MXPA00010464A - Treatment of mastitis - Google Patents

Abstract

The invention provides new fluorinated cephalosporin antibiotics of the Formula (I), processes for preparing these compounds and veterinary and pharmaceutical formulations containing a Formula I compound as an active ingredient.

Description

TREATMENT OF MASTITIS Description of the Invention This invention relates to new fluorinated cephalosporin antibiotics and to formulations comprising, methods for using these antibiotics to control susceptible pathogens. The invention relates particularly to veterinary formulations and methods for preventing or treating mastitis in a mammal using a fluorinated cephalosporin of this invention.

Mastitis is a serious problem, especially in the dairy industry. It is an inflammation of the udder caused by a variety of different pathogens, which include Staphylococcus species and Streptococcus species. There are, however, difficulties in effectively treating mastitis while still meeting the needs for the dairy industry. The agent used should be effective against the pathogen or pathogens that cause mastitis, should not adversely affect the animal undergoing treatment, and should be promptly purified from the animal's system so that the milk it produces is safe for a subsequent use. Ref: 123289 This invention provides a new group of fluorinated cephalosporin compounds of the formula I: wherein Ra, R ", Rc, R? and Re, each are, H, F or an alkyl group - (Z) n - of Ci-Cß which has at least one fluorine substitute; X is O or S, Y is S, 0, or -CH2-, Z is 0, S, -SO-, or -S02-, m and n are each 0 or 1, and R1 is H, C1-C6 alkyl, phenyl or benzyl, each of which may optionally have up to three substitutes selected from the halide, C1-C4 alkoxy, phenyl, N02, Ci-Cß alkanoyl, benzoyl, or Ci-Cß alkanoyloxy, or a physiologically acceptable salt thereof, provided that: 1) at least one of Ra, ",.,! ^, Rd or Re is different from hydrogen, and 2) when Rc is F, or one of Rb or Rd is CF3, at least one of the remaining Ra, R, Rc, Rd or Re is different from hydrogen The term "C 1 -C 8 alkyl" refers to a straight or branched chain alkyl group having a designated number of carbon atoms Examples include methyl, ethyl , isopropyl, n-pentyl, and the like The term "halide" refers to chlorine, iodine, bromine or fluorine The esters of formula I, ie those compounds wherein R 1 is anything other than hydrogen or a form Thus, these compounds are converted in vivo to the corresponding free acid which has the desired activity.Some esters of the formula I are preferred, these esters include in pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, 1- (acetyloxy) ethyl and phenacyl The physiologically acceptable salts of the compounds of the formula I are also part of this invention. These salts include the salts of metals alkaline, for example, potassium, sodium, etc., alkaline earth metal salts, for example, calcium, magnesium, etc., and salts with organic bases, such as organic amines, for example, benzathine, pyridine, triethylamine , tripropylamine, and triisopropylamine, etc. Triethylamine and sodium salts are the particularly preferred salts. The compounds of formula I are active both in vitro and in vivo against various pathogenic organisms. In one aspect, they are active against certain pathogens that cause mastitis in mammals, particularly in ruminants. A special benefit of the compounds of formula I is that they are active, at such levels, that they can provide improved control of mastitis unlike the products currently used in the dairy industry. Therefore, this invention provides a method for preventing or treating mastitis in a mammal comprising administering to the mammal an amount of the compound of formula I that effectively prevents or treats mastitis. This method is particularly useful for preventing or treating mastitis in a ruminant.

The compounds of formula I are especially useful for treating mastitis in cattle, goats and sheep. In one embodiment of the invention, the compound of formula I is administered while the animal is lactating ("wet cow" therapy). In this modality, the milk is discarded until the mastitis has been treated successfully and the drug has been purified from the animal, that is, the drug is not present in the milk. The compounds of this invention may also be useful for a "dry cow" therapy, that is when administration occurs after lactation, and therefore, the animal will be handled in the manner of a dry cow without another milking until the next delivery. In yet another aspect, the compound of the formula I can be used prophylactically when administered to a non-lactating animal, such as in a calving calf, in the period prior to delivery. The compounds of formula I can be administered by a variety of methods, such as intramuscular, subcutaneous, intravenous, intranasal, oral, or by intramammary infusion. When it is used to prevent or treat mastitis, are preferably administered by a mammary infusion. It is understood in the art that the amount of the compound of the formula I to be administered should be 1"amount that is effective to control the particular pathogen or pathogens in question.More, the type, size and condition of the host to be treated should be taken For example, when controlling a pathogen responsible for mastitis, the dose varies depending on the type and size of the ruminant to be treated.

