AU612468B2 - Zinc ceftiofur complexes - Google Patents

Description

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L_ OPI DATE 06/09/89 APPLN. ID 30458 89

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AOJP DATE 05/10/89 PCT NUMBER PCT/US89/0016 2 INTERNAT REATY (PCT) (51) International Patent Classification 4 (11) nteiona blication Number: WO 89/ 07600 C07D 501/36, A61K 31/545 Al (43) International Publication Date: 24 August 1989 (24.08.89) (21) International Application Number: PCT/US89/00162 (22) International Filing Date: 23 January 1989 (23.01,89) (31) Priority Application Number: 155,360 (32) Priority Date: (33) Priority Country: 16 February 1988 (16,02.88) (72) Inventors; and Inventors/Applicants (for US only) CAZERS, Alexander, R. [US/US]; 6030 North 35th Street, Richland, MI 49083 KOSHY, Thomas [US/US]; 5330 Glenharbor Drive, Kalamazoo, MI 49009 (US), (74) Agent: REYNOLDS, John, Patent Law Department, The Upjohn Company, Kalamazoo, MI 49001

(US).

(81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (European patent), DK, FI, FR (European patent), GB (European patent), IT (European patent), JP, KR, LU (European patent), NL (European patent), NO, SE (European patent), US.

Published With international search report.

Parent Applie',ttion or Grant (63) Related by Continuation

US

Filed on 156,360 (CON) 16 February 1988 (16.02,88) (71) Applicant (for all designated States except US) THE UPJOHN COMPANY [US/US]; 301 Henrietta Street, Kalamazoo, MI 49001 (US), (54) Title: ZINC C4FTIOFUR COMPLEXES (57) Abstract Zinc ceftiofur complexes, zinc ceftiofur hydrochloride and zinc ceftio'ur sodium complexes, have been found 'o provide the good antibiotic activity of ceftofur while providing low dissolution rate and long half-like properties enabling the formulation of improved long term therapy forms of ceftiofur, useful in antibiotic therapy of valuable warm-blooded animals, Pharmaceutical compositions of such zinc ceftiofur complexes and a method of using such zinc ceftiofur complexes if veterinary antibiotic applications are also provided, 367 Collins Street Melbourne, Australia 4397.P AUSAL 4 _.,,,Bft-a.-gttlaig--- WO 89/07600 PCT/US89/00162 -1- ZINC CEFTIOFUR COMPLEXES

INTRODUCTION

This invention relates to zinc derivatives of ceftiofu:, a cephalosporin antibiotic, presently of special interest as a veterinary antibiotic in valuable, warm-blooded animals. More particularly, this invention provides new and useful zinc complexes of ceftiofur which improve and expand the usefulness of ceftiofur in field use antibiotic therapy and provides a form of cetiofur that can be retained longer at the site of injection for longer term therapy of the animal being treated, BACKGROUND OF THE INVENTION The cephalosporin antibiotic ceftiofur, which can be named 7-[2- (2-amino-1,3-thiazol-4-yl) -2-methoxyimino)acetamido] fur-2ylcarbonyl)thiomethyl]-3-cephem-4-carboxylic acid, also named 7-[2- (2-amino- 3- thiazol-4-yl)- 2-methoxyimino)acetamido] (furanylcarbonylthiomethyl)-8-oxo-5-thia-l-azabicyclo[4,2,0]oct-2-ene-lcarboxylic acid, and its alkali metal, alkaline earth metal and amine salts of the carboxylic acid group, and easily hydrolyzable esters thereof are described and claimed in Labeeuw et al U.S. Patent 4,464,367.

Those free acid and alkali metal and amine salt and ester forms of this cephalosporin antibiotic, ceftiofur, are somewhat unstable chemically and are obtained as amorphous compounds which are difficult to purify, and are less desirable to work with in manufacturing pharmaceutical formulations containing them, Those patented salts create salt-solid isolation and salt-solid handling problems in a manufacturing plant which those in the pharmaceutical art would prefer to avoid, A hydrohalide salt of ceftiofur, particularly the hydrochloride salt thereof, was described and claimed in U.S. patent application Serial No, 664,651 filed 25 October 1984. A corresponding South African Patent No. 85/7613 has been published disclosing such ceftiofur hydrohalide salts.

Those in the art of ceftiofur therapy continue to search for S 35 improved forms of ceftiofur including forms of ceftiofur which will not only be acceptable to the health rand safety authorities for the contemplated antibiotic therapies but which will permit and provide higher bulk density forms of ceftiofur, easier dispersibility of the WO 89/07600 PCT/US89/00162 -2selected pharmaceutical form of ceftiofur in aqueous and organic diluents and still not be detrimental to the structure of the parent ceftiofur antibiotic itself.

OBJECTS OF THE INVENTION It is an object of the invention to provide zinc complexes of ceftiofur and salts thereof as new compounds per se.

It is another object of this invention to provide improved ceftiofur antibiotic zinc complexes which have lower aqueous solubility in water than the sodium and hydrochloride ceftiofur salts but which will provide more easily dispersible in water forms of ceftiofur than the hydrochloride salt of ceftiofur than previously known forms of ceftiofur.

It is another object of this invention to provide pharmaceutical compositions containing a zinc complex of ceftiofur as an active antibiotic ingredient therein.

