Field of the invention

The present invention relates to prodrugs of isoxazolyl penicillins, pharmaceutical compositions comprising same and their use in the treatment of microbial infections such as mastitis.

Background

Penicillin remains one of the most important antibiotics in the treatment and prophylaxis of a large number of infectious diseases. However, bacterial resistance is a major cause of treatment failure with natural penicillins such as benzylpenicillin (also known as penicillin G) and phenoxymethylpenicillin (also known as penicillin V). Resistance occurs because the natural penicillins are readily hydrolysed into an inactive form by /3-lactamase (also known as penicillinase), an enzyme produced by Gram-positive cocci such as Staphylococcus aureus (S. aureus). /3-lactamase causes opening of the /3-lactam ring resulting in deactivation of the penicillin (see Figure 2).

Resistance has resulted in a number of penicillins being ineffective against certain strains of S. aureus and also unsuitable for use in the treatment of many infectious diseases.

Given the prevalence of bacterial resistance, /3-lactamase-resistant antibiotics are now preferred for treating infections caused by /3-lactamase-producing micro-organisms. One of the first /3-lactamase-resistant penicillins to be developed was methicillin. Whilst methicillin is resistant to cleavage by /3-lactamase, it is still susceptible to acid hydrolysis. This has resulted in significant bacterial resistance to methiciliin and the need for more stable /3-lactamase-resistant antibiotics. Unlike methicillin, the isoxazolyl penicillins are /3-lactamase-resistant and also acid stable.

With reference to Figure 3, the isoxazolyl penicillins retain the penicillin nucleus 10 that is crucial to biological activity. However, unlike the natural penicillins the isoxazolyl penicillins have a bulky, electron-withdrawing side chain 50 that enhances the chemical stability of the molecule and improves /3-lactamase-resistance.

Another significant cause of treatment failure using benzylpenicillin and other antibiotics is insufficient contact of the antibiotic with pathogenic bacteria at the site of infection. After administration the concentration of antibiotic in various fluids and tissues can vary widely. Benzylpenicillin for example does not readily pass the lipid bilayer such as the blood-brain barrier, the blood-milk barrier and other phospholipid membranes. The inability of antibiotics to enter certain compartments of the body in sufficient concentration to be pharmaceutically effective is a known limitation of antibiotic treatment.

To address this issue prodrugs have been developed that have different pharmacokinetic profiles in comparison to the active drugs. Prodrugs are converted to the active drug in vivo following administration. The use of prodrugs is a known technique for improving absorption and/or bioavailability of an active drug or selectively targeting delivery of the active drug to the site of infection. A further advantage of using prodrugs is that the therapeutic effect can be achieved with lower dosages than when using the active drug. However, known penicillin prodrugs suffer from problems associated with stability. For example, penethamate hydriodide, a prodrug of benzylpenicillin, offers poor /3-lactamase resistance when used intramuscularly.

Given the prevalence of bacterial resistance to natural penicillins and the disadvantages associated with known penicillin prodrugs, there is an urgent need for alternative prodrugs of /3-lactamase-resistant antibiotics with pharmacokinetic profiles that enable the active drug to be well distributed throughout body compartments (that may be traditionally difficult for /3-lactamase-resistant antibiotics to reach) in effective concentrations. Such prodrugs would be beneficial in the treatment of infections (for example serious staphylococcal infections) that require treatment by /3-lactamase-resistant antibiotics where drug delivery to the site of infection is difficult. One example of such an infection is mastitis.

The consequences of mastitis in mammals caused by bacterial infection include: (a) discomfort and pain;

(b) reductions in the quality and quantity of milk produced, even when the infection is subclinical;

(c) inability to feed offspring or even rejection of offspring;

(d) direct economic impacts such as when the infection affects dairy animals.

Mastitis is reported to be among the most costly diseases affecting the dairy industry worldwide.

Significant causes (major pathogens) of mastitis include S. aureus, Streptococcus uteris (Strep, uberis), Strep, agalactiae and Strep, dysgalactiae. Traditionally, treatments for mastitis in dairy animals have involved the intramammary infusion of a suitable antibiotic. However, this is a difficult treatment to administer and causes significant pain and discomfort to the recipient animal. The use of penethemate hydriodide for the treatment of mastitis in dairy cattle by intramuscular injection suffers from the disadvantage of poor ^-lactamase resistance. Accordingly, there is a need for improved antibiotic treatments for mastitis.

Against this background, the present inventor has developed a class of /3-lactamase- resistant antibiotic prodrugs which offer significant advantages over known penicillin prodrugs and the active drugs themselves.

Summary of the Invention

In a first aspect, the present invention provides a compound of the formula (I):

- A -

wherein:

A is an optionally substituted phenyl group, wherein the optional substituents are selected from the group consisting of: halogen and Ci-C ₆ alkyl, R ₃ is selected from the group consisting of: O-B and NR ₄ -B, B is a monovalent hydrocarbon radical having between 1 and 20 carbon atoms which may optionally be substituted with one or more amino groups, and

R ₄ is selected from the group consisting of: hydrogen and CpC ₆ alkyl, the compound of formula (I) including pharmaceutically acceptable salts thereof.

