in Mycoplasma experimental broth incubated under aerobic conditions at 36 ° C.

Commercially available solid medium (Mycoplasma Experience Ltd.) is used to isolate Mycoplasma hyopneumoniae from lung tissue. Liquid medium (Mycoplasma Experience Ltd.) containing phenol red and glocos (pH 7.6) is used for MIC analysis.

Stock solutions of test compounds are prepared at a concentration of 1 mg / ml in deionized water, sterilized by filtration through a 0.2 μ pore size membrane filter (Sartorius Minisart, N) and stored at −20 ° C. For use in the MIC test, the stock solution is diluted in the liquid medium at twice the required initial concentration. The MIC test is performed according to the method of Tanner & Wu, Avian Diseases 36 (1992) 714-717. Actively growing challenge medium is prepared from 1 ml aliquots of broth culture stored at −70 ° C. or cultures stored in agar at −70 ° C. Challenge cultures are diluted to a desired titer of 10 ³ to 10 ⁵ color change units / ml. 0.1 ml aliquots of challenge inoculum are mixed with 0.1 ml aliquots of antibiotic diluent in microtiter wells. Each microtiter plate comprises a medium (Mycoplasma hyopneumoniae) (endpoint control) not inoculated at pH 6.8 and an inoculated challenge control without antibiotics. All plates are sealed with adhesive film and incubated aerobicly at 36 ° C. MIC is recorded when the color change in the challenge control well matches the pH of the endpoint control. MIC is the lowest concentration with no color change.

Results for valnemulin and two reference compounds, thiamulin and enrofloxacin, are shown in Table 2.

TABLE 2

All strains have been shown to be highly sensitive to balnemulin. Most strains showed very high sensitivity to enlofloxacin, but for individual strains, the MIC of Valnemulin is more than five times lower than the sensitivity to 0.0025 μg / ml enlofloxacin. These results indicate that the field isolates are very sensitive to balnemulin.

3. Generation of tolerance

Mycoplasma hyopneumoniae reference strain NCTC 10110 (obtained from the National Collection of Type Culture, London, UK) and the recent field isolate (MEVT G23), myco containing phenol red until color change by acid occurs Incubate aerobically at 36 ° C. in plasma glucose broth. After addition of sterile glycerol (5% v / v), the culture is divided into 1 ml aliquots and frozen at -70 ° C. These cultures are used to initialize additional broth cultures that are titrated to obtain the number of color change units (ccu) in the microtiter plate after culture (36 ° C.). Replication allows the challenge of antibiotic dilutions showing ccu number premeasured in primary culture in minimal inhibitory concentration (MIC) tests and habit studies.

A commercial medium (Mycoplasma Experience Ltd.) containing glucose and phenol red (MEGB) is used at pH 7.6.

Stock test compound solutions are prepared at 1000 μg / ml in deionized water. After shaking, the solution is sterilized by passing through a membrane filter (Sartorius Minisart, N) with a pore size of 0.2μ. For use in the MIC test, the test stock solution is diluted in mycoplasma broth to double the final concentration required. In habit studies, the stock solution is divided into 1 ml aliquots and frozen at -20 ° C. It is thawed and used at intervals of 5 to 7 days to prepare the concentration range of the preparation in MEGB in 1.9 ml aliquots containing MIC for two strains of Mycoplasma hyopneumoniae. Oxytetracycline hydrochloride is prepared immediately in each case. Each pass of the dilution range is gradually changed to allow resistance in mycoplasma to develop.

Test Methods:

The MIC test is performed according to the method of Tanner & Wu, Avian Diseases 36 (1992) 714-717 before conducting a habit study (below), and after completion of the 10th passage, the compounds in microtiter wells An aliquot of 0.1 ml of dilution is mixed with a 0.1 ml aliquot of challenge inoculum containing 10 ³ to 10 ⁵ color change units (ccu) per ml. Each microtiter plate comprises an uninoculated medium, a pH 6.8 medium (end point control) and an inoculated challenge control without preparation. All plates are sealed and incubated aerobicly at 36 ° C. MIC is recorded when the change in color in the challenge control well is consistent with the pH 6.8 control (orange-yellow). MIC is the lowest concentration at which no color change occurs.

Habit study: In the primary passaging test, 1.9 ml volume of antibiotic solution at 0.1 ml broth culture containing 10 ³ ccu / ml to 10 ⁵ ccu / ml at a concentration containing MIC of Mycoplasma hyopneumoniae strains. Inoculate. A growth control consisting of broth with no preparation and an uninoculated medium control inoculated with Mycoplasma hyopneumoniae is included in each subculture experiment. After 7 days of incubation (36 ° C.), two drug-containing broths with the highest concentration of acidic color change were collected and used for mycoplasma broth (0.1 ml culture in a 1.9 ml formulation containing broth). Inoculate a series of fresh preparation dilutions. This process is repeated every 5-7 days for passage up to 10 cultures. When mycoplasma strains are resistant to specific antibiotics or resistant in the 10th culture, each strain is grown once more in drug containing broth and the MIC is measured.

Results: MICs of mycoplasma hyopneumoniae strain "J" and field isolates before and after exposure to antibiotics are shown in Table 3.

TABLE 3

1) In vitro crab culture level

2) MIC after 8 passages in Mycoplasma broth containing tyrosine

When the MIC of the reference strain was 0.0025 μg / ml and the MIC of the field isolate was 0.001 μg / ml, the two strains prior to exposure were found to be highly sensitive to Valnemulin. This activity level is 50 to 100 times greater than the levels of tyrosine and oxytetracycline. The MIC results are used to select dilution ranges of drugs in habit studies.

After 10 exposures to Valnemulin, resistance development was minimal in both strains of Mycoplasma hyopneumoniae, and the MICs of the reference strain and the field isolates were 0.0025 μg / ml to 0.005 μg / ml, respectively, at 0.001 μg / ml. Increase to 0.0025 μg / ml. In contrast, the indicated resistance develops against tyrosine tartrate in both strains of Mycoplasma hyopneumoniae. In the reference strain, resistance occurs first within four to five passages in a tyrosine containing broth, and up to eight passages, the MIC rises above 500 μg / ml, which increases the resistance by more than 2000-fold for this antibiotic. Reflects the result (Table 3). The indicated tyrosine resistance occurred in the field isolates within 8 passages and MIC increased 500-fold to 62.5 μg / ml. This resistance level is maintained after 10-11 passages in tyrosine containing broth. During 10 cycles of exposure, both strains of Mycoplasma hyopneumoniae have a 4-fold increase in resistance to oxytetracycline and the MIC rises from 0.25 μg / ml to 1 μg / ml.

From these results, it can be seen that Balnemulin is much more active than mycoplasma hyopneumoniae than tyrosine or oxytetracycline and does not induce significant resistance to itself in mycoplasma hyopneumoniae strains.

4. Prevention of experimentally induced endemic pneumonia in vivo

The disease prevention effect of Valnemulin was evaluated using an experimental model of endemic pneumonia using passaged lung tissue induced by sterile pigs infected with Mycoplasma hyopneumoniae. Three tests are performed: Test 1 and Test 2 are dose titration studies of new compounds using the Mycoplasma hyopneumoniae standard challenge strain, and Test 3 assesses the effects of 200 ppm of compounds in feed on secondary challenge strains. It is. A large white Landrace male pig, 6-7 weeks old, raised in a stall not infected with Mycoplasma hyopneumoniae. The challenge substance is a lung with pneumonia containing Mycoplasma hyopneumoniae, administered intranasally as a homogenate for three days. The minimum inhibitory concentration (MIC) of Valnemulin for the strains present in the material used in Test 1 and Test 2 was 0.016 μg / ml, and the MIC for the strain isolated from the material used in Test 3 was 0.0078 μg / ml. . Challenge materials are induced by mycoplasma hyopneumoniae infection of sterile pigs and subsequent passage in SPF (no specific lesions) pigs and stored below −70 ° C.