It is another object of this invention to provide a method or process for treating warm-blooded animal patients in need of ceftiofur antibiotic therapy which comprises administering to such patient an effective amount of one of the zinc ceftiofur complexes of this invention in a pharmaceutical diluent to assist the patient to resist, ward-off or combat infections caused by bacteria susceptible to destruction, neutralization or elimination by ceftiofur from its zinc complex form of this invention.

SUMMARY OF THE INVENTION Briefly, this invention provides aew zinc complexes of ceftiofur and cationic and hydrohalide salts thereof, pharmaceutical formulations thereof, and a method for using these new complexes to take advantage of the chemical and physical properties thereof. The pharmaceutical compositions of this invention comprise the zinc complex of ceftiofur or salt thereof, hereinabove, mixed with one or more pharmaceutically acceptable diluent components. The method of use of this invention comprises administering to a valuable, warm-blooded animal an amount of the zinc complex of ceftiofur effective to assist the animal patient to ward-off, resist, combat or counteract undesired infections by bacteria susceptible to these zinc ceftiofur complex antibiotics.

DETAILED DESCRIPTION OF THE INVENTION According to this invention, we have discovered that zinc I ii I 1 WO 89/07600 PCTUS89/00162 -3complexes of ceftiofur or a hydrohalide or cationic salt thereof, preferably an alkali metal salt thereof, or a zwitterionic form of ceftiofur, are new useful forms of ceftiofur which extend the useful properties of ceftiofur to applications where lower solubility in standard pharmaceutical fluids and animal body fluids is desirable to lengthen the time particular tissues of the animal body are exposed to the antibiotic effect of the ceftiofur molecule.

Preferably, our invention comprises a complex of an ionic zinc form and a ceftiofur molecule selected from the group consisting of ceftiofur per se, a ceftiofur hydrohalide where the hydrohaide is preferably hydrochloride or hydrobromide, or a ceftiofur cationic salt such as an alkali metal salt, for example, a sodium or potassium salt or an alkaline earth metal salt such as a calcium or magnesium salt or an economical amine salt of ceftiofur such as a triethylamine salt, or the like. We prefer to make our zinc ceftiofur complexes from ceftiofur hydrohalide salts such as the ceftiofur hydrochloride salt or from an alkali metal salt form, the sodium ceftiofur salt, but they can be made from any of the possible ionic forms of ceftiofur such as the zwitterionic form of ceftiofur, or the like, The zinc ceftiofur complexes of this invention are not yet known to have a particular structure because it is not yet known whether the zinc of the complexes are bonded, electronically or co-valently, to any particular portion of the ceftiofur cephalosporin molecule.

[See, however, Page, "The Mechanisms of Reactions of B-Lacta)n Antibiotics" in Acc, Chem, Res,, 17, pp 144-151 (1984), particularly page 149, and Tomida, H. et al., "Kinetics and Mechanism of Zinc Ion- Mediated Degradation of Cephalosperins in Tromethamine Solution" in Pharmaceutical Research,, 4, No. 3, pp. 214-219 (1987) for zinc and metal complexes of other cephalosporins which are said to have more rapid rates of aminolysis and hydrolysis], In contrast the zinc ceftiofur complexes of this invention have been found to have enhanced stability relative to the above-referenced metallo cephalosporins, We are not sure of the reason why the zinc ceftiofur complexes of this invention are more stable but the presence of sulfur in the 3-position side chain of ceftiofur may favorably influence the stability of the zinc ceftiofur complexes of this invention. However, the evidence from how we made these complexes suggests that there is chemical bonding within the complex between r J _I WO 89/07600 PCT/US89/00162 -4the zinc and the ceftiofur molecules similar to a chelating-tpe bonding because when the zinc and the ceftiofur or ceftiofur de~'vative molecules are mixed, there is a precipitation from those mixtures of zinc and ceftiofur containing entities which we haVe found are antibiotically active and which have new desirable low solubility properties which suggest a new longer acting form of ceftiofur is obtainable with these complexes. For example, we have found that the zinc ceftiofur complex made from ceftiofur hydrochloride in non-micronized form is less soluble than the ceftiofur hydrochloride. The solubility of such zinc ceftiofur hydrochloride complex is about 85% of that of non-micronized ceftiofur hydrochloride bulk drug and is 73% of the solubility of the micronized ceftiofur hydrochloride. More interestingly, the rate of dissolution of the zinc ceftiofur hydrochloride complex is much slower compared to ceftiofur hydrochloride. For example, in 20 minutes under comparable equilibration conditions 74% of unmicronized ceftiofur hydrochloride is in aqueous solution compared to only 48% of the zinc ceftiofur hydrochloride complex. Similarly, the solubility of unmicronized zinc ceftiofur sodium complex in water after 20 minutes is 47% compared to 100% for ceftiofur sodium, under comparable equilibration conditions. For the above reasons we do not wish to be bound by any particular or possible chemical bonding structures in these complexes, We have not yet obtained a crystalline form of these zinc ceftiofur complexes which can be analyzed exactly to pinpoint the possible chemical structures of these complexes.

However, we do know that these complexes are of uniform elemental composition.

The invention here includes our finding that these zinc ceftiofur complexes are uniquely adapted for incorporation into pharmaceutical compositions, This finding appears to directly contradict J prior scientific nublications the Page et al publication, regarding metal complext, of other cephalosporins) that such metallocephalosporin complexes are inordinately labile, i.e, they decompose and lose their antibacterial properties, and thus are of singular interest only for chemical mechanistic studies of zinc dependent Blactamase cephalosporin degradation processos. Such references divert attention away from our discovery of the advantages to be had using zinc ceftiofur complexes in pharmaceutical formulations for WO 89/07600 PCT/US89/00162 treating valuable warm-blooded animal patients. We believe that the low solubility of the zinc ceftiofur complexes of this invention in water illustrates the great potential for the development of improved sustained release forms of the ceftiofur antibiotic for animal use.