A may be optionally substituted with one or more halogens selected from the group consisting of fluorine and chlorine. In an embodiment, A may be optionally substituted with 1 or 2 fluorine or chlorine substituents.

wherein R ₁ and R ₂ are independently selected from the group consisting of: hydrogen, chlorine and fluorine, or alternatively selected from the group consisting of: hydrogen and chlorine.

In one embodiment, R ₃ is O-B, wherein B is a monovalent hydrocarbon radical having between 1 and 10 carbon atoms which may optionally be substituted with one or more amino groups.

In a further embodiment, R ₃ is O-B, wherein B is a monovalent hydrocarbon radical having between 1 and 10 carbon atoms which is substituted with an amino groups.

In an alternative embodiment, B is a monovalent hydrocarbon radical of the formula - (CH ₂ ) _n NR ₅ R _ό , wherein n is an integer between 1 and 4 and R ₅ and R ₆ are CpC ₄ alkyl, or together with the nitrogen to which they are attached form a five or six membered saturated heterocyclic ring. In another alternative embodiment, B is a monovalent hydrocarbon radical of the formula -(CHb) _n NR ₅ R ₆ , wherein n is 1 or 2 and R ₅ and R ₆ are independently methyl or ethyl, or together with the nitrogen to which they are attached form a five or six membered saturated heterocyclic ring.

In yet a further alternative embodiment, B is a monovalent hydrocarbon radical of the formula -(CH ₂ ) _H NR ₅ R ₆ , wherein n is 2 and R ₅ and R ₆ are independently methyl or ethyl, or together with the nitrogen to which they are attached form a five or six membered saturated heterocyclic ring.

The compound of formula (I) may be in the form of a HX salt, wherein X is chloro, bromo or iodo.

The compound of formula (I) may have the following structure:

wherein A and R ₃ are as defined in the first aspect.

In a second aspect, the present invention provides a pharmaceutical composition comprising a compound of the formula (I), as defined in the first aspect, and one or more pharmaceutically acceptable diluents, carriers or excipients.

In a third aspect, the present invention provides the use of a compound of the formula (I) as defined in the first aspect, as a prodrug.

In a fourth aspect, the present invention provides a method for the treatment or prevention of a microbial infection in a subject in need of said treatment or prevention, the method comprising administering to the subject a therapeutically effective amount of a compound of the formula (I) as defined in the first aspect.

The microbial infection may be a bacterial infection.

The subject may be a mammal.

The method may involve administering to the subject an amount of the compound of formula (I) which corresponds to a lower dose of the active as compared to the dose of the active that would ordinarily be administered to the subject to treat or prevent the infection.

In a fifth aspect, the present invention provides a compound of the formula (I), as defined in the first aspect, for use in the treatment or prevention of a microbial infection.

hi a sixth aspect, the present invention provides the use of a compound of the formula (I), as defined in the first aspect, in the manufacture of a medicament for the treatment or prevention of a microbial infection.

hi a seventh aspect, the present invention provides a method for the treatment or prevention of mastitis in a mammal in need of said treatment or prevention, the method comprising administering to the mammal a therapeutically effective amount of a compound of the formula (I) as defined in the first aspect.

The mastitis may be caused by bacteria, for example Staphylococcus aureas, Streptococcus uteris, Streptococcus agalactiae or Streptococcus dysgalactiae.

The mammal may be a dairy animal, for example a cow, which may or may not be lactating.

The compound of formula (I) may be administered parenterally.

The method may involve administering to the mammal an amount of the compound of formula (I) which corresponds to a lower dose of the active as compared to the dose of the active that would ordinarily be administered to the mammal to treat or prevent mastitis. In an eighth aspect, the present invention provides a compound of the formula (I), as defined in the first aspect, for use in the treatment or prevention of mastitis.

In a ninth aspect, the present invention provides the use of a compound of the formula (I), as defined in the first aspect, in the manufacture of a medicament for the treatment or prevention of a mastitis.

In a tenth aspect, the present invention provides a method for the treatment or prevention of a microbial infection in a subject that is capable of being treated with, or prevented by, an isoxazolyl penicillin, the method comprising administering to the subject a therapeutically effective amount of a compound of the formula (I) as defined in the first aspect.

The subject may be a mammal.

The infection may be a bacterial infection.

The method may involve administering to the subject an amount of the compound of formula (I) which corresponds to a lower dose of the isoxazolyl penicillin as compared to the dose of the isoxazolyl penicillin that would ordinarily be administered to the subject to treat or prevent the infection.

The isoxazolyl penicillin may be selected from the group consisting of: cloxacillin, dicloxacillin, flucoxacilin and oxacillin.

In an eleventh aspect, the present invention provides a compound of the formula (I), as defined in the first aspect, for use in the treatment or prevention of a microbial infection in a subject that is capable of being treated with, or prevented by, an isoxazolyl penicillin.

In a twelfth aspect, the present invention provides the use of a compound of the formula (I), as defined in the first aspect, in the manufacture of a medicament for the treatment or prevention of a microbial infection in a subject that is capable of being treated with, or prevented by an isoxazolyl penicillin. Brief description of the drawings

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

Figure 1 depicts a generic structure of known penicillin antibiotics 10, including the /3-lactam ring 30 that is crucial to biological activity. The arrow 40 indicates the site of action of /3-lactamase which causes opening of the /3-lactam ring. The sidechain 50 determines much of the antibacterial, chemical and pharmacokinetic characteristics, and the carboxylic acid group attached to the thiazolidine ring 20 exerts effects on the lipophilicity of the compound.