By way of illustration, as mastitis is treated in cows, usually amounts from about 10 to 1000 milligrams per quarter are generally effective in controlling mastitis. Doses from about 50 to 300 mg per quarter are preferable; and doses of approximately 100 to 00 mg per quarter are the most effective. In goats, on the other hand, amounts from about 10 to about 100 milligrams per half are generally sufficient; doses from about 10 to 30 mg per half are preferred; and a dose of 20 mg per half is more preferred. An effective amount to be achieved by multiple dosages. This invention also provides a veterinary or pharmaceutical formulation comprising a compound of formula I and one or more pharmaceutically acceptable carriers. The veterinary formulations of this invention are particularly useful for preventing or treating mastitis in a mammal, especially in a ruminant. The preferred ruminants are cattle and goats. The invention also provides pharmaceutical formulations comprising a compound of the formula I and a pharmaceutically acceptable carrier. These pharmaceutical formulations are useful for treating a bacterial infection in a mammal, especially in a human .. The compounds of the formula I can be formulated for a veterinary or pharmaceutical administration according to methods that are understood in the art. When the compound to be used in a veterinary formulation for preventing or treating mastitis, preferably the formulation is one that can be administered by intramammary infusion. For this type of infusion, a compound can be formulated in an oily base, for example, in a vegetable oil such as peanut oil or in a non-vegetable oil such as mineral oil. The formulation may include a thickening agent and optionally also a surfactant agent. When a compound of formula I is to be administered to a mammal, for the treatment of other types of infections, it may be preferred to administer it in a pharmaceutical formulation comprising one or more pharmaceutically acceptable excipients. The preparation of these preparations is also understood in the clinic. See, for example, Remington the Science and Practice of Pharmacy, (Marck Publishing Co., Easton, PA, 1995). To prepare a veterinary formulation other than an intramammary infusion, or a pharmaceutical formulation, the compound of the formula I is usually mixed with an excipient, diluted with an excipient or included within a carrier which may be in the form of a capsule, sachet, or other container. When the excipient functions as a diluent, it can be a solid, semi-solid or liquid material that acts as a vehicle, carrier or medium for the active ingredient. Therefore, the compositions can be in the form of tablets, pills, powders, dragees, sachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (in a solid or liquid medium), ointments, soft or hard gelatine capsules, suppositories, sterile injectable solutions and sterile packaged powders. During the preparation of the formulation, it may be necessary to grind the active compound to provide the proper particle size before combining it with the ingredients. If the active compound is substantially soluble, it is usually ground to a particle size of less than 00 mesh. If the active compound is substantially water soluble, the particle size is usually adjusted by milling to provide a substantially uniform distribution in the formulation, for example, of about 40 mesh. When the compound is to be used in a formulation for intramammary infusion, it is preferred that the particle size be less than 100 microns and even more preferable that it be less than 10 microns. Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia gum, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. Formulations can also include: lubricating agents such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservative agents such as methyl- and propylhydroxybenzoate; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide a rapid, sustained or prolonged release of the active ingredient after administration to the host by methods that are known in the art. For oral administration, the compound can be mixed with carriers and diluents and molded into tablets or placed into gelatin capsules. Another aspect of this invention is a process for preparing a compound of the formula I which comprises reacting a 3-chloro-7-aminocephalosporanic acid, or a salt or ester thereof, with an acid halide of the formula II wherein Ra, R ", RC, Rd, Re, X and m are as defined supra, and the halo is Cl, Br, F or I to give the compound of formula I. This invention also provides a compound of the formula I when done by this process The following examples illustrate the compounds, methods and formulations of this invention.

Example 1 Step 1: Preparation of Ib Thionyl chloride [8.85 g (5.4 mL), 74.3 mmol] is added to the acid (15.2 g, 62.0 mmol) in a 25 mL round bottom flask. The mixture is heated to reflux (until it becomes a colorless solution and is held there for one hour.) The solution is cooled, and the excess thionyl chloride is removed at 25 mBar on a rotary evaporator.The remaining solution is distilled at 0.25-0.3 mBar at 56-60 ° C to give 14.9 g? s * Ib in a clear colorless liquid (92% yield).