Although, we do not yet have quantitative data on the solubility of the zinc ceftiofur complexes of this invention in various organic solvents, we know that these zinc ceftiofur complexes are practically insoluble in most common organic solvents [except dimethylformamide (DMF)] and pharmaceutically useful vegetable oils. These low solubility properties should make these zinc ceftiofur complexes an attractive form of ceftiofur for depot type administration formulations where it is desired to maintain dosages of the ceftiofur antibiotic close to the site of infection in the animals being treated.

Further, the zinc ceftiofur complexes of this invention have advantageous, heavier bulk density properties relative to the non-zinc complexed forms of the same ceftiofur entities. For example, the bulk density of the zinc ceftiofur sodium complex from water is about 0,19 g./ml. compared to 0,11 g./ml. for trftiofur sodium salt. The bulk density of zinc ceftiofur hydrochloriac of this invention is about 0.24 g,/ml, compared to 0.15 for the noncomplexed ceftiofur hydrochloride salt. This higher bulk density property is advantageous in pharmaceutical formulation prepaa:ticn.

The zinc ceftiofur sodium complex and the zinc ceftiofur hydrochloride complex materials are obtainable as granular powders. These granular powders are readily dispersible in water (whereas the precursor ceftiofur hydrochloride salt is less well adapted for distribution in pharmaceutical aqueous vehicle formulations) and in common pharmaceutical organic solvents and in vegetable oils.

The zinc ceftiofur complexes of this invention can be made by reacting the selected form of ceftiofur, preferably ceftiofur per se, an alkali metal salt of ceftiofur, the sodium or potassium ceftiofur salt, or a hydrohalide salt of ceftiofur, the ceftiofur hydrochloride salt, in solution with water or an organic diluent such as an alkanol having from one to six carbon atoms or similar diluent with a similar solution of a t!in salt of an organic acid, such as a zinc alkanoate salt having from one to six carbon atoms in the alkanoate moiety, zinc formate, zinc acetate, zinc 1- -1 i it' S WO 89/07600 PCT/1 -6- US89/00162 4 propionate, zinc butanoate, zinc hexanoate in their various isomeric forms, zinc benzoate, or a zinc methanesulfonate, and the like, preferably in slightly excess stoichiometric proportion of the zinc salt to ensure as complete a reaction as possible of the more expensive ceftiofur reactant to form the respective zinc ceftiofur complex which usually precipitates from the reaction mixture. The resulting precipitated zinc ceftiofur complex is then recovered from the reaction mixture by conventional methods such as filtration or centrifugation procedures and then purified to at least some degree by washing with one or more wash liquids or solvents for byproducts, dried to a constant weight and then such complex is ready for formulation into any of various possible pharmaceutical delivery vehicle compositions, which are exemplified by the detailed examples hereinbelow.

The term "dosage unit form" as used in this specification and in the claims refers to physically discrete units suitable as unitary dosages for mammalian subjects, each unit containing as the essential active ingredient a predetermined quantity of a compound of this invention with the required pharmaceutical means which adapt said ingredient for systemic or topical' administration. The specification for the novel dosage unit forms of this invention are dictated by and directly dependent on the physical characteristics of the essential active ingredient and the particular effect to be achieved in view of the limitations inherent in the art of compounding such an essential active material for beneficial effects in humans and animals as disclosed in detail in this specification. Examples suitable dosage unit forms in accordance with this invention are tablets, capsules, orally administered liquid preparations in suitable liquid vehicles, sterile preparations in suitable liquid vehicles for intramuscular and intravenous administration, suppositories and sterile dry preparations for the extemporaneous preparation (mixing just prior to administration) of sterile injectable preparations in a suitable liquid vehicle and topical ointments and creams. Suitable solid diluents or carriers for the solid oral pharmaceutical dosage unit forms are selected from the group consisting of lipids, carbohydrates, proteins and mineral solids, for example, starch, sucrose, lactose, kaolin, dicalcium phosphate, gelatin, acacia, corn syrup, corn starch, talc and the like. Capsules, both hard and soft, WO 89/07600 PCT/US89/00162 -7are filled with compositions of this antibiotic active ingredient in combination with suitable diluents and excipients, for example, edible oils, talc, calcium carbonate and the like and also calcium stearate. Liquid preparations for oral administration are prepared in water or aqueous vehicles which advantageously contain suspending agents, for example, methylcdllulose, alginates, tragacanth, pectin, kelgin, carragenan, acacia, polyvinylpyrrolidone, polyvinyl alcohol, and the like, to increase the viscosity of the composition. In the case of injectable forms, the injeccable formulation must be sterile and must be fluid to the extent that easy syrTngeability exists.