Figure 2 shows the effect of ring strain on the β- lactam ring. Because of ring strain penicillins are susceptible to hydrolysis leading to opening of the /3-lactam ring. Once the /3-lactam ring is opened all significant antibacterial activity is lost.

Figure 3 shows the structure of known isoxazolyl penicillin antibiotics, a class of /3-lactamase-resistant antibiotics. Rl and R2 are typically chlorine, fluorine or hydrogen.

Figure 4 depicts the structures of known isoxazolyl penicillin antibiotics. The substituents Rl and R2 do not contribute significantly to the electron withdrawing effect of the phenylisoxazolyl moiety, but do affect drug absorption and plasma protein binding.

Figure 5 shows the structure of an isoxazolyl penicillin prodrug in accordance with one embodiment of the present invention.

Figure 6 depicts isoxazolyl penicillin prodrugs in accordance with a further embodiment of the invention.

Figure 7 shows a ¹ H NMR spectrum of the hydroiodide salt of Prodrug 1 (dimethylaminoethyl cloxacillin).

Figure 8 shows a mass spectrum of Prodrug 1. Figure 9 shows a ¹ H NMR spectrum of the hydroiodide salt of Prodrug 2 (2-(pyrrolidin-l-yl) ethyl cloxacillin).

Figure 10 shows a mass spectrum of Prodrug 2.

Figure 11 shows a ¹ H NMR spectrum of the hydroiodide salt of Prodrug 3 (2-(piperidin-l-yl) ethyl cloxacillin).

Figure 12 shows a mass spectrum of Prodrug 3.

Figure 13 shows a ¹ H NMR spectrum of Prodrug 4 as the free amine (diethylaminoethyl cloxacillin).

Figure 14 shows a ¹³ C NMR of spectrum of Prodrug 4.

Figure 15 shows a ¹ H NMR spectrum of the hydroiodide salt of Prodrug 4.

Figure 16 shows a mass spectrum of Prodrug 4.

Figure 17 shows sample data from experiments to determine the pharmacokinetic characteristics of Prodrugs 1 to 4. Comparison is made with the known prodrug penethemate.

Figure 18 depicts a synthetic route for the preparation of Prodrug 4.

Definitions

The following are some definitions that may be helpful in understanding the description of the present invention. These are intended as general definitions and should in no way limit the scope of the present invention to those terms alone, but are put forth for a better understanding of the following description.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or" comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers, but not the exclusion of any other step or element or integer or group of elements or integers. Thus, in the context of this specification, the term "comprising" means "including principally, but not necessarily solely".

In the context of this specification, the term "Ci-C ₆ alkyl" is understood to include straight chain and branched chain monovalent saturated hydrocarbon groups having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, hexyl and the like.

In the context of this specification, the term "C ₁ -C ₄ alkyl" is understood to include straight chain and branched chain monovalent saturated hydrocarbon groups having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, tertiary butyl, and the like.

hi the context of this specification, the term "halogen" is understood to include fluoro, chloro, bromo and iodo.

In the context of this specification, the term "monovalent hydrocarbon radical" is understood to mean a monovalent hydrocarbon group that is either straight chain or branched chain and may include one or more carbon-carbon double bonds and/or one or more carbon-carbon triple bonds.

In the context of this specification, the term "pharmaceutically acceptable" means that the compound to which it refers is suitable for use in contact with tissues of the body without undue toxicity, incompatibility, instability, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts include organic acid addition salts and inorganic acid addition salts including, but not limited to, those formed from: acetic, ascorbic, aspartic, benzoic, benzenesulfonic, citric, cinnamic, ethanesulfonic, fumaric, glutamic, glutaric, gluconic, hydrochloric, hydrobromic, hydroiodic, lactic, maleic, malic, methanesulfonic, naphthoic, hydroxynaphthoic, naphthalenesulfonic, naphthalenedisulfonic, naphthaleneacrylic, oleic, oxalic, oxaloacetic, phosphoric, pyruvic, p-toluenesulfonic, tartaric, trifiuoroacetic, triphenylacetic, tricarballylic, salicylic, sulphuric, sufamic, sulfanilic and succinic acid. In the context of this specification, the terms "treatment" and "treating" refer to any and all uses which remedy an infection, condition, disease, disorder or symptoms thereof, or otherwise prevent, hinder or reverse the progression of an infection, condition, disease, disorder or symptoms thereof, in any way whatsoever. Treatment may be for a defined period of time, or provided on an ongoing basis depending on the particular circumstances of any given individual.

In the context of this specification, the terms "prevent" and "prevention" refer to any and all uses which prevent or delay the establishment or onset of an infection condition, disease, disorder or symptoms thereof in any way whatsoever.

In the context of this specification, the term "therapeutically effective amount" includes within its meaning a non-toxic amount of a compound of formula (I) sufficient to provide the desired therapeutic effect. The exact amount will vary from subject to subject depending on the age of the subject, their general health, the severity of the disorder being treated and the mode of administration of the compound. It is therefore not possible to specify an exact "therapeutically effective amount", however one skilled in the art would be capable of determining such an amount by routine trial and experimentation.

In the context of this specification, the term "dairy animal" includes animals which are farmed for their milk. Examples of dairy animals include but are not limited to, cows, sheep, goats and buffalo.