Step 2: Preparation of Compound 1 The cephalosporin nucleus (4.46 g, 19.0 mmol) is dissolved by suspending it in H20 (200 mL) and adding saturated aqueous K3P04 until the solution has a pH of 8. The resulting solution is cooled to 0 ° C and Ib acid chloride (4.95 g, 19.0 mmol) is added in portions over a period of 30 minutes. K3P0 is also added to keep the pH at about 8. The solution is allowed to slowly warm up until the solids begin to precipitate. EtOAc (150 mL) is added, and the resulting solution is acidified to a pH of 1 with IN HCl. The aqueous layer is back extracted with EtOAc (2 x 150 mL).

The organic extracts are stirred for one hour with activated charcoal; the carbon is removed by filtration; and the solution is then evaporated in a vacuum to give 8.33 grams of an off-white solid. The solid is suspended in diethyl ether and filtered to give 4.66 of 1, m.p. 164 ° C (dec); ms = 457.9.

Analysis: Calculated: C, 39.27; H, 1.76; N, 6.11 Found: C, 39.35; H, 1.75; N, 5.87.

Examples 2-10 Other illustrative compounds of formula I (R1 = H) are prepared by using procedures analogous to those described in Example 1. Tables 1 and 1 summarize these compounds and some of their physical characteristics. Table 1: Illustrative Compounds of Formula I R Table 1 a: Physical Characteristics of the Compounds of Formula I * FD-MS (m + 1) Example 5 Tables 2 and 2 summarize the minimum inhibitory concentrations (MIC) at which the illustrative compounds of formula I and cephalothin inhibit certain microorganisms that are associated with mastitis, such as Staphylococcus aureus 10092 (strain Ne bould).

Table 2: Comparison of the ± n vitro activity of the Compounds of Formula I and Cefalotin against Staphylococcus aureus and Streptococcus sp.

Incubated at 37 ° C during the night. Table 2 a: Comparison of the In Vitro Activity of the Compounds of Formula I and Cefalotin against Staphylococcus sp and Streptococcus sp.

The compounds of the formula I are also active against certain Gram negative species. Tables 3 and 3 a show the MICs of the compounds of the formula I against certain Gram negative species.

Table 3: Comparison of the In Vitro Activity of Compound 1 and Cefalotin against Gram-negative Organisms Incubated at 37 C overnight.

Table 3 a Comparison of the in vitro activity of the compounds of the formula I against Pasteurella haemolytica "MIC compound" 5 16 6 4 7 32 8 32 9 4 Cefalotin 1"Strain 128K b Incubated at 37 ° C overnight.

Example 13 Efficacy of the Compounds of Formula I in an Experimental Model of Mastitis Induced by Staphylococcus aureus in Lactating Goats Sixteen lactating goats are selected based on the absence of Staphylococcus aureus in pre-challenge milk cultures. The maintenance portion of the animals was maintained throughout the study. The goats are milked twice each day using a portable milker for goats.

Milk samples are collected during morning milking. One day before the challenge, the milk culture mixes are collected from each half of the animals that undergo the experimental test to determine if there is a pre-existing infection. In addition, a sample of each half is collected for the determination of the Somatic Cell Count (SCC). A sample of a milk culture and a sample for SCC are collected from each half 24 hours after challenge and on days +4 to +6, +8, +11, +13, and +15. The grade of the udder is performed during each milking using the following table: Degree of Severity Description 1 Normal milk - no inflammation of the udder 2 Normal milk - inflammation of the udder 3 Abnormal milk - with little or no inflammation 4 Abnormal milk and inflamed, soft, or hot udder. Acute clinical mastitis. 5 Acute clinical mastitis with systemic complication.