Such preparations must be stable under the conditions of manufacture and storage, and ordinarily contain in addition to the principal solvent or suspending liquid, preservatives in the natre of bacteriostatic and fungistatic agents, for example, parabens, chlorobutanol, benzyl alcohol, benzoic acid, phenol, thimerosal, and the like to preserve the composition against microorganisms, in many cases, it is preferable to include osmotically active agents, for example, sugars or sodium chloride in isotonic concentrations in water pharmaceutical diluent systems. Carriers and vehicles include vegetable oils, dimethylacetamide, dimethylformmaaide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols, for exa 'ie, glycerol, propylene glycol, liquid polyethylene glycol, and the like. Any solid preparations for subsequent extemporaneous preparation of sterile injectable preparations are sterilized, by exposure to steam, cobalt 60 irradiation, or by exposure to a sterilizing gas, for example, ethylone oxide. The aforesaid carriers, vehicles, diluents, surfactartts, excipients, preservatives, isotonic agents and the like constitute the pharmaceutical means which adapt the preparations for systemic administration.

In these pharmaceutical compositions it may be desirable to include a viscosity-increasing agent such as sodium carboxymethylcellulose (sodium CMC), Other suitable viscosity-increasing agents can be substituted for sodium CMC, The pharmaceutical dosage unit forms of the compounds of this invention are prepared in accordance with the preceding general description to provide from about 1 mg. to about 500 mg. of the essential active ingredient per dosage unit form, which as aforesaid may be in the form of a semi-solid or solid, topical, oral jr rectal WO 89/07600 PC-T/US89/00162 -8preparation, a liquid oral preparation, an injectable preparation including l4,,quid preparations and solid dry preparations for extemporaneous r~econstitu~tion to a liquid injectable preparation. T he amount of the essential aptive ingredient provided in the pharmaceutical dosage unit forms is that amount sufficient to obtain antibiotic effects within the aforesaid effective non-toxic range.

Expressed otherwise, when used systemically, an amount of the essential active ingredient is provided to a recipient within a range frowu about 0.2 mg./kg. to about 100 mg./kg. of body weight of the recipient.

Preferred dosages for most applications are 0,2 mg./kg. to 10.0 mg,/kg. of body weight depending upon the animal being treated. In a topical semi-solid ointment formulation the concentration of the active ingredient may be preferably 5%-10% in a carrier, such as a pharmaceutical cream base.

The useful pharmaceutical dosage unit forms of these compounds in pharmaceutical formulations are preferably adapted for systemic administration to obtain antibiotic effects comprising an effective, non-toxic amount of the Formula I salt.

Further, the invention relates to methods of obtaining antibiotic effects in maimmals, for example, Valuable warm-blooded animals such as dogs, cats, horses, and other commercially valuable animals, by administortng systemically to the mammals the aforesaid pharmaceutical dosage units forms supplying an effective, non-toxic amount for antibiotic effects, The invention is further exemplified and described by the following detailed examples, which are not intended to be limiting, B1amnp1§ I Zinc Ceftiofur Complex From Sodium Ceftiofur (Compound I) From Water A 0,22 g, (0.4 mmole) portion of sodium ceftiofur was dissolved in 8 ml, of water with the aid of a few drops of 0. 1 M sodium bicarbonate solution., The mixture was cooled in an ice bath. To this cooled solution there was added dropwise with stirring five ml..

of a solution containing 0,44 g, (2 mmole) of zinc acetate, pre- Viously cooled in, an ice bath, A white precipitate resulted. The reaction mixture was allowed to stand for 1.5 hours in an acetone/ice bath and then the mixture was centrifuged at about 2000 R.PN and the supernatant liquid was discarded, The separated precipitate warj 0 WO 89/07600 PCT/US89/00162 -9washed with cold water three times, discarding the supernatant wash water each time. The resulting zinc ceftiofur sodium complex was suspended in water and lyophilized to obtain as product 120 mg. of the zinc ceftiofur sodium complex as an off-white powder having a purity of about 76% by an HPLC analytical method.

Example 2 Zinc Ceftiofur Sodium Complex From Methanol (Compound I) A 11 g. (20 mmole) por ;ion of sodium ceftiofur was dissolved in a mixture of 50 ml. of water and 150 ml. of methanol. Separately, a g. portion of zinc diacetate dihydrate was dissolved in a mixture of 20 ml. of water and 100 ml. of methanol. The zinc diacetate dihydrate solution was added with stirring to the sodium ceftiofur solution contained in a 500 ml. beaker. The resulting reaction mixture was allowed to stand for about 45 minutes in an ice bath and then filtered through a coarse sintered glass funnel to separate the precipitate which had formed. The separated precipitate was washed with about 120 ml. of cold methanol and then dried in a vacuum at room temperature for 64 hours. There was thus obtained 6 g, of the zinc ceftiofur sodium complex as a granular powder, having a purity of 94,8% (HPLC method), from methanol, Examle 32 Zinc Ceftiofur Hydrochloride Complex (Compound II) A 11 g. (20 mmole) portion of ceftiofur hydrochloride salt was dissolved in 150 ml, of methanol. Separately, a 20 g, portion of zinc ditcetate dihydrate 4as dissolved in 20 ml, of water plus 150 ml. of methanol. While stirring, the zinc diacetate dihydrate solution was added to the ceftiofur hydrochloride solution in a 500 ml, beaker, The reaction mixture was allowed to stand at 0C, for minutes and then filtered through a coarse sintered glass funnel, The separated precipitate was washed with 120 ml, of cold methanol and then dried in a vacuum for 64 hours. The resulting zinc ceftiofur hydrochloride complex weighed 9 g, and was a granular powder, having a purity of about 94% (HPLC method).

The formulation of the zinc ceftiofur complexes of this invention into pharmaceutical compositions can be done by conventional methods. The following examples illustrate various useful formulations for these new complexes, Example_ Sterile Parenteral Suspension Sterile Vehicle Part I Polysorbate 80, N.F, 0,i to 10 gms, -4 WO 89/07600 PCT/US89/00162 Sodium Carboxymethylcellulose low viscosity 2 to 20 gms.

Benzyl Alcohol 9.1 gns.

Benzoic Acid 0.2 to 2.0 gms.

Povidone 1 to 10 gms.

Sodium Chloride, Fine Crystals Reagent, if needed, up to 9 gms.

Hydrochloric Acid, Reagent Grade adjust pH to approx. Water for Injection q.s. to 1000 cc.

Part II Compounds I and II from Example 1, 2 or 3, powder 1,0 to 100 gns.

Vehicle Part I q.s. adjust 1000 cc, Directions: Part I All of the ingredients are dissolved in water and the vehicle sterilized by filtration, Part II Asepticaily add sterile Compound I or II from Example 1, 2 or 3 in sufficient vehicle to make 900 mis. Stir the suspension and pass through colloid mill to a- uniform consistency, Add sufficient vehicle to make 1000 mis, Example 5 Sterile Parenteral Suspension Sterile Vehicle Part I PEG 3350 NF 5 to 120 gms.

Benzyl Alcohol 9,1 gis, Betizoic Acid 0.2 to 2,0 gas.

Polysorbate 80 NF Food Grade 1 to 5 gas.

Sodium Chloride Fine Crystals Reagent 0,5 to 10 gis.

Hydrochloric Acid, Reagent Grade q.s. adjust pH to approx, Water for Injection q.s. to 1000 cc, Compounds I or II from Example 1, 2 or 3 powder 1 to 100 gms, Vehicle Part I q,s. adjust 1000 cc.

Directions All of the ingredients are dissolved in water and pH adjusted to approximately 3.0, and the vehicle sterilized by filtration, Part I Aseptically add sterile Compound I or Compound II from Example 1, 2 or 3 in sufficient vehicle from ?art I to make 900 mls, Stir WO 89/07600 PCT/US89/00162 -11the suspension and pass through a colloid mill to a uniform consistency. Add sufficient vehicle to make 1000 mis.

Example 6 Sterile Extemporaneous Parenteral Suspension (Aqueous) Sterile Vehicle Part J Benzyl Alcohol 9.1 gms.

or Benzoic Acid 0.2 to 2.0 gms.

Carboxymethylcellulose Sodium USP 1.0 to 20.0 gms, low viscosity or any other viscosity inducing agent Sodium Chloride Fine Crystals, Reagent Grade 0,5 to 10 gms.

Hydrochloric Acid, Reagent Grade q.s. adjust pH to approx, Water for Injection Part II Amount per Vial Sterile Compound I or II from Example 1, 2 or 3 in a 10 to 100 ml. glass vial 0,01 to 1,5 gm, Directions: Part 1 All of the ingredients are dissolved in water, and pH adjusted to approximately 5.6 to 6.1, preferably about 6,0, Vehicle sterilized by filtration and packaged in appropriate glass vials, Sterile powdered Compound I or II is packaged aseptically in sterile vials and the final container(s) are sterilized by Cobalt irradiation, Example 7 Sterile Extemporaneous Parenteral Suspension Sterile Vehicle Part 1 Methylparaben 1.0 to 2,7 gms, Propylparaben 0.1 to 0,5 gm, Povidone 1 to 10 gms, Sodium Chloride Fine Crystals Reagent Grade 0,5 to 10 gms, Solution Hydrochloric acid q.s. adjust pH to approx, Water for Injection qs. to 1000 cc, Part II Amont P al Sterile Compound I or II from Example 1, 2 or 3 in a 10 to 100 ml. glass vial 0.01 to 1,5 gm, Directions £=lIe I _1 _I WO 89/07600 PCT/US89/00162 -12- Methylparaben and propylparaben are dissolved in boiling water.

Then all of the ingredients dissolved in water, and F-Y -adjusted to approximately 5.6 to 6.2, preferably about 6.0. Vehicle sterilized by filtration and pa'ck.ged in appropriate glass vials.

Sterile Compound I or I is packaged aseptically in sterile Example 8 xepoaeu Parentetal Suspension (Aqueous) Sterile Vehicle Part .1 Polyethylene Glycol 3350 NF 5 to 120 gins.

Polyvinyl Pyrrolidone 1 to 10 gins., QuatresinO myristyl gamma picoliniui chloride 0. 1 to 2, 0 gins.

Sodium Chloride, Fine Crystals Reagent Grade 0. 5 to 10 gins.

Solution Hydrochloric Acid q.s, adjust pH to approx. Water for Injection q~s. to 1000 cc, ~jAmount Per Vial Sterile powdkred Compound I or II from Example 1, 2 or 3 (milled otr mnicronized) in a 10 to 100 ml. glass vial 0.01 to 1,5 gnms.

DIRECTIONS

All of the vehicle ingredients are dissolved in water and pH adjusted to approximately 5.6 to 6,1, preferably About Vehicle sterilized by filtration and packaged in appropriate glass vials, Part 11 Sterile powdered Compound I or It is packaged aseptically i sterile vials and sterilized by Cobalt 60 irradiation.

Thereafter, just prior to use, the vehicle and drug components are mixed and then administered to the animal.