Detailed description

The present invention relates to a compound of the formula (I):

wherein:

A is an optionally substituted phenyl group, wherein the optional substituents are selected from the group consisting of: halogen and CpC ₆ alkyl, R ₃ is selected from the group consisting of: O-B and NR ₄ -B,

B is a monovalent hydrocarbon radical having between 1 and 20 carbon atoms which may optionally be substituted with one or more amino groups, and

R ₄ is selected from the group consisting of: hydrogen and Q-C ₆ alkyl, the compound of formula (I) including pharmaceutically acceptable salts thereof.

The compounds of formula (I) may have one or more chiral centres. The present invention includes all enantiomers and diastereoisomers, as well as mixtures thereof in any proportions. The invention also extends to isolated enantiomers or pairs of enantiomers. Enantiomers and diastereoisomers may be separated according to methods well known to those skilled in the art. hi one embodiment, A is:

in R ₁ and R ₂ are selected from the group consisting of: hydrogen, chlorine and fluorine, and B is a monovalent hydrocarbon radical of the formula -

(CH ₂ ) _n NR ₅ R _ό , wherein n is an integer between 1 and 6 and R ₅ and R ₆ are Ci-C ₆ alkyl, or together with the nitrogen to which they are attached form a five or six membered saturated heterocyclic ring.

In another embodiment, A is:

Ri and R ₂ are selected from the group consisting of: hydrogen, chlorine and fluorine, and B is a monovalent hydrocarbon radical of the formula - (CH ₂ ) _P NR ₅ R ₆ , wherein n is an integer between 1 and 4 and R ₅ and R ₆ are Ci-C ₄ alkyl, or together with the nitrogen to which they are attached form a five or six membered saturated heterocyclic ring.

In a further alternative embodiment, A is:

n Ri and R ₂ are selected from the group consisting of: hydrogen and chlorine, and B is a monovalent hydrocarbon radical of the formula -(CH ₂ ) _H NR ₅ R ₆ , wherein n is 1 or 2 and R ₅ and R ₆ are methyl or ethyl, or together with the nitrogen to which they are attached form a five or six membered saturated heterocyclic ring.

In an alternative embodiment, A is:

R2 wherein Ri and R ₂ are selected from the group consisting of: hydrogen and chlorine, and B is a monovalent hydrocarbon radical of the formula -(CH ₂ ) _n NR ₅ R ₆ , wherein n is 2 and R ₅ and R ₆ are methyl or ethyl, or together with the nitrogen to which they are attached form a five or six membered saturated heterocyclic ring..

The compound of the formula (I) may have the following structure:

wherein A and R ₃ are as defined herein. In this structure, the stereochemistry is equivalent to that of the cloxacillin. In one embodiment, the compound of the formula (I) is not (2S,5R,6R)-6- {[3-(2- chlorophenyl)-5-methyl-oxazole-4-carbonyl]amino}-3,3-dimethy l-7-oxo-4-thia-l- azabicyclo[3.2.0]heptane-2-carboxylic acid N,N-diethylaminoethyl ester (cloxacillin 2- N,N-diethylaminoethyl ester).

In another embodiment of the invention the compounds of formula (I) are selected from the group consisting of:

Prodrugs 1 to 4 may be in the form of HI salts.

The compounds of formula (I) may be prodrugs of cloxacillin, dicloxacillin, flucoxacilin and oxacillin, wherein the group R ₃ is as defined in the first aspect. The compounds of the formula (I) may be conveniently prepared in accordance with the synthetic route depicted in Figure 18, which shows the preparation of prodrug 4. Referring to Figure 18, reaction of cloxacillin (compound 100) with ethylchloro formate provides the intermediate ethoxyformic anhydride 110, which is subsequently reacted with N _^ /V-diethylethanolamine (compound 110a) thereby affording the desired aminoester free base (compound 120). The free base is then conveniently converted to the HI salt (compound 130) by treatment with aqueous acetic acid followed by aqueous sodium iodide.

hi order to prepare alternative compounds of the formula (I), one need only substitute the hydroxyamine HOa with an alternatively functionalised hydroxyamine. For example, in order to prepare Prodrug 1, hydroxyamine HOa is replaced by N _^ /V-dimethylethanolamine. In order to prepare prodrugs 2 and 3, hydroxyamine 110a is replaced by l-(2- hydroxyethyl)pyrrolidine or l-(2-hydroxyethyl)piperidine.

Other compounds of the formula (I) may be conveniently obtained by starting with an alternative penicillin derivative, for example oxacillin, dicloxacillin or floxacillin.

The compounds of the formula (I) are isoxazolyl penicillin esters which on administration to a subject are hydrolysed, thereby releasing the antimicrobially active isoxazolyl penicillin. Accordingly, in one embodiment, the invention relates to the use of a compound of the formula (I) as a prodrug.

The compounds of formula (I) are resistant to ^-lactamase and possess a favourable pharmacokinetic profile. As a result, administration of the compounds of formula (I) provides advantages over administration of the corresponding active compounds and other prodrug compounds in that the compounds of formula (I) can more effectively target the site of infection and also more effectively reach problematic sites of infection in sufficient concentration to be pharmaceutically effective.