Trial 1: Dosing with Valnemulin hydrochloride in the gastrointestinal tract (feeding) in an amount of 0 (control), 2.5, 5, 7.5 or 10 mg / kg body weight once daily. 6 pigs per group.

Trial 2: Dosing with Valnemulin hydrochloride added to the feed at 0, 100, 200, 300 or 400 ppm (corresponding to 0, 5, 10, 15 and 20 mg / kg / day). 6 pigs per group.

Trial 3: Dosing with Valnemulin hydrochloride added to the feed at 0 or 200 ppm (equivalent to 0 and 10 mg / kg / day). 8 pigs per group. Dosing is performed from 1 day after challenge infection and autopsy is performed about 3 weeks after challenge infection. The disease was determined by measuring lung-weight ratio and isolation of Mycoplasma hyopneumoniae from the lung [Cambridge Lesion Scoring System (Goodwin, RFW and Whittlestone, P., J. Hyg. 69 [1971] 391-397). ), This score is the approximate percentage of lungs affected by pneumonia.

The results are shown in Table 4.

TABLE 4

1): MIC μg / ml of Valnemulin hydrochloride for Mycoplasma hyopneumoniae isolated from challenge material.

2) approximate daily dose of Valnemulin hydrochloride per pig

In Trial 1, the average lesion score in undosed challenge pigs was 13.2. Valnemulin reduced lesions by 71% and 87% at doses of 7.5 and 10 mg / kg, respectively, while being somewhat less effective at reducing lesions at doses of 2.5 and 5 mg / kg. Mycoplasma hyopneumoniae is trimmed (1 vs. 4 pigs) from a few pigs treated with 10 mg / kg compared to pigs treated with 2.5 mg / kg.

In Trial 2, the average lesion score in untreated challenge pigs was 22.1. In pigs treated with 200, 300 or 400 ppm of Valnemulin, lesions were found to be reduced by 46%, 43% and 71%, respectively. Lung weight reflecting not only total lesions but also microscopic lesions showed a more dramatic effect with significantly reduced levels below 200 ppm. The lesions were not reduced in pigs treated with 100 ppm. Mycoplasma hyopneumoniae was trimmed from all pigs not treated at necropsy, but not from pigs treated with 400 or 300 ppm of Valnemulin. Mycoplasma ioniomoniae was trimmed from pigs 3 and 4 treated with 200 and 100 ppm, respectively.

In Trial 3, the mean lesion score in untreated pigs was 10.8. Feeding a drug containing 200 ppm of Valnemulin in the diet reduced the lesion score by 79%. The levels of mycoplasma hyopneumoniae detected at necropsy did not differ between treated and untreated pigs.

Thus, Valnemulin has been proven effective in the prevention of experimentally induced endemic pneumonia in pigs in individual experiments with challenge materials containing two different Mycoplasma hyopneumoniae strains.

Accordingly, the medicaments of the present invention are useful for treating endemic pneumonia in pigs caused by Mycoplasma hyopneumoniae infection. Effective dosages for these uses will of course depend on the particular salt used, the route of administration, the size and age of the animal and the desired effect; For example, prophylactic treatment will require relatively small doses to be administered over a long period of time. In general, however, satisfactory results are obtained when the drug is administered at a dosage of about 5 mg to 15 mg per kg body weight of the animal per day, suitably in divided doses 2 to 4 times per day, or in a sustained release form. Obtained. For most animals, the total daily dose is about 100 to about 1000 mg, preferably about 100 to about 500 mg, given once or twice daily.

It can advantageously be administered as a single therapy.

Preferred doses in drinking water are from 0.01 to 0.05% by volume, in particular from 0.01 to 0.025% by volume, with a preferred dosage in the feed being from 100 to 400 ppm (g / metric ton), in particular from 100 to 200 ppm (g / metric ton).

B) Serpulina High Odysenteria Infection:

Serpulina hyodicenteria infection can be diagnosed by conventional methods, for example, as described in the Veterinary Manual. Taylor, D. J., in Pig Diseases, 6th Ed. (1995), Publ. D. J. Taylor, Glasgow, U. K. on page 143-144. The beneficial activity of the medicaments of the invention in this use is measured as follows:

1. MIC measurement

From the onset of dysentery in pigs. Nine field isolates of high odysenteria, and strains of this type (ATCC 31212) and ATCC 29796 (Serpullina innosense, group 3) [Fellstrom, C., Res. Vet. Sci. 59 [1995] 1-4]. 5% bovine blood is used to identify hemolysis patterns, indole production, and hypofury hydrolysis (Rosco Diagnostic Tablets, Taastrup, DK) in Tryticase soy agar (TSA). Bacteria were transferred from agar plates to 0.9% saline, turbidity was adjusted to 1.0 on McFarland, and then 10 μl of each isolate was doubled to valemulin hydrochloride, thiamulin hydrogen fumarate, dimethidazole, linco Inoculate agar plates with mycin hydrochloride and tyrosine. Growth and hemolysis are recorded after 4 days of growth under anaerobic conditions, and MIC is measured as the lowest concentration of antibiotic in which the spirochete does not grow.

s. The MIC measurement results for the high odyscenteria are shown in Table 5:

TABLE 5

All s. Hiodicenteria strains exhibit high susceptibility to Valnemulin, which exhibits a MIC value of 2 to 32 times less than the MIC value of thiamulline. s. Because of the high sensitivity of Valnemulin to high odyscenteria, the compounds of the present invention can be used for clinical treatment in infected livestock herds.

2. MIC Measurement:

The minimum inhibitory concentration (MIC) was determined from S. aureus isolated from porcine feces. Ten strains of Hiodicenteria are measured by agar dilution. National Collection of Type Cultures (NCTC) strains or reference strains are used as controls in all MIC tests. s. The agar medium for high odyscenteria consists of BAB2 (Unipath CM271) containing 7% sterile amounts of whole blood depleted of fibrin. All antibiotics are tested in double dilutions. The prepared antibiotic dilution was previously cooled to 50 ° C., mixed and added to a suitable volume of MH agar poured in 20 ml each. All s. Incubation of the high odyscenteria is carried out at an anaerobic working station (Don Whitley Scientific) at 37 ° C. (+/− 0.5 ° C.) providing a strict anaerobic atmosphere comprising 80% nitrogen, 10% hydrogen and 10% carbon dioxide. All other incubations are performed at 37 ° C. All strains are incubated at 37 ° C. for 24 hours under aerobic atmosphere. The number of microorganisms is adjusted to an optical density (OD) corresponding to 1 × 10 ⁸ colony forming units (cfu) per ml using MH broth as blank. Antibiotic containing plates are inoculated twice using a multipoint inoculator (Denley Instruments) which transports approximately 1 μl of plate inoculum providing 10 ⁴ to 10 ⁵ cfu of inoculum per spot for all strains. Amon, J. Antimicrob. Chemother . 21 [1988] 701-710: Ericsson et al., Acta Pathol. Microbiol. et Immunol. Scand . 217 [1971] (B) Suppl., 1-90. The control plate is inoculated under the same conditions as the test plate. The MIC is read after 24 and 48 hours and the MIC after 48 hours is the final reading. MIC is defined as the lowest concentration of antibiotic without microbial proliferation, ignoring single colonies or faintly turbid sites caused by inoculum [National Committee for Clinical Laboratory Standards [1989], Antimicrobial Susceptibility Testing , NCCLC Publications SC3, Villanova, PA, USA].

MICs for Serpulina high odyscenteria in μg / ml are Balnemulin (0.1), Thiamulin (0.3), Lincomycin (50.0), Tylosin (200.0) and Dimethidazole (30.0). s. All strains of hyodicenteria are susceptible to balnemulin, which is most active in formulations tested 3 to 600 fold.

3. Challenge Attempts :

Two tests were performed to compare valemulin with thiarmulin for the prevention of swine dysentery supplied at varying levels of inclusion. The severity of the disease is assessed by evidence of clinical disease, as determined by clinical scores on the condition, fecal hardness and composition of the body. The excretion of serpulina or high odysenteria in feces, lesions at necropsy, growth rate and feed conversion ratio are also measured.

For the first test, Valnemulin hydrochloride supplied at 20, 30 and 40 ppm is compared to thiamulline hydrogen fumarate and untreated drug supplied at 30 ppm. Typically, five groups of nine pigs (5-6 weeks old) are used as treatment groups, respectively. Pigs were fed untreated drug for 14 days and then S. Challenge with standard strains of Hiodicenteria (P18A). Challenge is performed by the oral route for two consecutive days. Drug treated feeds are fed on the day of the second challenge. For the second test, a similar procedure is followed, except that four groups of approximately nine are used. Valnemulin hydrochloride at 5, 10 and 20 ppm is fed to four groups of pigs and compared to the untreated drug control. In this test, the pigs used were taken from field breeding herds and isolated from the outdoor herds and were expected to be able to develop pig dysentery ahead. Challenge with strains of high odystheria. In both trials, evidence of clinical disease is assessed daily and assigned clinical scores and S. Isolates of high odystheria are collected twice a week, and all pigs are weighed at regular intervals. Feed intake is also recorded. An autopsy is performed 21 days after challenge and the colon is checked for the presence of the lesion. The mucosal wounds were taken from four parts of the large intestine. Incubate to isolate the high odystheria.

In the first trial, clinical swine dysentery first appeared in the non-dose control group 8 days after challenge, with 8 of 9 pigs diseased. Clinical disease also occurs in one pig in the group fed with 30 ppm of balnemulin, and in two out of nine animals in the thiamulin group. Evidence of disease is not seen in the 20 and 40 ppm balnemulin treatment groups. The average clinical score per pig is 36 for the control group and 6 for the thiamulin treatment group. The difference between the scores recorded in all treatment groups is statistically different from the control group (p <0.001). s. Hiodicenteria is isolated from rectal specimens taken from all pigs in the control group and also two diseased pigs from the thiamulin treatment group. s. Hiodicenteria is also isolated from two pigs fed with 30 ppm of Valnemulin, consistent with clinical signs of swine dysentery following an incidental infection in this group. s. Hiodicenteria are not isolated from the 20 and 40 ppm groups. The weight gain or feed conversion ratios for all groups fed the drug-treated feed were slightly different. Pigs in the control and thiamulin-treated groups with clinical signs of disease at necropsy show typical lesions of swine dysentery consisting of adherent mucus with necrosis of the colonic mucosal surface. The lesions also appeared in one pig and control pigs in the thiamulin treatment group, both of which previously had no clinical signs of swine dysentery. s. Hiodicenteria is isolated from mucosal wound sites taken from all of these diseased animals. One pig in the group fed 30 ppm of Valnemulin also had lesions of swine dysentery, but S. Hiodicenteria are not isolated from mucosal wounds. Lesions do not appear at necropsy of the groups fed with 20 and 40 ppm of Valnemulin.

In the second trial, clinical swine dysentery was first observed in non-dosed control pigs 5 days after challenge, with all 9 pigs in this group diseased, 6 of which died from the severity of the disease. Clinical signs of swine dysentery were first observed 8 days after challenge in groups fed 5 ppm of Valnemulin, 5 of 10 animals became ill. However, the number of diseases in the 5 ppm group of Balnemulin was much less than that of the control group (p <0.05). Clinical signs of the disease are not observed in the group fed with 10 or 20 ppm of balnemulin. The mean clinical score in the 5 ppm group of Valnemulin was much smaller than the score 77 of the control group (p <0.005). s. High odyscenteria is isolated from rectal specimens taken from all control pigs during the test. However, only from 2 out of 5 animals affected in the Balnemulin 5 ppm group. High Odysenteria is isolated. Pigs fed Valnemulin at all concentrations weighed significantly more than the control group at the end of the test (5 ppm, p = 0.05, 10 and 20 ppm, p <0.05, respectively). At necropsy, a typical lesion of swine dysentery is seen in the colonic mucosa of the three remaining control pigs. In the group fed with 5 ppm of Valnemulin, out of 5 pigs showing signs of inherent disease, only 2 pigs showed lesions at necropsy. Hiodicenteria were not isolated from these animals. However, S. Hiodicenteria are isolated from mucosal wounds taken from two other animals with typical lesions at necropsy but no clinical signs of the disease. Lesions do not appear in pigs fed with 10 or 20 ppm of balnemulin, Hiodicenteria are not isolated from these groups.

Therefore, at a content rate lowered to 10 ppm, balnemulin is effective for the development of clinical signs of swine dysentery, the separation of spirochetetes in manure and the presence of lesions at necropsy. At 30 ppm some pigs showed clinical signs of swine dysentery, but this coincided with an incidental infection that could affect the intake of antibiotics. The effects of the disease concurrent with the intake of antibiotics and the development of swine dysentery have already been described by Burch, D. G. S., Vet. Rec. 110 (1982) 244-246. Analysis of the weight gain data shows that there are no differences between treatment groups, indicating that antibiotics do not affect the taste of the feed. Valnemulin at 5 ppm does not completely prevent clinical signs or the division of the spirochete, but significantly lowers the clinical score and the division of the spirochete when compared to the control group. In this trial, thiamulin supplied at 30 ppm does not prevent swine dysentery, but when compared to the control group, the clinical score is reduced while reducing the incidence of the disease. However, in these trials, feed dosing with 10 ppm of Valnemulin is clearly more effective than 30 ppm of thiamulin in the prevention of swine dysentery.

4. Additional Challenge Exams

A group of 60 pigs (3.5 to 4 weeks old) typically fed untreated feed for 14 days before S. Challenge by the oral route using standard strains of Hiodicentriae (P18A). If clinical signs of the disease appear, pigs are divided into six treatment groups of eight per group, antibiotic-containing feeds are fed for 10 days and then non-drug feeds are additionally fed for 14 days. Valnemulin hydrochloride is fed at 50 ppm, 75 ppm, 100 ppm and 150 ppm and compared to untreated control and 100 ppm of thiamulline hydrogen fumarate. Pigs showing clinical signs of disease are included in each treatment group. After assigning treatment groups, daily evidence of clinical disease is observed and clinical scores assigned, S. Rectal samples are taken twice weekly to isolate the high odyscenteria and all pigs are weighed at regular intervals. Feed intake is also recorded. An autopsy is performed 24 days after challenge and the presence of lesions in the colon of each pig is observed. Mucosal wounds are also taken from four parts of the large intestine. Incubate to isolate the high odystheria.

After challenge and before assignment, 7 days after the clinical porcine dysentery was infected and the first two treatment groups (control and thiamulin) were formed 8 days after challenge. The other group is formed 12, 15, 20 and 28 days after challenge in the order of 75 ppm, 50 ppm, 100 ppm and 150 ppm of Balnemulin. Each group includes pigs with blood and / or mucus in feces as well as animals with only mild clinical signs of disease, ie soft feces. All pigs in the non-drug control group died with severe clinical signs of the disease on day 4 of testing. During the treatment, a total of three animals in the thiamulin group had to die or be euthanized due to severe swine dysentery. All pigs treated with Valnemulin survived until the end of the test. In the group fed all levels of balnemulin, clinical signs of the disease are quickly cleared and no clinical disease is observed until five days after testing. By day 8, it was observed that pigs fed thiamulin survived. The 4-10 average clinical scores recorded for the Balnemulin group are compared with the 30 average scores recorded for the Tiamulin group. Statistical analysis of clinical scores for 3 to 10 days indicated a significant difference (p <0.001) between all groups fed valemulin and the thiamulin treated group. All pigs fed with Valnemulin continued to gain weight during the treatment period and had a higher daily survival weight gain (DLWG) than the thiamulin group (range of 0.4 to 0.9 compared to 0.1). Feed conversion rate (FCR) is much greater in the thiamulline treated group (6.4) compared to the balnemulin group (range from 1.9 to 2.5) during the treatment. s. Hiodicenteria are isolated from most pigs of each treatment group when assigned. Five days after the assignment, S. Hiodicenteria is not detected in any rectal specimens taken from pigs in the treatment group.

During the post-treatment period, one pig in the group fed with 50 ppm of balnemulin showed clinical signs of disease 4 days after discontinuation of the drug-treated feed, and also in one pig in the thiamulin treated group. Pig dysentery is observed on the last day of the test. However, S. Hiodicenteria are not isolated from the feces of these pigs. In this post-treatment period, a slight difference for DLWG or FCR is observed between all treatment groups. At necropsy performed at the end of the test, lesions of swine dysentery are not observed in all pigs fed with 75, 100 or 150 ppm of Valnemulin. Among the groups fed 50 ppm of valemulin, clinical pig dysentery was seen in one pig, and the colon was slightly reddened in the other pigs of the group at necropsy. s. Hiodicenteria are also isolated from the mucosal wound site from the two pigs and from three other animals in the group that do not have lesions of swine dysentery. One pig in the 75 ppm dose of Valnemulin was treated with mucosal wounds from the mucosal wounds, although no clinical porcine dysentery was observed. High odysenteria is obtained.

Thus, 50, 75, 100 and 150 ppm of balnemulin reduces the number of days needed to recover from clinical porcine dysentery when compared to thiamulin. In addition, animals in these Balnemulin treatment groups do not die or require euthanasia. There is a significant difference (p <0.001) in clinical scores for the Balnemulin treated group compared to the Tiamullin group. S in feces. The division of high odystheria is also inhibited by all concentrations of balnemulin after withdrawal of the administered feed. However, S. Hiodicenteria is isolated from mucosal wound sites from pigs injected with 50 and 70 ppm of Valnemulin. 35 to 40 ppm of thiamulin is obtained from feces. The high odyscenteria is removed but from the mucosal wound site. It does not remove the high odystheria. See Taylor, DJ, Vet. Rec . 106 [1980] 526-528. 100 ppm of thiamulline is a test condition for treating swine dysentery in animals that are so severely affected by anorexia that the animal will not lose food. See Taylor, DJ, Proc. It was successfully used under the 7th IPVS Congress Mexico [1982]. In this trial, 100 ppm of thiamulin did not prevent some deaths, but reduced mortality compared to non-dose controls, which may be due to the severity of the disease derived from this experimental challenge.

In these trials, 50, 75, 100 and 150 ppm of balnemulin are fed to the feed to successfully treat experimentally developed swine dysentery while suppressing mortality and eliminating clinical signs. After treatment, relapse is stopped only in one animal treated with 50 ppm and no S. Hyodysenteric excretion is inhibited during treatment. 50 to 75 ppm of balnemulin was obtained from all pigs. While not completely removing high odystheria, 100 ppm is very effective.

Thus, the formulations of the present invention are useful for the treatment of swine dysentery resulting from serpulina hyodicenteria infection. For this use, the effective dosage will of course depend on the particular salt used, the dosage form, the size and age of the animal and the desired effect, for example for a prophylactic treatment a relatively small dosage will have to be administered for a long time. In general, however, the daily dosage of the formulation may be administered in divided doses of 2 to 4 times daily, optionally in admixture with food or water, or in sustained release form, in a daily dosage of about 1 to about 5 mg per animal body weight. Satisfactory results are obtained. For most animals, the total daily dose is about 10 to about 400 mg, for example about 10 to about 200 mg for prophylaxis, or about 20 to about 400 mg for therapeutic purposes, optionally mixed with food or water, Or divided doses once or twice daily. Preferred dosages in drinking water are 0.001 to 0.05% by weight, in particular 0.001 to 0.005% by weight, and the dosage in the feed is 20 to 100 g / metric tons, in particular 20 to 75 g / metric ton.

C) Porcine colitis associated with serulina phylloccoli infections:

Colitis is described, for example, in Taylor, D. J., in Pig Diseases, 6th Ed. (1995), Publ. D. J. Taylor, Glasgow, U. K. on pages 148-149 can be diagnosed by conventional methods. The beneficial activity of the formulations of the invention for this use is measured, for example, as follows:

1. MIC value

Among the herds with or without diarrhea, nine field isolates of WBHS and ATCC 29796 (Serpulina innocens, Group 3) from dysentery pigs (Fellstrom, C., Res, Vet. Sci. 59 [1995] 1-4) This includes. The identification is based on hemolysis patterns on Trypticase soy agar (TSA) containing 5% bovine blood, and indole production and hypofury hydrolysis tests (Rosco Diagnostic Tablets, Taastrup, DK). One of the nine weak beta-hemolytic spirokits is designated as Group 2, 4 as Group 3, and 5 as Group 4 (Fellstrom, ibid). Bacteria are transferred from agar plates to 0.9% saline, the turbidity is adjusted to 1.0 on the McFarland scale, and 10 μl of each isolate is inoculated into agar plates containing twice the concentration of the following antimicrobial agents: Valnemulin hydrochloride, thia Mullin hydrogen fumalate, dimethidazole, lincomycin hydrochloride and tyrosine. Growth and hemolysis are recorded anaerobicly for 4 days and then recorded, and MIC is measured as the minimum concentration of antibiotic in which the spirochete does not grow.

Table 6 shows the results of the MIC measurements for WBHS. In general, WBHS is sensitive to all five antimicrobial agents. There is no difference in sensitivity between the three groups of WBHS for all antimicrobials during the trial. The MIC values obtained for Valnemulin are minimal in 9 of 10 strains, while this MIC value is much higher for most strains containing all four reference compounds.

Because of its high sensitivity to both WBHS, it is very useful for use in clinical treatment in infected herds.

TABLE 6

Therefore, the formulations of the present invention are useful for the treatment of swollen colitis associated with serulina or phylloccoli infection. In this use, the effective amount will of course vary depending on the particular salt used, the mode of administration, the size and age of the animal, and the desired effect; For example, for prophylactic treatment, relatively low doses are administered for a long time. Generally, however, the reagent is in a daily dosage of about 1 mg to about 5 mg per kg body weight, suitably divided into two to four divided doses per day or optionally mixed with feed or water. Or when administered in sustained release, satisfactory results are obtained. The total daily dose for most animals is about 10 mg to about 400 mg, for example, about 10 mg to about 200 mg for prophylaxis or about 20 mg to 400 mg for therapeutic use and optionally in mixed form with feed or water. Dose once or twice daily.

Preferred dosages in drinking water are 0.001 to 0.05% by weight, in particular 0.001 to 0.005% by weight and dosages in the feed are 20 to 100 g / metric tons, in particular 20 to 75 g / metric ton.

D) Spleen in pigs associated with Lausonia intracellular infection

Lausonia intracellulitis infection is a common method, for example see Taylor, D. J., in Pig Diseases, 6th Ed. (1995), Publ. D.J. Taylor, Glasgow, U.K. Diagnosis can be made by methods described in the Veterinary Manual, such as on pages 154-157. In this use, the beneficial activity of the agent of the present invention is measured, for example, as follows.

1. In vitro activity

See, Lawson, G.H.K. et al., J. Clin. Microbiol. 31 (1993) 1136-1142 is performed with a slight modification (the standard Kirby- Bauer disc method cannot be applied to intracellular bacteria).

Cells: Monolayer of IEC-18 rat enterocyte culture (ATCC CRL 1589) is established and maintained by standard cell culture methods. At certain times, aged monolayers (about 30% confluence) of dividing cells are prepared for infection on glass coverslips in small culture vials on the first day.

Inoculum: A batch of three Lausonia intracellularis strains is used as the inoculum. They are derived from pigs with a form of proliferative bowel disease, proliferative hemorrhagic bowel disease (PHE) and swine enteromatosis (PIA) that develop in swine intestine and are passaged in cell cultures as described in the Lawson reference above. . See McOrist, S. et al., Infect. Immun. 61 (1993) 4286-4292 two PHE strains are partially tested for pathogenic in pigs as described. Inoculation batches are harvested after several cell passages and stored frozen at −70 ° C. in 1 ml vials. Preliminary tests establish appropriate dilutions of thawed vials to generate cell infections that are readily identifiable in the 5 day test.

Antibiotics are prepared in 100-fold stock solutions for each use. Antibiotic activity of each batch is confirmed by standard Kirby-Bauer disc methods using laboratory strain Escherichia coli.

Infection: Each strain vial is thawed on the first day and diluted to 1/8 to 1/32 using culture medium for use in groups of 1 to 4.

In group 4 (continuous treatment), the initial inoculation is incubated for one hour at 37 ° C. in culture medium containing varying concentrations of antibiotics prior to addition to the cells.

For group 3 (extracellular activity), the inoculum is prepared in culture medium containing various concentrations of antibiotics added immediately prior to addition to the cells.

For groups 1 (control) and 2 (intracellular activity), inoculum is prepared in antibiotic-free culture medium and added to the cells. Culture Vials (Tracs) containing cells with added inoculum (+/- antibiotic) in 0.5 ml of medium were placed in a still bottle and evaporated to 40% atmospheric pressure (8% O ₂ ) and allowed to stand for 2 minutes. Hydrogen and finally 10% CO ₂ are fed back. Tracs are removed from this steel bottle and placed in an incubator to supply wet air of 8% O ₂ , 8.8% CO ₂ and residual nitrogen at 37 ° C.

All vials were taken one and two days and groups 2 and 4 were fed with medium containing antibiotics and groups 1 and 3 were fresh medium without antibiotics and placed back into the incubator. On day 5, all cover glasses are removed and stained for Lausonia intracellularis via indirect immunoperoxidase staining incorporating specific monoclonal antibodies. The number of cells heavily infected with Lausonia intracellularis (> 30 per cell, heavily infected cells = HIC) on each cover slip is used as a measure of infection. The lesion and total cell counts of HIC are also shown. The HICs in the control group (Group 1), intracellular active antibiotic assay (Group 2), extracellular active antibiotic assay (Group 3) and continuous treatment assay (Group 4) are compared by triple assay for up to three strains each. Certain assays are repeated at various inoculum concentrations. The percentage after induction of the mean HIC of the control group for the test group is calculated. The average control HIC is the total number of HICs in the control track divided by the number of infected control tracks. The percentage of test tracks is then calculated by dividing the HIC values of these test tracks by the average control HIC and multiplying by 100 to convert to percentages. That is, the test tracks where antibiotics have no effect will have a percentage of about 100, and the percentage of test tracks where antibiotics will completely inhibit growth is zero.

The results obtained using Valnemulin hydrochloride are as shown in Table 7 showing the percentage of uninfected cells after treatment.

TABLE 7

The results indicate that the minimum concentration of Valnemulin hydrochloride that can significantly inhibit (<1% growth) the growth of Lausonia intracellularis is <2 μg / ml.

2. Challenge Study:

35 piglets are described in Lawson et al., J. Clin . Microbiol . 31 (1993) 1136-1142, Lausonia intracellularis, a isolate of the inducer of swine proliferative intestinal disease obtained by culturing in the rat enterocyst cell line IEC-18 (ATCC CRL 1589). Challenge with the same toxic inoculum of strain LR 189/5/83). Seven control pigs are administered buffer. Seven of the 21 challenged pigs are left untreated. The seven pigs showed a decrease in weight gain, all of which developed proliferative bowel disease lesions detected in the intestinal tract collected at the necropsy 3 weeks after challenge. Six of these seven had significant visible lesions and three had mild to severe diarrhea two weeks after challenge. To test the "prophylactic" dosing, administration of 25 ppm and 75 ppm, respectively, via the given premix (ie, 1.25 mg / kg and 3.7 mg / kg) to the other two groups of challenged pigs with Valnemulin hydrochloride two days before challenge The dose is administered orally and continued until euthanasia. To test the "treatment" approach, 25 ppm and 75 ppm of Valnemulin were administered orally to the other two groups of challenged pigs via a given premix 7 days after challenge and continued until euthanasia.

Proliferative bowel disease lesions in the intestinal sections collected at necropsy were not observed in 3 of 7 in the preventive group and 5 of 7 in the treatment group. The remaining control pigs are normal. Thus, Balnemulin prevents or treats diseases even with low dose drug treatment in most challenged pigs.

Thus, the medicaments of the present invention are useful for treating ileitis in pigs associated with Lausonia intracellulitis infection. For this use, the effective dosage may of course depend on the particular salt used, the mode of administration, the size and age of the animal, and the desired effect, for example for relatively prophylactic treatment, relatively low dosages may be administered over a long period of time. May be administered. Generally, however, the medicament is generally administered at a dosage of about 1.5 mg / kg to about 6.5 mg / kg animal body weight per day, suitably optionally in water or feed, or in divided doses of 2 to 4 times per day or sustained. Satisfactory results are obtained when administered in a release form. For most animals, the total daily dose is about 10 mg to about 1000 mg, preferably about 15 mg to about 500 mg, administered once or twice daily.

Preferred dosages in drinking water are 0.001 to 0.05 volume percent by weight, in particular 0.001 to 0.005% and dosages in the feed are 20 to 400 g / metric tons, especially 20 to 200 g / metric ton.

E) Chronic Respiratory Diseases and Arthritis in Poultry Associated with Mycoplasma Gallicecticum Infection

Infections, i.e. mycoplasma galilicepticum infection, are routinely described, for example, in the Veterinary Manual [Diseases of Poultry, 8th Ed. (1984), Ed, Hofstad et al., Lowa State University Press, on pages 196-198. The beneficial activity of the formulations of the invention in this use is measured, for example, as follows.

1. In vitro activity: MIC

The antimicrobial effect of Valnemulin hydrochloride is measured by a series of microdilution standard techniques comparing to tyrosine (tilane soluble) and thiamullin hydrogen fumarate.

12.8 mg of test compound are dissolved in 100 ml of distilled water to obtain a stock solution, which is stored at -20 ° C until use. Mycoplasma galilicepticum reference strains used are X95, S6 Holland, S6 Bench (UK), MS-16 (Japan), MK-7 (Japan) and 1226, and various new field isolates.

The antibiotic-sensitivity of the strains is a modified microbroth dilution process [Stipkovits et al., Vet. Microbiol. 15 (1987) 65-70. Add 100 μl of test medium to all wells of the microtiter plate using double dilution of antibiotics. Inoculum (100 μl) contained 10 ⁵ cfu / ml. The plate is sealed with cello tape and incubated at 37 ° C. over 3 days. At day 3 readings, the lowest concentration of antibiotic that completely prevents the color change of the medium is the minimum inhibitory concentration (MIC). The viability of mycoplasma is examined by plating wells without color range on agar medium. All mycoplasma strains are grown in 10 ml of medium B. Erno, H. and Stipkovits, L., Acta Vet. Scand. 14 (1973) 436-449. After incubation at 37 ° C. to initial log phase growth, 1 ml of culture aliquots are suspended in tubes as seed cultures and stored at −20 ° C. Glucose-fermented mycoplasma strains were tested in modified medium Bg with glucose supplemented with 1% glucose (pH 7.8), and arginine hydrolytic strains with medium Ba containing 1% arginine (Erno, H. and Stipkovits, L). , supra, 450-463, and glucose- and arginine-negative strains are tested in medium B containing 1% triphenyltetrazolium chloride.

Dilution is taken into account to calculate average MIC values for different strain groups and antibiotics.

The MIC values of the reference strains are shown in Table 8 and the MIC values of Mycoplasma isolates are shown in Table 9:

TABLE 8

TABLE 9

The results obtained using reference mycoplasma strains show that the three compounds exhibit somewhat the same range of activity. However, it reflects a clear difference between test substances in MIC values of poultry field isolates: uniformly high sensitivity to Valnemulin (0.03-0.06 μg / ml), but slightly reduced sensitivity to thiamulline (0.06- 1 μg / ml), very resistant strains were observed when tyrosine was used (0.06-32 μg / ml).

2. Challenge Investigation :

2.1. prevention

Groups of male roasted chickens were infected with the pathogenic strain Mycoplasma gallisepticum (MG), and then each group was administered with one of the following drugs selected: Valnemulin (concentration 0.00312%, 0.00625%) , 0.0125% and 0.025%), thiamulin (0.00625%, 0.0125% and 0.025%) and tyrosine (0.05%). 0.1 ml of mycoplasma galilicepticum culture containing about 10 ⁶ cfu of viable organisms was injected into the lungs of each chicken on day 2 and then infected (Jordan, FTW, The Veterinary Record 127 (1990) 502). The drug is administered within 1 hour after infection and then continued for 3 days. There are also groups that are not infected and groups that are infected but not treated with drugs.

Satisfactory results in both clinical symptoms and prevention of death in the group receiving high concentrations of Balnemulin (0.0125% and 0.025%), Thiamulin (0.00625%, 0.0125% and 0.025%) and Tyrosine (0.05%) Got. High doses of Valnemulin (0.0125% and 0.025%) after treatment with tyrosine followed by thiamulin reduced the number of chickens with lesions and the symptoms were less severe. In this respect, efficacy was low when low concentrations of Balnemulin (0.00312 and 0.00625%) were administered. The group that gained the most weight was the group treated with tyrosine; Groups with significantly smaller weight gains differed (p <0.05) but were treated with the uninfected group, with thiamulline, and with concentrations other than the lowest (0.00625%, 0.0125% and 0.025%). Mullin was administered. The smallest weight gain group was the one treated with the lowest concentration of balnemulin and the group infected but not treated with drugs. There was no significant difference between them. MG was not recovered during survival or during necropsy from the uninfected group, the highest concentration of thiamulin and tyrosine, and was isolated from only one chicken in the highest dose of banemulin. More was isolated from all other infection groups. These serologic results do not fully reflect the results for tyrosine, because there are relatively many chickens positive in this group.

Thus, it was found that Valnemulin administered at high concentrations (0.0125% and 0.025%) was as effective as the reference compound in preventing young chickens from spreading to Mycoplasma galilicepticum.

2.2. process

Male broiler chickens were infected with the pathogenic Mycoplasma gallicepticum (MG) strain and each group had 0.0125%, 0.025%, 0.05% concentration of Balnemulin, equal concentration of Tiamulin, 0.05% concentration of Tyrosine, and 0.083% Concentrations of lincosfectin and spectinomycin combinations (linco-spectin) are administered. There is also one untreated and one uninfected group. On day 2, chickens are infected by injecting 3.4 × 10 ⁵ organisms into each lung. Drug administration in drinking water, with the exception of linco-spectin, begins after 24 hours and is performed for three consecutive days and this treatment is continued for five days.

The results obtained indicate that control of lethality of 5% or less is best for all valemulin at three concentrations (0.0125%, 0.025% and 0.05%) and thiamulline at 0.05% concentration. Only the chickens given the highest and lowest doses of Valnemulin and a few chickens given thiamulin and tyrosine did not find lesions. When all concentrations of balnemulin were administered, weight gain was higher than in the thiamulin treated group and comparable to the tyrosine group. Mycoplasma was not recovered from the three groups of algae administered with balnemulin and the group administered with thiolin at the end of the 23 day experiment. In serological results this time there were some positive responders in all infected groups.

Given the results of lethality, weight gain, isolation of MG, and serum aggregation tests, the high doses (0.025% and 0.05%) of Valnemulin provided in drinking water are as good or better than at least 0.05% of thiamulin or 0.05% of tyrosine. Excellent and far superior to rinco-spectin.

Thus, the formulations of the present invention are useful for the treatment of chronic respiratory diseases and arthritis in poultry associated with mycoplasma galilicepticum infection. The effective amount for this use may of course be very variable depending on the particular salt used, the mode of administration, the size and age of the animal and the desired effect, for example when a relatively low dose is administered over a prolonged period of time for prophylactic treatment. do. Generally, however, when the formulation is administered in a beverage at a dose of about 0.005 to about 0.05% by weight, in particular 0.01 to 0.03% by weight, and in the feed at 20 to 400 g / ton, in particular 20 to 200 g / ton, Satisfactory results are obtained.

F) Secondary infections caused by Pasteurella multocida, Actinobacillus (Hemophilus) fluoroneumoniae and / or Haemophilus parasuis infection :

Secondary infections with Pasteurella multocida, Actinobacillus (Hemophilus) Pluronomymoniae and / or Haemophilus parasus are described, for example, in Taylor, DJ, in Pigs Diseases, 6th Ed. , (1995), Publ. D. J. Talor, Glasgow, U.K. on pages 185-187; 188-194; and 194-197, as described in the veterinary manual, can be diagnosed by conventional methods. The advantageous activity of the formulations of the invention for this use is measured, for example, as follows:

Minimum inhibitory concentrations of Valnemulin (MIC) on recent isolates of Pasteurella multocida, Haemophilus pluronyumoniae, and Haemophilus parasus resulting from porcine respiratory substances are measured and compared to thiamulins. Pasteurella multocida 10, h, isolated within 5 years from infected pigs as assessed at autopsy. Pluronuemonia 10 and H. Bacterial isolate cultures comprising Parasus 3 are used. Three of the Pasteurella isolates are toxic producing strains of Pasteurella multocida. Haemophilus pluronyumoniae and h. For Parasus, the day before testing, each pure culture was inoculated using an inoculation loop and Todd & Hewitt broth containing 1% calf serum and 2 mg / ml nicotinamide adenine dinucleotide (NAD). broth; Unipath CM 190) 10 ml single inoculation. For Pasteurella multocida, on the day of testing, each pure culture is inoculated once in 10 ml of nutrient broth No. 2 (Unipath CM67) containing 1% of calf serum using an inoculation loop. Each of the prepared broth cultures is incubated at 37 ° C. for 18 hours under 10% CO ₂ . After incubation, broth culture 23 is adjusted to no more than 10 ⁵ to 10 ⁶ organisms per ml of sterile physiological saline on the day of testing for inoculation of MIC culture medium. For each test compound, a standard control culture of Oxford Staphylococcus aureus NCTC 6571 was also set to verify the results of the MIC test. In each case, the broth culture established from the control organism is identical to the culture of the organism under test. Valnemulin is hydrochloride and thiamulin is used as hydrogen fumarate. On the day of testing, stock solutions of test compounds containing 3.2 mg / ml of active ingredient are prepared in sterile distilled water. Nine 2-fold dilutions are then prepared in sterile distilled water and lowered to 1.25 μg / ml from 320 μg / ml of each antibiotic for incorporation into MIC medium. Sensitive agar (Oxide CM409) containing lysed horse blood (Oxoid SR50) is prepared according to the manufacturer's recommendations and adjusted to pH 7.4 if necessary. 2 mg of NAD per ml is added to every batch of MIC medium prepared for MIC analysis for two Haemophilus species to provide the necessary growth factors. 18 ml of molten agar (50 ° C.) is mixed with 2 ml of each test compound dilution (10-fold dilution) made in a 90 mm Petri dish. Four replicate plates are prepared from each dilution. For each MIC analysis, four replicate plates containing 18 ml of agar and 2 ml of sterile distilled water are prepared to act as growth indicators.

The dried MIC plate was incubated for 18 hours at 37.degree. H. pleuropneumoniae and H. p. H. parasuis is inoculated with 0.2 μl of test organism prepared by incubating for 18 hours at 37 ° C. under 10% CO ₂ . After inoculation, the growth of the organism is first examined by eye, followed by a stereo zoom microscope. The MIC value is considered to be the lowest concentration of test compound whose growth cannot be detected at the inoculation point on all four plates by microscopic examination.

The results obtained are as shown in Table 10.

TABLE 10

Compared with thiamulin, balnemulin on average has a 2.5-fold increase in activity against P. multocida, H. 5.5 times and H activity against Pluronyumoniae. It appears to have a threefold increase in activity against parasuis.

Accordingly, the medicaments of the present invention are useful for treating secondary pneumonia in pigs associated with Pasteurella multocida, actinobacillus (hemophilus) fluronimoniae and / or Haemophilus parasus infection. When used, the effective amount will of course vary depending on the particular salt used, the mode of administration, the size and age of the animal and the desired effect, but for example in the case of prophylactic treatment a relatively low dose is administered for a long time. Generally, however, the medicament is optionally administered in a daily dose of about 5 mg / kg to about 15 mg / kg per animal body weight, optionally in admixture with water or feed, divided doses 2-4 times a day, or sustained release. When administered in sexual form, satisfactory results are obtained. For most animals, the total daily dose is about 100 mg to about 1000 mg, preferably about once or twice a day, about 100 mg to about 500 mg.

It may be advantageous to administer as a monotherapy.

Preferred doses in drinking water are 0.01 to 0.05% by weight, in particular 0.01 to 0.025% by volume, and 100 to 400 ppm (g / metric ton), especially 100 to 200 ppm, in feed.

Pneumonia of lambs, sheep and cattle associated with G. Pasteurella haemolytica infection

Pasteurella haemolytica infection is described in Veterinary Medicine, 8th Ed. (1994), Eds. Radostits, O.M., Blood, D.C. and Gay, C. C., Publ. Diagnosis is by conventional methods as described in Bailliere Tindall, London, U.K., on pages 748-770. The beneficial activity of the formulations of the invention in this use is measured in vivo as follows.

The clinical effect of two valnemulin hydrochloride formulations in the treatment of pneumonia of calves due to controlled infection with Pasteurella hemolytica A1 is measured.

On day 6, weigh the 24 calves that were wiped their nose and found to be free of Pasteurella haemolytica A1 and distributed into 4 treatment groups of 6 each, depending on body weight with a uniform gender distribution among the groups. Balance The calves are housed in individual stalls in a regular calf rearing unit that they have been accustomed to for 33 days before they are distributed. Blood samples are taken twice during the preliminary study period for leucotoxin antibody analysis, a possible prerequisite before exposure to Pasteurella haemolytica A1.

Treatment begins on day 1, approximately 27 hours before challenge.

Group 1-control;

Group 2-Injectable 2.5% Valnemulin Hydrochloride (2 times 2.5 mg / kg daily)

Group 3-Valnemulin hydrochloride 10% premix administered orally via calf milk changer (2x 5.0 mg / kg daily).

All calves are challenged on day 0 by depositing 30 ml of Pasteurella haemolytica A1 broth culture (M4 / 1/3, collected study group-1.25 × 10 ⁻⁷ cfu / ml) into the bronchus via a fiber optical bronchoscope. Clinical tests are performed once a day on days -3 and -2 and twice a day on days -1 to 3. Clinical scores are allocated to each of the four parameters: rectal temperature, respiratory rate, respiratory status and modality. The injection site is also investigated. 20% (w / v) of pentobarbitone sodium BP (Vet) is given to calves that show signs of depression and / or depression and / or dyspnea or whose total clinical score is greater than 5 in a single test from 0 to 3 days. Intravenously, euthanize calves immediately and autopsy within 24 hours. Calf surviving until 4 days after Pasteurella haemolytica challenge is euthanized on day 4 in the same manner as calf euthanized on days 0-3. Lungs are removed from all calves and evaluated visually for hardened lesions and pleural adhesions. Lung tissue samples are excised from eight standard sites and tested for the presence of Pasteurella hemolytica to determine the value for the separation index. A cardiac blood swab is taken from the calf and tested for the presence of Pasteurella haemolytica.

Each calf and group total, group mean and group median are measured for all parameters: total clinical score, sclerotic lesion score, sequestration index, pleurisy score and total score. Each calf and group total, group mean, and group median are calculated for all categories that make up the total clinical score: rectal temperature, respiratory rate, respiratory status, and modality. If there is a difference between the group scores for each parameter, it is determined by applying the basic analysis by the Kruskal-Wallis test (small table): total clinical score, sclerotic lesion score, sequestration index , Pleurisy score and total score. The meaning of the Kruskal-Wallis test indicates that the groups are not all the same but they are not all different. In tests where the group differs, pairs of groups are tested by the Mann-Whitney test (small Table 9).

The total clinical score (p = 0.013) was higher than that of untreated calves. Significantly lower in calves treated with a 5.0 mg / kg / dose of Balnemullin 10% mixture twice daily orally via a calf milk changer for 5 days, starting 27 hours before challenge with hemolytica A1 Turned out.

In addition, the total clinical score is statistically insignificant but not statistically significant. It decreases in infants treated intramuscularly with 2.5% of Valnemulin, which can be administered at 2.5 mg / kg / dose twice daily for 5 days, starting 27 hours before challenge with Hemolytica A1.

When comparing total clinical scores, there is no significant difference between groups treated.

Thus, the agents of the present invention are useful for treating pneumonia in lambs, sheep and cattle associated with Pasteurella haemolytica infection. For use herein, of course, the effective dosage may vary depending on the particular salt used, the mode of administration, the size and age of the animal, and the desired effect; For example, for prophylactic treatment, relatively small dosages may be administered for a long time. However, when the medicament is administered in a daily dosage of about 5 to about 15 mg / kg body weight, suitably optionally in admixture with the feed, divided into two to four times daily, or administered in sustained release form, Satisfactory results are obtained. For most animals, the total daily dose administered once or twice daily or optionally in admixture with the feed is about 250 to about 3000 mg, preferably about 250 to about 1000 mg.

H) Poyarthritis in pigs associated with mycoplasma hyosinobia infection

Mycoplasma hyosinobia infection can be diagnosed by conventional methods as described in the Veterinary Manual. Taylor, D.J., Pig Diseases, 6th Ed. (1995), Publ., D. J. Taylor, Glasgow, U.K., pages 172-173. The advantageous activity of the preparations of the invention in this use is measured, for example, as follows.

1. Activity of Tissue Isolates in Vitro (MIC Analysis)

Tissue samples are taken from the lungs of swine swine with various endemic pneumonia (EPP). Samples are placed and transported on dry ice for incubation. After culturing mycoplasma hyosinobia, after confirming that many colonies in the form of typical mycoplasma hyosinobia have grown in some samples (restored at -70 ° C), mycoplasma hyosinobia Four isolates from lung samples initially isolated for recovery of sorrow are trimmed for Mycoplasma hyosinobia. In addition, three isolates are recovered from other lung samples provided. All isolates are serologically identified by disc growth inhibition with specific rabbit antiserum generated against Mycoplasma hyosinobia. Mycoplasma hyosinobia is isolated from lung tissue using a commercially available solid medium (Mycoplasma Experience Ltd.). Liquid medium containing phenol red and arginine (Mycoplasma Experience), pH 7.00, is used for MIC analysis.

A stock solution of the test compound was prepared at a concentration of 1 mg / ml in deionized water, sterilized by filtration through a membrane filter (Sartorius Minisart N) having a pore size of 0.2 μm, and then at −20 ° C. Save it. For use in the MIC test, the stock solution is diluted twice in liquid medium to the required final concentration. MIC tests are performed by the methods described in the literature (Tanner and Wu, Avian Diseases 36 (1992) 714-717). Actively growing challenge cultures are prepared from 1 ml of broth culture stored at −70 ° C. or cultures stored on agar at −70 ° C. The challenge culture is diluted to obtain the desired titer of 10 ³ to 10 ⁵ color change units / ml. 0.1 m of challenge inoculum is mixed with 0.1 ml of antibiotic dilution in microtiter wells. Plates of each microtiter contain uninoculated medium and antibiotic-free inoculation challenge control at pH 7.6 (endpoint control). All plates are sealed with adhesive film and incubated aerobicly at 36 ° C. MIC is recorded when the color change in the challenge control well matches the endpoint control pH. MIC is the minimum concentration that does not exhibit color change. The results obtained for Valnemulin hydrochloride and two reference compounds, thiamullin hydrogen fumarate and enlofloxacin, are shown in Table 11.

TABLE 11

The sensitivity of the seven field isolates of Mycoplasma hyosinobia to Balnemulin, indicating that recent field isolates are very sensitive to Valnemulin, is much higher than the sensitivity to the other two compounds.

Thus, the medicaments of the present invention are useful for treating multiple arthritis in pigs associated with mycoplasma hyosinobia infection. To this end, of course, the effective dosage will vary depending on the particular salt used, the dosage form, the size and age of the animal, and the desired effect, eg, in prophylactic treatment, relatively low doses are administered over a long period of time. Generally, however, the medicament is administered in a daily dosage of about 5 mg to about 15 mg per kg of body weight, preferably in admixture with water or feed, divided into two to four divided doses daily, or in sustained release form. If so, satisfactory results are obtained. For most animals, the total daily dose is about 100 mg to about 1000 mg, preferably about 100 mg to about 500 mg and is administered once or twice daily.

It may advantageously be administered as a monotherapy.

Preferred dosages in drinking water are about 0.01 to 0.05% w / v, preferably 0.01 to 0.025% w / v, and the dosage in the feed is 100 to 400 ppm (g / metric ton), in particular 100 to 200 ppm (g / meter) Ton).

In all such uses, the compounds can be used in free base form or in veterinary acceptable salt form, for example in the form of quaternary salts or, in particular, acid addition salts. Such salt forms exhibit the same degree of activity as the free base form. Examples of suitable acid addition salts are hydrogen fumarate, fumarate, naphthalin-1,5-sulfonate and especially hydrochloride.

The agents of the present invention may be administered orally, topically or parenterally and may be mixed with chemotherapeutic acceptable diluents and carriers, and optionally, with other excipients, in the form of tablets, capsules or injectable preparations. May be administered. In addition, they form excellent additives for feed mixtures (eg premixes) or for drinking water.

Veterinically acceptable preferred carriers include, for example, pharmaceutical excipients commonly used, such as sugars, corn starch, lactose, cellulose, as well as grain carrier systems and grain by-products (e.g. milled rice bran, coarse flour and soy flour). And further solid diluents such as milled limestone, sodium sulfate, calcium carbonate and kaolin and liquid substances such as veterinary acceptable oils (vegetable oils or mineral oils), propylene glycol and polyethylene glycol. Suitably, these preparations are administered orally to animals, preferably mixed orally administered to the feed in the form of a dosed feed or dosed pellets.

For application to drinking water, aqueous solutions with or without solvents such as ethanol, propylene glycol, polyethylene glycol, applied liquid surfactants and sorbitol are used.

For example, preparations for intramuscular injection are typically prepared in solution in water, which may contain a solvent such as ethanol or propylene glycol, or in solution in a veterinary acceptable oil. Examples of acceptable oils include sesame oil, medium chain triglycerides (eg migliol), isopropyl myristate and ethyl oleate. The formulation may contain preservatives, buffers and other conventional excipients.

Veterinary formulations for use in the present invention can be prepared by mixing the respective components in a predetermined ratio. The formulation is packaged in a suitable container ready for administration.

The following examples illustrate the invention:

The formulations of the present invention are widely accepted. For rats, the acute toxicity of the compound of formula 1 in the form of the hydrochloride salt is determined by oral administration of 5 male rats and 5 female rats at a single dose of 1000 mg / kg and 2000 mg / kg per dose group. Lethality occurs within 1 to 8 days. LD ₅₀ values obtained are above 1000 mg / kg upon oral administration.

Claims (9)

A compound of formula I in the form of a free base or a veterinary acceptable salt, occurring in animals with secondary infections due to an increase in breeding density, Serpulina pilosicoli, Lausonia intra Bacteria selected from the group of Cellularis, Pasteurella multocida, Pasteurella multocida, Pasteurella haemolytica and mixed strains thereof. Veterinary preparations for treating veterinary diseases associated with infection by. Formula I The veterinary preparation according to claim 1, wherein the veterinary disease is porcine colitis associated with Serpulina pilosicoli infection. The veterinary preparation according to claim 1, wherein the veterinary disease is swine ileitis associated with infection with Lausonia Intracellularis. The veterinary agent according to claim 1, wherein the veterinary disease is associated with Pasteurella multosida infection. The veterinary preparation according to claim 1, wherein the veterinary disease is pneumonia of the lamb, sheep and cattle associated with Pasteurella haemolytica infection. The veterinary preparation according to claim 1, wherein the compound of formula I is in the form of hydrochloric acid addition salt. The veterinary preparation according to claim 1, wherein said animal in need of treatment is a pig. Animals with secondary infections due to an increase in breeding density, comprising mixing a compound of formula (I) in the form of a therapeutically effective amount of free base or veterinary acceptable salt with one or more veterinary acceptable carriers or diluents In the field, Serpulina pilosicoli, Lausonia intracellularis, Pasteurella multocida, Pasteurella haemolytica and mixtures thereof A method of making a veterinary agent for treating veterinary diseases associated with infection by bacteria selected from the group of strains. Formula I The veterinary preparation according to claim 1 comprising the compound of formula (I) and at least one veterinary acceptable carrier or diluent.