Example -Sterile Non-aqueous Parentzeral Suspenoion P'owdered Compound I or 11 from Example 1, 2 or 3 (milled or micronized) 1 to 100 gxs., Chlorobtutanol Anhydrous -prese.rvative S,.25 gins, Bflnzyl Alcohol 9.25 gmsa Corn Oil Glyceryl Monostearate Cml or Cottonseaei Oil Glyceryl, Morlostearate Gel qos. to 1000 cc.

DIEflIQ 4 WO 89/07600 PCT/US89/00162 -13- Preservative is dissolved in sufficient oily gel to make 800 cc. Compound I or II is added and the suspension is colloid milled to a uniform consistency. Add sufficient gel to make 1000 mls.

After packaging into glass vials, the suspension is sterilized by Cob<,t irradiation or by anr other suitable method.

Example 10 Sterile Non-aqueous Parenteral Suspension Compound I or II from Example 1, 2 or 3 (milled or micronized) 1 to 100 gms.

Chlorobutanol Anhydrous 5.25 gms.

or Benzyl Alcohol 9.25 gms.

Corn Oil USP q.s. adjust 1000 cc.

or Cottonseed oil q.s. adjust 1000 cc,

DIRECTIONS

Preservative is dissolved in sufficient oil to make 800 cc.

Compound I or II is added and the suspension is colloid milled to a uniform gonsi&tency to break the aggregates, Add sufficient amount of oil to make 1000 mis, Stir and package into glass vials, The suspension can be sterilized by Cobalt 60 irradiation or sterile powdered Compound I or II can be added to sterile vehicle and manufactured Eollowing aseptic procedure(s), Exaim.? ii Sterile Extemporaneous Parenteral Suspension (Nonaqueous Gel) Controlled Release Formulation ri..le Vehicle Part I I000 Benzyl Alcohol preservative 9,0 to 9.25 gms, or Chlorobutanol 5.0 to 5.25 gas.

Cor Oil Glyceryl Monostearate Gel 1000 cc, Cottonseed Oil Glyceryl Monostearate Gel 1000 cc.

SPM I 100 Vials Powdered Compound I or II from Example 1, 2 or 3 (milled or micronized) 1 to 100 gms,

DIRECTIONS

Preservative is dissolved in sufficient gel and the gel is filled into vials asceptically and the vials sealed, These vials I I i WO 89/07600 PCT/US89/00162 -14will be packaged with the vials of Part II as companion package.

Part II 0.01 to 1.0 gm, of powdered Compound I or II or sterilized powdered Compound I or II is packaged in a sterile glass vial and the vials sealed. If the powdered Compound I or II is non-sterile, then the packaged vials will be sterilized by Cobalt 60 irradiation.

Prior to dosing appropriate amounts of Part I diluent will be added to Part II sterile powder and shaken until homogeneous.

Example 12 Sterile Extemporaneous Parenteral Suspension (Nonaqueous) Sterile Vehicle Part I 1000 Benzyl Alcohol preservative 9.0 to 9,25 gms.

or Chlorobutanol 5.0 to 5.25 gms.

Corn Oil, USP q.s. adjust 1000 cc, or Cottonseed Oil, USP qs. adjust 1000 cc.

Part II 100 Vials Compound I or II from Example 1, 2 or 3 (milled and micronized) 50 to 100 gms.

Part T.

Preservative is dissolved in the oil, and the solution sterilized by filtration. The sterile solution is filled into vials and the vials sealed. These vials will be packaged with the vials of Part II as companion package, Part II to 1.0 gm, of Compound I or II or sterilized Compound I or II is packaged in a sterile glass vial and the vials sealed. If the Compound I or II is non-sterile, then the packaged vials will be sterilized by Cobalt 60 irradiation.

Prior to dosing appropriate amounts of Part I diluent will be added to Part II sterile Compound I or II and shaken until uniformly mixed.

Example J6 Suppositories Formulation for a 2 gm. suppository containing 62.5 mg. of powdered Compound I or II La given. However, any size suppository can be manufactured using any amount of Compound I or II and appropriate amounts of excipients at the same ratio as indicated below.

WO 89/07600 PCT/US89/00162 Lot Size 12.

Compound I or II from Examples 1, 2 or 3 (milled or micronized) 7.5 gm, PEG-400 144 ml.

PEG-8000 96 gm.

Directions Measure out 144 ml. of PEG-400 and place in a container suitable for heating. Add 96 gms. of PEG-8000 (melting point 140*F.) to the PEG-400 solution and melt over a hot water bath approximately two minutes or until there is a clear solution.

Add the 7,5 g. of the Compound I or II and stir until dispersed. Pour the mix into the mold and let set. Chill the mold.

,emove suppositories after they set up 15-30 minutes at room temperature. Sterile suppositories can be manufactured with sterile raw materials and observing aseptic conditions during manufacturing, or can be sterilized by Cobalt 60 irradiation, 'Example 14 Suppositories Suppositories can also be manufactured from excipients suec' as cocoa butter, SuppocireTM AM, Suppocire T

AS

2 and SuppocireTM AT, Suppocire BT or Suppocire CT brand of C 8 to Cl 0 -saturated fatty acid glycerides, Formula for a 2 gm. suppository containing 62.5 mg. of Compound I or II is given- however, any size suppository can be manufactured using any desired amount of powdered Compound I or II and appropriate amount of excipient, Lot Size H Compound I or II from Example 1, 2 or 3 (milled or miaronized) Sterile 0.750 gm.

Suppocire AM or AS 2 or AT, or BT or CT 23.25 ga, Directions Weigh the Suppocire TM diluent in a container suitable for heating, Melt (45"C temperature) over a hot water bath for approximately two minutes or until there is a clear solution (microwave oven can also be used instead of the water bath), Sterilize by fil.

tration. Add sterile Compound I or II and stir until dispersed.

Pour the mix into the cold mold. After two to four minutes, the surplus of the casting is eliminated by scraping, The temperature and time of cooling must be governed according to the type of :4 -z WO 89/07600 PCT/US89/00162 -16formula. The circulating cold air should come in contact with all faces of the mold. Release from the mold must be gentle. Sterile suppositories can be manufactured with sterile raw materials and observing aseptic conditions during manufacturing, or can be sterilized by Cobalt 60 irradiation.

Example 15 Capsules One thousand two-piece hard gelatin capsules for oral use, each containing 50 mgs. of activity of the Compound I or II, are prepared from the following types and amounts of materials: 1000 Compound I or II from Example 1, 2 or 3 (50 gms. equivalent of ceftiofur) or Coated with Carnauba Wax or 75 gm, White Wax Talc and/or Magnesium Stearate 2.5 gms.

Wax coated powdered Compound I or II will have controlled release properties. The materials are thoroughly mixed and then encapsulated in the usual manner. Different strength capsules can be prepared by changing the amounts of powdered Compound I or II.

Example 16 Antibiotic Activity of the Zinc Ceftiofur Complexes of This Invention A zinc ceftiofur hydrochloride complex of this invention was tested and compared for antibiotic potency against ceftiofur hydrochloride per se and against sodium ceftiofur in standard mouse challenge tests against the representative bacterial organisms Salmonella typhimurium UC6164 and Pasteurella multocida UC9581 to determine their ED 5 0 numbers.

Each test compound was suspended or dissolved in sterile Vehicle 122 (0,025% carboxymethylcellulose in water) to a starting test concentration sufficient to provide in the first set of test animals a concentration of the test drug of 2 mg,/kg. Portions of each such test drug formulation were diluted seriatim 50:50 v/v with water to reduce the concentration of the test drug in the test animal to 7, mg./kg., then to 0.5 mg./kg., to 0.25 mg./kg, and finally to 0,125 mg./kg. Sets of mice were injected with the test dilutions of the test drugs subcutaneously once or three times after injecting i: WO 89/07600 j ILY )I 1 1^ PCT/US8900162 -17challenge doses lethal doses of one of the above-named bacterial organisms. The sets of animals were observed over six days to determine the number of surviving mice. The mortality ratios were noted and the ED 5 0 values were calculated for each test drug by standard methods. The test results are summarized in the table which follows.

TABLE I Organism No. of UC6164 1000 579 1000 No. of Treatments 3X

IX

IX

C-HCl 2.3 (1.5-4.0) 1.7 (1.0-2.8) 1.3 (0.7-2.1) 0.3 (0.2-0,4) 0.3 (0.2-0.4) Zn-C-HC1 C-Na 2.1 ND (1.4-3.1) 1.0 ND (0.6-1.7) 3.3 1.1 (0.6-1.9 0.4 ND (0.2-0.5) 0.3 0,3 (0.2-0.4 UC9581 Footnotes: values are within 95% confidence limits.

All values are adjusted for drug potency as provided, C-HC1 means ceftiofur hydrochloride, Zn-C-HCl means zinc ceftiofur hydrochloride complex of this invention.

C-Na means ceftiofur sodium salt.

ND means not determined, These above ED 5 0 number values indicate a potent antibiotic protection by the zinc ceftiofur hydrochloride complex of this invention, similar to that of the ceftiofur hydrochloride and the sodium ceftiofur salts.

In addition, dissolution rate curves for percent of the ceftiofur complexes of this invention dissolved over time compared to the dissolution rates of sodium ceftiofur and ceftiofur hydrochloride over the same time periods indicate that the zinc ceftiofur complexes of this invention generally take longer to dissolve, particularly over the first part of the dissolution test time, than do the ceftiofur hydrochloride and sodium ceftiofur salts. The sodium I. WO 89/07600 PCT/US89/00162 -18ceftiofur salt itself dissolves quite quickly in water. These dissolution rate test comparisons suggest that the zinc ceftiofur complexes of this invention would be a better slow release form of ceftiofur and that it has a longer half-life than do the ceftiofur hydrochloride and the ceftiofur sodium salts. Knowing this enables formulators of ceftiofur to pick a preferred ceftiofur sat or complex entity and an appropriate pharmaceutical formulation that wil, allow the drug to be retained at the site of injection or application longer for extended drug treatment.

Claims (5)

1. 2. A complex according to Claim 1 which is a complex of bivalent zinc and a ceftiofur hydrochloride.
2. 3. A complex according to Claim 1 which is a complex of bivale.:t zinc and an alkali metal salt of ceftiofur.
3. 4. A pharmaceutical composition comprising a zinc ceftiofur complex according to Claim 1 as an essential antibiotic ingredient therein and one or more pharmaceutically acceptable diluent carrier ingredients, A method for treating a warm-blooded animal patient to resist, ward-off, or combat undesired biological pathogen infections in said animal which comprises administering to said animal patient an effective amount of a pharmaceutical composition contning a zinc ceftiofur complex as defined in Claim 1, 4 INTERNATIONAL SEARCH REPORT international Application No PCT!US 89 /002.62 1. CLASSIFICATION OF SUBJECT MATTER (it several classifle3tion symqois aooiy, Indicate all) According to International Fatent Classification (IPC) or to both National Claaaitication and IPC IPC 4 C 07 D 501/36; A 61 K 31/545
4. 11. FIELDS SEARCHED Minimum Documentation Searched Ciasaification Systemn Iclassification Symbol& IPC 4C 07 D 501/00 Documentation Searched other than Minimum Documentation to the Eittent that such Documents are Included In the Fields Searched I 1ll. DOCUMENTS CONSIDERED TO BE RELEVANT$ Category citation of Document, I' with Indication, wnere approorlitte, of the relevant Passages 12 R elevant to Claim No. A EP, A, 0036812 (SANOFI) 30 September 1981, 1-4 see the whole document, US, 1~4464367 (cited. in tho application) A EP, A, 0180372 (UPJOHN) 7 May 1986, 1- 4 see the whole document &ZA, A, 85/7613 ('cited ini the application) A WO, A, 87/01117 (UPJOHN) 26 February 1987 1-4 see the whole documenit Special categories ot citd documental iS later document published aft4r the ternet's~nat fiiing data document defining the general state of the art which is not ot Priority Oate end not In Colitlict with t' A application but considered to be of particular relevance iten tondrtn )$Pnikoftl.yu eligth earlier document but published on or atter the International document of particular rsievance, the Claimed Invention tI'ling date cannot be consideredt novel or cannot be conaldated to document which may throw dowits on Priority clainn(O t or nVotve an Inventive ase which Is cited to estabili the P Ubi.tation date of another document of particuilar relevance: Ithe clitmed Invention Citation or other silecial zemo'o las atteClIled) cannot tie considered to Involve en liittve $too wrler the "0D" document relerring lo an oral dicltosure, use, exhibition i~r document Is comained with On$e Or more oilier such doCU- Other means menta, Such combination being obvious to a person skilled 4'P" document Pubished prior to the International tIling date but In the art, later than the Priority date climed document member Of the same Patent tamily IV, CERTIFICATION Date of the Actual Completion of the international &earch Datl of Miilllno of this International Search fiepurt April 1989
5. 16. E5 89 International Searching Aiuthority Sgsu f-3Q(I Ono EUROPEAN PAT.ENTr OFFICE Form PCTIISA/210 (aecond sheet) (January 1111115 Sterile Vehicle Part I Polysorbate 80, N.F. 0.1 to 10 gns International Application No, PCT/US 89/00162 FUFKTNER INFORMATION CONTINUED FROM THE SECOND SHEET VD 0O3SERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHADLE I This International search report has not been established In respect of certain claims under Article 17(2) for the following reasonst IK] Claim numbers because they relate to subject malter not rspulrc.d to be searhod by this Authority, namely; SEE PCT RULE 39.1(Iv) Methods for treatment of the human or animal body by surgery or therapy, as well. as diagnostic methods, 2.7 claim. numbers,*., ,j because they relate to parts of the International application that do not comply with the prescribed require. ments to such an extent that no meaningful International search can be carried opun, "pectcaiiy: Clai18M nUMbers. o b4141aU1e theyf are deperxiorit claims and ljro not dratted In accordance with the second and tmrd setences of PCT Rule 6,4(a), VIQE OWSRVATIONS WHER!E UNITY OF INVENTION IS LkCKING I This International Searchlng Authority foundi multiple, lnvirntlons In this Inistrnetlixr.i application as followst I.rl AsI all required additional search lees were timely paid by the applicant, this Inismalional search report -covers all searchable claims of the International application. 2.]As only some of the required additional search foes were timely paid by the applicant, this International search report covers only those claims of the International application for which lees were pald. apecltically cialmets 3,E) No reovired additional search, lees were timely paid by the rippllcant, Consequently, this International search report Is restricted to the lntlotio first mentioned In the 0lalmel It ls covered by claim numbefsi 4f As all searchable claims Could be searched without effort justifying an additional fee, the lntsmatoni Searching Authrity dl~t not Invite, Payment ot any additional tea, Remark on Protest SThe additional search fees ware, accontpailied by applicat's prtroest. SNo protest accompanied the payment of 'dKtloIs ec nh loes, Form IOCTASAWOt (supplemental shoot (january leall) ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. US 8900162 SA 26646 This annex lists the patent family members relating to the patent dnctments cited in the abo~ve-mentioned ijiternational search report. The memhcrs arc as contained in~ the European Patent Office EDP ile on 02/05/89 The European Patent Office is in n~o way liable for these particulars which are merely given for the purpose of informacion. Patent document Publication Patent family Publiiation cited in search report date member(s) Tdate EP-A- 0036812 30-0q-81 FR-A,B 2479229 02-10-81 JP-A- 57254192 22-09-82 CA-A- 1140114 25-01-83 US-A- 4464367 07-08-84 AT-B- E11541 15-02-85 EP-A- 0180372 07-05-86 AU-A- 4817085 01-05-86 JP-A" 61106583 24-05-86 CA-A- 1248088 03-01-89 WO-A- 8701117 26-02-87 EP-A- 0233255 26-08-87 ~JP-T- 63500455 18-02-88 EP-A- 0263901 20-04-88 P or more detaills about this anniex see offial Journal of the Europeain Pantent offlee so, 12/8%