The favourable pharmacological properties of the compounds of formula (I) may also enable them to be administered in an amount which corresponds to a lower dose of the active as compared to the dose of the active that would ordinarily be administered to the subject to treat or prevent an infection. By utilising the compounds of the formula (I) the dosage of the active may be able to be reduced by up to about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 80% of the dose of the active that would ordinarily be administered, or alternatively between about 2% and 50%, or between about 2% and 30%, or between about 2% and about 20%, or between about 2% and about 15%, or between about 2% and about 10%, of the dose of the active that would ordinarily be administered. For example, if the treatment of a particular infection ordinarily called for a dose of cloxicillin of about 25 mg/kg of body weight per day, the use of a prodrug compound of formula (I) of cloxcillin may permit administration of an amount of cloxicillin corresponding to about 20 mg/kg of body weight per day, or even less. Such a reduction in dosage is clearly advantageous in that any associated side effects would be reduced, or possibly even eliminated.

In embodiments of the invention, the compounds of the formula (I) may be used in the treatment of bacterial infections in mammals. The infection may be, for example, an infection of the skin, an ear or throat infection, a respiratory infection, an infection of a joint, or any other bacterial infection of the mammalian body. The infection may be an infection caused by an organism which is susceptible to treatment with an isoxazolyl penicillin.

The pharmacokinetic profiles of the compounds of the formula (I) are such that the active drug is able to be well distributed throughout difficult to reach body compartments in therapeutically effective concentrations. Accordingly, the compounds of the formula (I) are particularly useful in the treatment or prevention of mastitis, where in the case of cows, the active compound is required in the udder.

Accordingly, in another embodiment of the invention, the compounds may be used in the treatment or prevention of mastitis in a mammal, for example a dairy animal such as a cow. The animal may or may not be lactating. hi the treatment or prevention of mastitis, the compounds of the formula (I) may be administered by any suitable route, such as parenterally (for example intramuscularly), orally or by infusion (for example intramammary infusion). The mastitis may be clinical or sub-clinical mastitis.

Compositions and routes of administration

The compounds of formula (I) find use in the treatment and/or prevention of microbial infections, and in particular bacterial infections such as mastitis. The compounds of formula (I) may be administered to subjects in the form of pharmaceutical compositions.

Pharmaceutical compositions include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), inhalation (including use of metered dose pressurised aerosols, nebulisers or insufflators), rectal and topical (including dermal, buccal, sublingual and intraocular) administration. The most suitable route may depend upon, for example, the condition of the recipient and the nature and/or severity of the infection being treated.

The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing one or more compounds of the formula (I) into association with a carrier which constitutes one or more accessory ingredients.

Generally, an effective dosage of a compound of the formula (I) may be in the range of about O.OOOlmg to about lOOOmg per kg body weight per 24 hours; about O.OOlmg to about 750mg per kg body weight per 24 hours; about O.Olmg to about 500mg per kg body weight per 24 hours; about O.lmg to about 500mg per kg body weight per 24 hours; about O.lmg to about 250mg per kg body weight per 24 hours, or about l.Omg to about 250mg per kg body weight per 24 hours. More typically, an effective dose range is expected to be in the range of about l.Omg to about 200mg per kg body weight per 24 hours; about l.Omg to about lOOmg per kg body weight per 24 hours; about l.Omg to about 50mg per kg body weight per 24 hours; about l.Omg to about 25mg per kg body weight per 24 hours; about 5.0mg to about 50mg per kg body weight per 24 hours; about 5.0mg to about 20mg per kg body weight per 24 hours, or about 5.0mg to about 15mg per kg body weight per 24 hours. Altematively, an effective dosage may be up to about 500mg/m ² . Generally, an effective dosage is expected to be in the range of about 25 to about 500mg/m ² , about 25 to about 350mg/m ² , about 25 to about 300mg/m ² , about 25 to about 250mg/m ² , about 50 to about 250mg/m ² , or about 75 to about 150mg/m ² .

5 The frequency of administration of the compounds of formula (I) is also likely to depend on the stability of the compound in vivo. For compounds of formula (I) which are stable in vivo, it is possible that administration will not be necessary on a daily basis, but rather may only be required on a weekly or even monthly basis. Those skilled in the art would, by routine trial and experimentation, be able to determine an appropriate dosage io frequency.

Compositions suitable for buccal (sublingual) administration include lozenges comprising a compound of the formula (I) in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising a compound of the formula (I) in an inert base such as gelatine and glycerin or sucrose and acacia.

ι ₅ Compositions suitable for oral administration may be presented as discrete units such as gelatine or HPMC capsules, cachets or tablets, each containing a predetermined amount of a compound of formula (I) as a powder, granules, as a solution or a suspension in an aqueous liquid or a non-aqueous liquid, or as an oil-in-water liquid emulsion or a water-in- oil liquid emulsion. The compound of formula (I) may also be present as a paste.