General health observations and body temperature are recorded once a day after milking during challenge and treatment periods. The milk samples are subjected to an analysis for SCC using an electronic somatic cell count system. Milk culture samples are titrated for a bacterial culture by placing in culture plates 1.0 mL and 0.1 mL of the milk sample directly on plates in duplicate blood agar. In addition, 10"1 dilutions of the milk sample are grown in plates.The plates are incubated overnight at 37 ° C. Staphylococcus aureus 10092 (Newbould) is used as the challenge strain.The challenge culture is incubated in soy triptych broth (TSB) from a liovial strain overnight (16 hours) at 37 ° C. TSB (100 mL) is inoculated with 1 mL of the culture grown overnight and incubated at 37 ° C. C with shaking for 4 hours.The culture is centrifuged at 9,000 rpm for 10 minutes at 10 ° C, the supernatant is removed and the pellet is resuspended in phosphate buffered saline (PBS). nm is approximately 0. 035. A series of 10-fold dilutions is prepared with saline to determine the CFU / mL, plating 10"5, 10" 6, and 10"7 dilutions on duplicate plates of blood agar. (approximately 10 CFU / mL) is used for the challenge material, resulting in a challenge of 50 CFU / half with a 5 mL infusion, and 16 goats are used for the trial. Milk samples are collected and temperatures are taken during the morning milking.The milk / udder evaluations are documented during the morning and evening milkings Staphylococcus aureus 10092 is infused into both halves in each of the goats. They challenge each other after morning milking and are milked in the afternoon on the day of challenge.The challenge inoculum volume is 5mL / half which contains approximately 50 CFU of S. aureus.For the 24 hour period after the challenge they are collected milk samples during the milking morning, and evaluations of milk / udder are done during the morning and afternoon milking.

The treatment begins hours after the challenge.

Milk samples are collected from the crop and SCC 24 hours after the challenge, before the morning milking, and the treatment period begins after the milking in the afternoon. Eight halves (8 goats) are treated during 2 consecutive milkings 12 hours apart, after milking.

Each designated compound is formulated in peanut oil with 45 μmol of compound per 5 grams of formulation to give approximately 25 mg / mL of dose. After the treatment, the goats are milked and observed for 15 days. Milk samples are collected on days +4 to +6, +8, +11, +13 and +15 to determine if the infection has cleared. The establishment of mastitis is determined by a bacterial culture, degree of severity of the udder, results of the somatic cell count and body temperature. On days +2 to +6, milk samples are collected for a colorimetric assessment to determine if the antibiotic is still present in the milk.

Table 4 summarizes the activity of the typical compounds of formula I in this assessment.

Table 4: In Vivo Efficacy of Compounds of Formula I against Goat Mastitis Induced by Staphylococcus aureus Compound% of Bacteriological Cures 1 75 * 2 50 5 100 6 75 7 13 8 50 9 63 10 57 • Dosage is of 100 g / 5 mL per dose It is noted that in relation to this date, the best known method for the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (10)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A compound of the formula I wherein: Ra, R ", R °, Rd and Re, are each, H, F or an alkyl group - (Z) n - of Ci-Cß having at least one fluorine substituent; Z is O, S, -SO-, or -so2-; m and n are each 0 or 1, and R1 is H, Ci-Cß alkyl, phenyl or benzyl, each of which may optionally have up to three substituents selected from halo, C1-C4 alkoxy, phenyl, N02, C? -C6 alkanoyl, benzoyl, or Ci-Cd alkanoyloxy; or a physiologically acceptable salt thereof; provided that: 1) at least one of Ra, Rb, Rc, Rd or Re is different from hydrogen; and 2) when Rc is F, or a Rb or Rd is CF3, at least one of the remaining Ra, Rb, Rc, Rd or Re is different from hydrogen.

2. A compound of the formula I as claimed in claim 1, characterized in that m is 0.

3. A compound of the formula I as claimed in claim 1, characterized in that m is 1.

4. A compound of formula I as claimed in any one of claims 1 to 3, characterized in that R1 is H or its sodium salt.

5. A compound of the formula I as claimed in any of claims 1 to 4, characterized in that Ra R ", Rc, Ra and Re are F.

6. A compound of the formula: where: m is 0 or 1; and R1 is H, C? -C6 alkyl, phenyl or benzyl, each of which may optionally have up to three substituents selected from halo, C? -C4 alkoxy, phenyl, N02, C? -C6 alkanoyl, benzoyl, or C? -C6 alkanoyloxy; or a physiologically acceptable salt thereof.

7. A pharmaceutical formulation containing as active ingredient a compound according to any of claims 1 to 6, characterized in that it is associated with one or more pharmaceutically acceptable carriers.

8. A veterinary formulation adapted for the treatment of mastitis, characterized in that it contains a compound as claimed in accordance with any of claims 1 to 6.

9. A process for preparing a compound of formula I as claimed in any one of claims 1 to 5, characterized in that it comprises reacting 3-chloro-7-aminocephalosporanic acid, or a salt or ester thereof, with a composed of formula II: wherein halo is Cl, Br, F or I to give the compound of formula I.

10. A compound of the formula I as described in any of the Examples or when made by the process according to claim 14.