-0 When compounds of the formula (I) are formulated as capsules, the compound may be formulated with one or more pharmaceutically acceptable carriers such as starch, lactose, microcrystalline cellulose, silicon dioxide and/or a cyclic oligosaccaride such as cyclodextrin. Additional ingredients may include lubricants such as magnesium stearate and/or calcium stearate. Suitable cyclodextrins include α-cyclodextrin, β-cyclodextrin, γ-

₂ 5 cyclodextrin, 2-hydroxyethyl-β-cyclodextrin, 2-hydroxypropyl-cyclodextrin, 3- hydroxypropyl- β-cyclodextrin and tri-methyl-β-cyclodextrin. The cyclodextrin may be hydroxypropyl-β-cyclodextrin. Suitable derivatives of cyclodextrins include Captisol® a sulfobutyl ether derivative of cyclodextrin and analogues thereof as described in US patent No. 5,134,127. Tablets may be prepared by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the compound of formula (I) in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant (for example magnesium stearate or calcium stearate), inert diluent or a surface active/dispersing agent. Moulded tablets may be made by moulding a mixture of the powdered compound of formula (I) moistened with an inert liquid diluent, in a suitable machine. The tablets may optionally be coated, for example, with an enteric coating and may be formulated so as to provide slow or controlled release of the compound of formula (I) therein.

Compositions for parenteral administration include aqueous and non- aqueous sterile injectable solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the compositions isotonic with the blood of the intended recipient, and which may include suspending agents and thickening agents. A parenteral composition may comprise a cyclic oligosaccaride such as hydroxypropyl-β-cyclodextrin. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example saline or water- for-injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Dry powder compositions for topical delivery to the lung by inhalation may, for example, be presented in capsules and cartridges of, for example gelatine, or blisters or for example laminated aluminium foil, for use in an inhaler or insufflator. Compositions generally contain a powder mix for inhalation of the one or more compounds of the formula (I) and a suitable powder base (carrier substance) such as lactose or starch. Use of lactose is preferred. Each capsule or cartridge may generally contain between 20μg-10mg of a compound of formula (I), optionally in combination with another therapeutically active ingredient. Alternatively, the compound or compounds of the formula (I) may be presented without excipients. Packaging of the composition may be for unit dose or multi- dose delivery. Spray compositions for topical delivery to the lung by inhalation may, for example be formulated as aqueous solutions or suspensions or as aerosols, suspensions or solutions delivered from pressurised packs, such as a metered dose inhaler, with the use of a suitable liquefied propellant. Suitable propellants include a fluorocarbon or a hydrogen-containing chlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, especially 1,1,1,2-tetrafluoroethane, 1,1,2,2,3,3,3-heptafluoro-n-propane or a mixture thereof. Carbon dioxide or other suitable gas may also be used as propellant. The aerosol composition may be excipient free or may optionally contain additional composition excipients well known in the art, such as surfactants e.g. oleic acid or lecithin and cosolvents e.g. ethanol. Pressurised compositions will generally be retained in a canister (e.g. an aluminium canister) closed with a valve (e.g. a metering valve) and fitted into an actuator provided with a mouthpiece.

Compositions for administration by inhalation desirably have a controlled particle size. The optimum particle size for inhalation into the bronchial system is usually 1-10 μm, preferably 2-5 μm. Particles having a size above 20 μm are generally too large when inhaled to reach the small airways. When the excipient is lactose it will typically be present as milled lactose, wherein not more than 85% of lactose particles will have a MMD of 60-90 μm and not less than 15% will have a MMD of less than 15 μm.

Compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Such patches suitably comprise the compound of the formula (I) as an optionally buffered aqueous solution of, for example, 0.1 M to 0.2 M concentration with respect to the compound.

Compositions suitable for transdermal administration may also be delivered by iontophoresis, and typically take the form of an optionally buffered aqueous solution of the active compound. Suitable compositions comprise citrate or Bis/Tris buffer (pH 6) or ethanol/water and contain from 0.1 M to 0.2 M of a compound of the formula (I). Compositions for rectal administration may be presented as a suppository with carriers such as cocoa butter or polyethylene glycol, or as an enema wherein the carrier is an isotonic liquid such as saline. Additional components of the compositions may include a cyclic oligosaccaride, for example, a cyclodextrin, as described above, such as hydroxypropyl-β- cyclodextrin, one or more surfactants, buffer salts or acid or alkali to adjust the pH, isotonicity adjusting agents and/or anti-oxidants.

Compositions suitable for topical administration to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include Vasoline, lanoline, polyethylene glycols, alcohols, and combinations of two or more thereof. The compound of the formula (I) is generally present at a concentration of from 0.1% to 5% w/w, or from 0.5% to 2% w/w. Examples of such compositions include cosmetic skin creams.

The production of pharmaceutical compositions for the treatments herein described are typically prepared by admixture of the compounds of the formula (I) with one or more pharmaceutically or veterinary acceptable carriers and/or excipients as are well known in the art.

The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the composition and must not be deleterious to the subject. The carrier or excipient may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose, for example, a tablet, which may contain up to 100% by weight of the active compound, preferably from 0.5% to 75% by weight of the compound of the formula (I).

The present invention will now be described with reference to specific examples, which should not be construed as in any way limiting the scope of the invention. Examples

Example 1 - Synthesis of Prodrug 4

Prodrug 4 was prepared in accordance with the synthetic route set out in Figure 18 as follows.

To an ice/salt cooled (approximately -1O ⁰ C) suspension of cloxacillin sodium (compound 100, around 10 g, 21.8 mmol) in dry dichloromethane (approximately 300 mL) is added ethylchloroformate (around 2.5 g, 23 mmol) dropwise, followed by dry pyridine (around 30 drops). The mixture is stirred (stirrer fitted with drying tube) at around -1O ⁰ C for about 90 minutes. A clear solution is observed with precipitated sodium chloride. The precipitated sodium chloride is filtered off and the filtrate comprising the ethoxyformic anhydride intermediate (compound 110) is again cooled at around -1O ⁰ C by an ice/salt bath. ΛyV-diethylethanolamine (around 2.6 g, 22 mmol) in dry dichloromethane (10 mL) is added in a dropwise manner to the cooled solution which is stirred for about 30 minutes and then allowed to reach room temperature and stirred for a further 22 hours. The mixture is then washed with water (1 x 250 mL), 10% aqueous NaHCO ₃ solution (6 x 200 mL), water (1 x 250 mL), brine (1 x 250 mL) and dried over anhydrous sodium sulfate (all amounts approximate). The solution is then passed through a celite pad (Fluka, Celite ^® 503) and washed with a little dry dichloromethane. The solvent is then removed under rotary vacuum at room temperature to obtain the free cloxacillin aminoester (compound 120) as a yellow oil.

The structure of aminoester 120 is confirmed by ¹ H NMR (Figure 13), ¹³ C NMR (Figure 14) and mass spectrometry (+ESI) LC/MS spectra, m/z 535 (M+H) (Figure 16).

The aminoester is then dissolved in acetic acid/water (4:1; 20 mL) at -1O ⁰ C and stirred for about 90 minutes under these conditions. A solution of sodium iodide (approximately 10 g in 10 mL of water) is added dropwise at around -5 ⁰ C and the mixture is stirred under these conditions for a further 90 minutes. The compound is then extracted with a little dichloromethane, washed with water (2 x 250 mL) and brine (1 x 100 mL) and then dried over anhydrous sodium sulfate. The solvent is concentrated to ~70 mL under rotary vacuum without warming. The solution is then added slowly to dry diethylether (500 mL) at O ⁰ C with continuous stirring. The precipitated HI salt is then collected by filtration and washed with a little cold, dry ether and finally dried under high vacuum to obtain the hydroiodide salt of the cloxacillin aminoester as an off-white powder (9.4 g, 80 % yield).

Example 2 - Investigation of pharmacological properties

In vitro experiments have been performed in order to investigate the pharmacological properties of Prodrugs 1 to 4. A summary of the experimental data is provided in the table in Figure 17. Comparative data was also obtained using the known benzylpenicillin prodrug penethemate.

Penethemate was selected for the comparison because it is currently the only prodrug of a penicillin antibiotic indicated for the treatment of mastitis in dairy cattle and available for administration by intramuscular injection. However, penethamate has a number of drawbacks, including:

(a) poor /3-lactamase resistance, making it unsuitable in the treatment of certain infections, including infections caused by S. aureus, one of the major causes of mastitis in dairy animals;

(b) a short half-life due to instability which limits its potential to be used as "dry cow" treatment; and

(c) limited availability of the active drug (benzylpenicillin) for migration to the site of infection - this is because only a relatively small percentage of the administered drug remains in non-ionised form in serum and is therefore easily able to pass out of the blood across lipoprotein membranes (e.g. the blood-milk barrier).

Referring to the table in Figure 17, Prodrugs 1 to 4 show favourable pharmacokinetic characteristics compared with penethemate, as described in detail below.

p Ka and "ion trapping" of the active ingredient A differential in the pH of aqueous solutions on either side of a cellular membrane serves as a drug transport mechanism, as molecules with suitable pKa values are driven down the pH gradient. Where the pH of any aqueous solution is lower than that of blood, drug molecules (of suitable pKa) will exit the blood.

Ion trapping of a non-polar antibiotic molecule can occur whenever there is a pH difference across any cellular membrane. This is because as soon as the non-polar molecule passes from serum into an aqueous solution with a lower pH, the larger fraction of molecules will become charged and no longer able to enter the phospholipid membrane. The pKa of Prodrugs 1 to 4 is sufficiently high (7.33, 8.17, 7.88 and 8.35, respectively, at 39 ⁰ C - the body temperature of cows) to enable "ion trapping" of the active drug (i.e. cloxacillin) within certain body compartments (e.g. the udder).

In the example of mastitis, the pH of milk in the udder gets closer to the pH of serum because of inflammation thereby reducing ion trapping. However, because of the composition of milk (being 4 to 5% fat), the active drug molecules that do enter the udder are trapped in the fat of the milk in increasing concentrations as they penetrate the phospholipid membrane.

The pKa of Prodrugs 1 to 4 is closer to the pH of blood (7.2-7.4) than penethemate (pKa = 8.4). This results in a greater fraction of Prodrugs 1 to 4 being in a non-charged state, which in turn leads to more efficient ion trapping in cases of both acute and non-clinical mastitis.

Polarity and lipophilicity

Passage across membranes is further assisted if a molecule is non-polar because the molecule is able to pass through the lipid membrane by passive diffusion. Non-polar molecules are readily soluble in milk fat which serves as a second drug transport mechanism across the lipid membrane.

Known penicillins are hydrophilic, polar molecules with limited ability to penetrate lipoprotein membranes. By derivatisation of the carboxylic acid moiety of cloxacillin, it has been possible to control the polarity of Prodrugs 1 to 4 (depending on the nature of R ₃ ), and thereby their ability to penetrate the phospholipid membrane. Prodrugs 1 to 4 are able to pass phospholipid membranes. The counterbalance for good lipophilicity is to also maintain sufficient hydrophilicity to enable the molecule to enter aqueous solutions such as any bodily or intracellular fluid. The reason for this is that drug solubility is important for absorption.

Prodrugs 1 to 4 have increased, but also balanced lipophilicity as compared to penethamate. This fact was confirmed by computerised calculation of Log P values in octanol/water, which is an accepted model for in vivo lipophilicity. Improved lipophilicity facilitates transport across the lipid membrane. This is particularly important where inflammation is present (e.g. in acute mastitis), since the pH gradient driving drugs across the membrane and ion trapping will be reduced. The prediction software ADME Boxes predicted that each of Prodrugs 1 to 4 will be transported solely by passive diffusion. The same software also correctly predicts that penethamate is solely transported by that mechanism.

The table in Figure 17 shows that at the pH of blood and the pH of milk Prodrugs 1 to 4 will have a higher fraction of non-ionised molecules than will penethemate. Take for example, Prodrug 1. The amount of Prodrug 1 that can be captured in milk by ion trapping is far greater than that for penethamate. The reason for this is that only about 7.4% of penethemate is available in non-ionised form at pH 7.3. Only the non-ionised fraction can readily penetrate the lipoprotein membrane (e.g. the blood-milk barrier or any other cellular membrane). This represents a small proportion of the dosage administered being able to migrate to the site of infection. By contrast, 48.3% of Prodrug 1 is non-ionised at pH 7.3 meaning that Prodrug 1 is able to pass cellular membranes and deliver considerably more therapeutically relevant quantities of the active to difficult to reach body compartments (for example, the udder of a cow).

This improvement in the ratio of non-ionised to ionised compound at the pH of plasma enables lower initial doses of Prodrugs 1 to 4 to be administered compared with penethemate (doses used for dairy cattle are about 10 to 15 mg/kg). This is because a greater fraction of Prodrugs 1 to 4 are able to be absorbed from the site of administration, penetrate the lipid membrane and then reach the site of action or a biological fluid from which the compound can reach the site of action.

Half-life

Prodrugs 1 to 4 have a longer half- life (between 31 and 99 minutes) under physiological conditions (39°C and pH 7.3) compared with penethemate, which has a half-life of 22 minutes under the same conditions. This improvement in half-life enables the implementation of different dosing regimens which may provide advantages over current regimens.

Antimicrobial activity

The spectrum of antimicrobial activity of Prodrug 1 has been demonstrated to be identical to the antimicrobial activity of non-derivatised cloxacillin. This has been confirmed by in vitro experiments to assess the spectrum of antibacterial activity of Prodrug 1 and minimum inhibitory concentrations (MIC) against various micro-organisms. The data for Prodrug 1 has also been compared to similar data for non-derivatised cloxacillin and ceftiofur. Ceftiofur was chosen in order to enable a comparison with a cephalosporin antibiotic possessing excellent /3-lactamase resistance and also because, like cloxacillin, ceftiofur is used (via the intramammary route) in the treatment of bovine mastitis.

The microorganisms tested were Streptococcus dysgalacteae, Streptococcus agalacteae, Streptococcus uberis, Staphylococcus aureus and Staphylococcus aureus (coagulase negative) - all major causes of mastitis in dairy animals. In vitro testing confirmed that Prodrug 1 has the same MICs as cloxacillin against the organisms listed. Therefore, Prodrug 1 has the same spectrum of activity and potency as underivatised cloxacillin.

A scientific study of dairy heifers (Salmon, S. A., et al. J. Dairy ScL (1998) 81, pp 570- 578) underlines the importance of cloxacillin in the treatment of bovine mastitis, since that study indicated that while cloxacillin is primarily active against penicillinase-producing staphylococci, it: (a) also has similar activity to penicillin against the enterococci and Gram-negative bacilli;

(b) is more potent than ceftiofur against Staphylococcus aureus and the wide group of Staphylococcus spp.\ and

(c) is active against all streptococcal species , including Strep, dysgalactiae and

Strep, uberis, which are major causes of mastitis in dairy cattle, and is effectively more potent than Ceftiofur.

Prodrugs 1 to 4 provide advantages over non-derivatised cloxacillin in that Prodrugs 1 to 4 can effectively target the site of infection and reach sites where it is difficult for cloxacillin to reach in sufficient concentration following parenteral administration to be pharmaceutically effective.

The pharmacological properties of Prodrugs 1 to 4 may also enable a reduction in the dosage of active antibiotic administered, and also broaden the range of treatment regimens available for use in certain problematic infections, for example the treatment of mastitis in non-lactating dairy animals (e.g. "dry" cows).

At body temperature, penethemate fully degrades in 10 to 12 hours and needs to be administered on a daily basis. The standard "dry" period in the dairy industry is two months. A single course of penethemate is not therefore an effective prophylaxis in non-lactating dairy animals (e.g. "dry" cows) because there is no coverage for the full two months.

In contrast, cloxacillin is chemically very stable and degrades at a rate of approximately 3% per day at body temperature. A single dose could therefore sit in residual milk in the udder and degrade over a much longer period than penethemate (e.g. one month). Accordingly, the present invention finds particular application in the prevention of mastitis in dairy animals, for example non-lactating dairy animals. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications.