WO2017019943A1

WO2017019943A1 - Antimicrobial formulations and applications thereof

Info

Publication number: WO2017019943A1
Application number: PCT/US2016/044661
Authority: WO
Inventors: Susanne Gardner
Original assignee: Susanne Gardner
Priority date: 2015-07-29
Filing date: 2016-07-29
Publication date: 2017-02-02

Abstract

Described herein are new approaches to antimicrobial therapy, which includes the development of new combinations of antibiotic agents as well as their use for specific therapeutic purposes. These specific therapeutic purposes may apply to clinical situations inherently different from treatment of infections that require systemic antibiotic administration. More localized approaches offer a number of advantages. The advantages of such antibiotic combinations include, but are not limited to, targeting a broader spectrum of microbes; faster microbial eradication; sparing the subject systemic exposure to the individual antimicrobial agents; enhancing the antimicrobial activity against microbes considered resistant to individual agents; and enhancing the antimicrobial activity against microbes considered resistant to individual agents at levels appropriate for systemic administration.

Description

ANTIMICROBIAL FORMULATIONS AND APPLICATIONS THEREOF

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority upon U.S. Provisional Application Serial No. 62/198,303, filed July 29, 2015. This application is hereby incorporated by reference in its entirety for all of its teachings.

BACKGROUND

Standard assessments of antibiotic antimicrobial effects are generally centered around antibiotic concentrations that are safely achievable in serum, reflecting scenarios of multiple systemically delivered doses over time. Clinical situations where altogether different scenarios prevail are fewer in number and may represent "orphan" areas of clinical needs. Little to no data is available for

antibiotics/combinations utilized and delivered in categorically atypical

concentrations or methods for localized needs, or non-systemic needs. In addition, the microbial composition of localized infections warrants new approaches to local delivery of antibiotics or antibiotic combinations, where issues of local vs. systemic toxicity are separated, and where additive, potentiating or synergistic actions of the antimicrobials are explored for specific clinical needs. For example, such a clinical need is exemplified by intraocular or periocular injections that take into account the unique pharmacokinetic/pharmacodynamic (PK PD) aspects of the eye, and its surrounding tissues. These interventions may represent prophylactic or treatment measures. Similar unique approaches apply to other localized infections or applications where unique PK/PD factors apply, but are not well served under current circumstances. No single antibiotic agent, or available compound, is currently indicated to eradicate both the highly pathogenic Gram-positive microbes (such as methicillin-resistant staphylococci, Bacillus species such as B. cereus, or

Enterococcus species such as E. faecalis) and Gram-negative microbes (such as Pseudomonas aeruginosa, species of Proteus, Enterobacter, Klebsiella, or Escherichia such as E. coli) that are encountered in certain circumstances, or that eradicates these microbes in rapid, unique time frames that may be needed in organs such as the eye. The compositions and methods described herein offer sight-saving interventions and reductions in morbidity under specialized circumstances that are currently not served.

SUMMARY

Described herein are new approaches to antimicrobial therapy, which includes the development of new combinations of antibiotic agents as well as their use for specific therapeutic purposes. These specific therapeutic purposes may apply to clinical situations inherently different from treatment of infections that require systemic antibiotic administration. More localized approaches offer a number of advantages. The advantages of such antibiotic combinations include, but are not limited to, targeting a broader spectrum of microorganisms; faster microbial eradication; sparing the subject to systemic exposure of the individual antimicrobial agents; enhancing the antimicrobial activity against microbes considered resistant to individual agents; and enhancing the antimicrobial activity against microbes considered resistant to individual agents at levels appropriate for systemic administration. Further advantages and applications are described below.

DETAILED DESCRIPTION

Before the present compounds, compositions, and/or methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific compounds, synthetic methods, or uses as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like. "Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

References in the specification and concluding claims to parts by weight, of a particular element or component in a composition or article, denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

The term "treat" as used herein is defined as maintaining or reducing the symptoms of a pre-existing condition when administered the antimicrobial agents described herein when compared to the same symptom in the subject prior to the administration of the antimicrobial agents to the subject, or reducing the number of colonizing micro-organisms.

The term "prevent" is defined herein as reducing or eliminating the growth rate of bacteria or micro-organisms in or on a subject when administered the antimicrobial agents described herein when compared to the growth rate in the same subject prior to the administration of the antimicrobial agents to the subject. The term "prevent" is also defined as eliminating the onset of any bacterial growth in a subject, where no bacteria or microorganisms existed in the subject. The term "prevent" is also defined as eliminating the onset of one or more symptoms in a subject when administering the antimicrobial agents described herein when compared to the same symptom in the subject prior to the administration of the antimicrobial agents to the subject.

The term "subject" as defined herein is any organism that is in need of bacteria/micro-organism eradication or prevention of bacteria/micro-organism growth. In one aspect, the subject is a mammal including, but not limited to, humans, domesticated animals (e.g., dogs, cats, horses), livestock (e.g., cows, pigs), and wild animals.

Described herein are combinations of antimicrobial agents and their applications thereof. As discussed in detail below, the antimicrobial agents used herein are useful in rapidly killing bacteria in or on a subject. The nature of the antimicrobial agents, the modes of administration, applications, and the numerous advantages of the invention are described in detail below.

Antimicrobial Agents

The concentrations of antimicrobial agents described herein are considerably higher than serum levels achieved after systemic administration. In one aspect, at least one antimicrobial agent described herein has a concentration at least two multiples of customary human serum concentrations achieved after systemic administration of that agent. In another aspect, at least one antimicrobial agent described herein has a concentration of two to three multiples, two to four multiples, or two to five or more multiples of customary serum concentrations achieved after systemic administration of that agent. The significance of the selection of the antimicrobial agents and their concentrations thereof is discussed in detail below.

In one aspect, the subject is administered (a) at least one cephalosporin and (b) at least one polymyxin. Examples of cephalosporins useful herein include, but are not limited to, cefazolin, cefuroxime, ceftazidime, cephalexin, cephaloridine,

cefamandole, cefsulodin, cefonicid, cefoperazine, cefoprozil, ceftriaxone, or any combination thereof. In one aspect, the cephalosporin is cefazolin, cefuroxime, ceftazidime, or any combination thereof.

The concentration of the cephalosporin can vary depending upon the application. In one aspect, the cephalosporin has a concentration from 0.1 mcg/ml to 4,000 mcg/ml. In another aspect, the cephalosporin has a concentration of 10 mcg/ml to 4,000 mcg/ml, 100 mcg/ml to 4,000 mcg/ml, 250 mcg/ml to 4,000 mcg/ml, 500 mcg/ml to 4,000 mcg/ml, or 1,000 mcg/ml to 4,000 mcg/ml. In another aspect, the cephalosporin has a concentration from 0.001 mg/ml to 200 mg/ml, 0.001 mg/ml to 50 mg/ml, 0.01 mg/ml to 50 mg/ml, 0.1 mg/ml to 50 mg/ml, 0.2 mg/ml to 50 mg/ml, 1 mg/ml to 50 mg/ml, 2 mg/ml to 50 mg/ml, 5 mg/ml to 50 mg/ml, 5 mg/ml to 50 mg/ml, 5 mg/ml to 40 mg/ml, 5 mg/ml to 30 mg/ml, or 5 mg/ml to 20 mg/ml.

Examples of polymyxins useful herein include, but are not limited to, polymyxin B, colistin, or a combination thereof. In one aspect, the polymyxin has a concentration from, 0.0001 mcg/ml to 1,000 mcg/ml, 0.001 mcg/ml to 1,000 mcg/ml, 0.01 mcg/ml to 1,000 mcg/ml, 0.1 mcg/ml to 1,000 mcg/ml, 1 mcg/ml to 1,000 mcg/ml, 10 mcg/ml to 1,000 mcg/ml, 50 mcg/ml to 1,000 mcg/ml, or 100 mcg/ml to 1,000 mcg/ml. In another aspect, the polymyxin has a concentration from 0.0001 mg/ml to 50 mg/ml, 0.0001 mg/ml to 5 mg/ml, 0.001 mg/ml to 5 mg/ml, 0.1 mg/ml to 5 mg/ml, or 0.5 mg/ml to 5 mg/ml.

In one aspect, the cephalosporin has a concentration of 0.001 mg/ml to 200 mg/ml, 0.01 mg/ml to 200 mg/ml, 0.01 mg/ml to 50 mg/ml, 0.1 mg/ml to 50 mg/ml, 0.2 mg/ml to 50 mg/ml, 1 mg/ml to 50 mg/ml, 2 mg/ml to 50 mg/ml, 5 mg/ml to 50 mg/ml, 5 mg/ml to 50 mg/ml, 5 mg/ml to 40 mg/ml, 5 mg/ml to 30 mg/ml, or 5 mg/ml to 20 mg/ml and the polymyxin has a concentration of 0.0001 mg/ml to 5 mg/ml.

In certain aspects, one or more additional antimicrobial agents can be administered to the subject in combination with the cephalosporin and polymyxin. In one aspect, the subject is further administered a penicillin. Examples of penicillins useful herein include, but are not limited to, ampicillin, amoxicillin, oxacillin, carbenicillin, ticarcillin, piperacillin, and/or concomitant use with beta-lactamase inhibitors such as clavulanic acid, or any combination thereof, or macrolide antibiotics. In one aspect, the penicillin has a concentration from 0.0001 mcg/ml to 100 mg/ml. In another aspect, the penicillin has a concentration from 0.001 mcg/ml to 1,000 mcg/ml, 0.01 mcg/ml to 1,000 mcg/ml, 0.1 mcg/ml to 1,000 mcg/ml, 0.5 mcg/ml to 1,000 mcg/ml, 1 mcg/ml to 1,000 mcg/ml, 10 mcg/ml to 1,000 mcg/ml, 100 mcg/ml to 1,000 mcg/ml, 250 mcg/ml to 1,000 mcg/ml, 500 mcg/ml to 1,000 mcg/ml. In another aspect, the penicillin has a concentration from 0.1 mg/ml to 20 mg/ml, 0.5 mg/ml to 20 mg/ml, or 1 mg/ml to 20 mg/ml. In one aspect, the cephalosporin has a concentration of 0.001 mg/ml to 50 mg/ml, 0.01 mg/ml to 50 mg/ml, 0.1 mg/ml to 50 mg/ml, 0.2 mg/ml to 50 mg/ml, 1 mg/ml to 50 mg/ml, 2 mg/ml to 50 mg/ml, 5 mg/ml to 50 mg/ml, 5 mg/ml to 50 mg/ml, 5 mg/ml to 40 mg/ml, 5 mg/ml to 30 mg/ml, or 5 mg/ml to 20 mg/ml, the polymyxin has a concentration of 0.0001 mg/ml to 5 mg/ml, and the penicillin has a concentration from 0.1 mg/ml to 20 mg/ml. In another aspect, the subject is further administered a fluoroquinolone in combination with the cephalosporin, polymyxin, and optionally a penicillin. Examples of fluoroquinolones useful herein include, but are not limited to, levofloxacin, norfloxacin, ofloxacin, ciprofloxacin, perfloxacin, lomefloxacin, fleroxacin, sparfloxacin, grepafloxacin, trovafloxacin, clinafloxacin, gemifloxacin, enoxacin, sitafloxacin, nadifloxacin, tosulfloxacin, cinnoxacin, rosoxacin, miloxacin, moxifloxacin, gatifloxacin, cinnoxacin, enoxacin, fleroxacin, lomafloxacin, lomefloxacin, miloxacin, nalidixic acid, nadifloxacin, oxolinic acid, pefloxacin, pirimidic acid, pipemidic acid, rosoxacin, rufloxacin, temafloxacin, tosufloxacin, trovafloxacin, besifloxacin, or any combination thereof. In one aspect, the fluoroquinolone has a concentration from 0.0001 mcg/ml to 100 mg/ml. In another aspect, the fluoroquinolone has a concentration from 0.01 mcg/ml to 5,000 mcg/ml, 0.1 mcg/ml to 5,000 mcg/ml, 1 mcg/ml to 5,000 mcg/ml, 10 mcg/ml to 5,000 mcg/ml, 100 mcg/ml to 5,000 mcg/ml, 500 mcg/ml to 5,000 mcg/ml, or 1,000 mcg/ml to 5,000 mcg/ml. In another aspect, the fluoroquinolone has a concentration from

0.05 mg/ml to 20 mg/ml, 0.1 mg/ml to 20 mg/ml, 0.5 mg/ml to 20 mg/ml, 1 mg/ml to 20 mg/ml, 2 mg/ml to 20 mg/ml, 3 mg/ml to 20 mg/ml, 3 mg/ml to 15 mg/ml, or 3 mg/ml to 10 mg/ml.

In another aspect, the subject is further administered a lincosamide antibiotic such as clindamycin, in combination with a cephalosporin, a polymyxin, a fluoroquinolone, and optionally a penicillin.

In another aspect, the subject is administered a combination of a

cephalosporin, a penicillin, a fluoroquinolone and a polymyxin.

In another aspect, the subject is administered a lipopeptide antibiotic such as daptomycin, a fluoroquinolone, a cephalosporin, a polymyxin, and optionally a penicillin.

In one aspect, the lincosamide antibiotic, such as clindamycin, has a concentration from 0.001 mcg/ml to 1,000 mcg/ml, 0.01 mcg/ml to 1,000 mcg/ml, 0.1 mcg/ml to 1,000 mcg/ml, 0.5 mcg/ml to 1,000 mcg/ml, 1 mcg/ml to 1,000 mcg/ml, 10 mcg/ml to 1,000 mcg/ml, 100 mcg/ml to 1,000 mcg/ml, 250 mcg/ml to 1,000 mcg/ml, 500 mcg/ml to 1,000 mcg/ml. In another aspect, the cyclic lipopeptides antibiotic, such as daptomycin, has a concentration from 0.0001 mg/ml to 100 mg/ml, 0.001 mcg/ml to 1,000 mcg/ml, 0.01 mcg/ml to 1,000 mcg/ml, 0.1 mcg/ml to 1,000 mcg/ml, 0.5 mcg/ml to 1,000 mcg/ml, 1 mcg/ml to 1,000 mcg/ml, 10 mcg/ml to 1,000 mcg/ml, 100 mcg/ml to 1,000 mcg/ml, 250 mcg/ml to 1,000 mcg/ml, 500 mcg/ml to 1,000 mcg/ml.

In another aspect, the lincosamide antibiotic has a concentration from 0.05 mg/ml to 20 mg/ml, 0.1 mg/ml to 20 mg/ml, 0.5 mg/ml to 20 mg/ml, 1 mg/ml to 20 mg/ml, 2 mg/ml to 20 mg/ml, 3 mg/ml to 20 mg/ml, 3 mg/ml to 15 mg/ml, or 3 mg/ml to 10 mg/ml. In another aspect, the lipopeptide antibiotic has a concentration from 0.05 mg/ml to 20 mg/ml, 0.1 mg/ml to 20 mg/ml, 0.5 mg/ml to 20 mg/ml, 1 mg/ml to 20 mg/ml, 2 mg/ml to 20 mg/ml, 3 mg/ml to 20 mg/ml, 3 mg/ml to 15 mg/ml, or 3 mg/ml to 10 mg/ml.

In one aspect, the cephalosporin has a concentration of 0.001 mg/ml to 50 mg/ml, 0.01 mg/ml to 50 mg/ml, 0.1 mg/ml to 50 mg/ml, 0.2 mg/ml to 50 mg/ml, 1 mg/ml to 50 mg/ml, 2 mg/ml to 50 mg/ml, 5 mg/ml to 50 mg/ml, 5 mg/ml to 50 mg/ml, 5 mg/ml to 40 mg/ml, 5 mg/ml to 30 mg/ml, or 5 mg/ml to 20 mg/ml, the polymyxin has a concentration of 0.0001 mg/ml to 5 mg/ml, the penicillin has a concentration from 0.1 mg/ml to 20 mg/ml, the fluoroquinolone has a concentration from 0.05 mg/ml to 20 mg/ml

In another aspect, the subject is administered (1) cefazolin, (2) polymyxin B, and (3) ampicillin, wherein the cefazolin is from 0.001 mg/ml to 20 mg/mL, the polymyxin B is from 0.001 mg/ml to 5 mg/ml, and the penicillin is from 0.001 mg/ml to 20 mg/ml.

In another aspect, the subject is administered (1) cefazolin, cefuroxime, ceftazidime or a combination thereof, (2) polymyxin B, (3) ampicillin, and (4) levofloxacin, wherein the cefazolin is from 0.001 mg/ml to 20 mg/mL, the polymyxin B is from 0.001 mg/ml to 5 mg/ml, the amikacin is from 0.01 mg/ml to 10 mg/ml, and the levofloxacin is from 0.05 mg/ml to 10 mg/ml.

In another aspect, the subject is administered (1) cefuroxime and (2) polymyxin B, wherein the cefuroxime is from 0.01 mg/ml to 20 mg/mL and the polymyxin B is from 0.001 mg/ml to 5 mg/ml.

In another aspect, the subject is administered (1) cefuroxime, (2) polymyxin B, and (3) ampicillin, wherein the cefuroxime is from 0.01 mg/ml to 20 mg/mL, the polymyxin B is from 0.001 mg/ml to 5 mg/ml, and the ampicillin is from 0.001 mg/ml to 10 mg/ml.

In another aspect, the subject is administered (1) cefuroxime, (2) ceftazidime, and (3) polymyxin B, wherein the cefuroxime is from 0.01 mg/ml to 20 mg/mL, the ceftazidime is from 0.001 mg/ml to 10 mg/mL, and the polymyxin B is from 0.001 mg/ml to 5 mg/ml.

In another aspect, the subject is administered (1) cefuroxime, (2) ceftazidime, (3) polymyxin B, and (4) levofloxacin, wherein the cefuroxime is from 0.01 mg/ml to 20 mg/mL, the ceftazidime is from 0.001 mg/ml to 10 mg/mL, the polymyxin B is from 0.001 mg/ml to 5 mg/ml, and the levofloxacin is from 0.05 mg/ml to 10 mg/ml.

In another aspect, the subject is administered (1) cefuroxime, (2) ceftazidime,

(3) polymyxin B, (4) levofloxacin, and (5) ampicillin, wherein the cefuroxime is from 0.01 mg/ml to 20 mg/mL, the ceftazidime is from 0.001 mg/ml to 10 mg/mL, the polymyxin B is from 0.001 mg/ml to 5 mg/ml, the levofloxacin is from 0.05 mg/ml to 10 mg/ml, and the ampicillin is from 0.001 mg/ml to 10 mg/ml.

In another aspect, the subject is administered (a) at least one cephalosporin and (b) at least one penicillin. Any of the cephalosporins and penicillins described above as well as the concentration ranges can be used in this aspect. In one aspect, the cephalosporin is cefuroxime and the penicillin is ampicillin, wherein the cefuroxime is from 0.01 mg/ml to 20 mg/mL and the ampicillin is from 0.001 mg/ml to 10 mg/ml. In another aspect, the subject is administered (a) at least one cephalosporin and (b) a lincosamide antibiotic, (c) a fluoroquinolone, (d) an aminoglycoside, and optionally, (e) a polymyxin.

Examples of aminoglycosides useful herein include gentamicin, amikacin, tobramycin, debekacin, kanamycin, neomycin, netilmicin, paromomycin, sisomycin, spectinomycin, streptomycin, or any combination thereof. In one aspect, the aminoglycoside has a concentration of from 0.0001 mg/ml to 100 mg/ml. In another aspect, the aminoglycoside has a concentration of from 0.001 mcg/ml to 1,000 mcg/ml. In a further aspect, the aminoglycoside has a concentration of from 0.1 mg/ml to 20 mg/ml.

In one aspect, the subject is administered at least one cephalosporin, at least one polymyxin, at least one fluoroquinolone, and at least one penicillin. In another aspect, the subject is administered cefuroxime, polymyxin B, levofloxacin, and ampicillin. In a further aspect, the subject is administered cefuroxime having a concentration of from 0.2 mg/ml to 50 mg/ml, polymyxin B having a concentration from 0.0001 mg/ml to 5 mg/ml, levofloxacin having a concentration from 1 mg/ml to 20 mg/ml, and ampicillin having a concentration of 0.1 mcg/ml to 10 mg/ml.

In one aspect, the subject is administered at least one cephalosporin, at least one polymyxin, at least one aminoglycoside, and at least one lipopeptides antibiotic. In another aspect, the subject is administered cefuroxime, polymyxin B, daptomycin, and amikacin. In a further aspect, the subject is administered cefuroxime having a concentration of from 0.2 mg/ml to 50 mg/ml, polymyxin B having a concentration from 0.0001 mg/ml to 5 mg/ml, daptomycin having a concentration from 0.1 mg/ml to 10 mg/ml, and amikacin having a concentration of 0.1 mg/ml to 20 mg/ml.

Depending upon the application, one or more additional biological agents can be administered with the antimicrobial agents. In one aspect, the subject is further administered corticosteroid agents and congeners including, but not limited to, betamethasone, dexamethasone, fludrocortisone, hydrocortisone, tixorcortol, prednisolone, methylprednisolone mometasone, amcinonide, budesonide, desonide, fluocinonide, halcinonide, fluocortolone, flunisolide, fluocorotolone, fluticasone, fluprednidene, beclomethasone, budesonide, clobetasone, prednicarbate, fluticasone, or any combination thereof.

Applications and Modes of Administration

The antimicrobial agent combinations and concentrations used herein are effective in rapidly killing bacteria in a subject. In one aspect, upon administration of the antimicrobial agents, 10 to 100% of the targeted bacteria (i.e., the bacteria intended to be killed upon administration of the agents described herein) in the subject are killed within 1 to 16 hours, 1 to 14 hours, 1 to 12 hours, 1 to 10 hours, 1 to 8 hours, 1 to 6, 1 to 4 hours, or 1 to 2 hours. In another aspect, 10 to 100%, 25 to 100%, 50 to 100%, 75 to 100%, or 95 to 100% of the targeted bacteria in the subject are killed within 1 to 2 hours after administration of the antimicrobial agents.

The antimicrobial agents described herein are useful in clinical situations inherently different from treatment of infections that require systemic antibiotic administration. The antimicrobial agents used herein permit the localized

administration of the agents to a specific site in the subject. There are numerous advantages to this approach including, but are not limited to: localized targeting of microbes; local use of antimicrobials that are associated with toxicity after systemic administration; avoidance of allergic reactions; sparing of antibiotics that are deemed critical for systemic use in the treatment of serious infections; use of antibiotic concentrations that reduce the propensity for microbial resistance development.

Additionally, in certain aspects, the use of the antimicrobial agents at the

concentrations and combinations described herein only require one, two, three, or four, or a limited number of dose applications where multiple applications are not practical. Moreover, the ability of the antimicrobial agents to rapidly kill bacteria or prevent the growth of bacteria reduces or prevent local tissue damage caused by replicating bacteria during an infection is enhanced.

Additionally, the antimicrobial agent combinations and concentrations described herein permit the use of two or more antibiotics in combination, where the combination targets a broader spectrum of microbes; where the combination allows faster microbial eradication; where the combination spares systemic exposure to the individual antimicrobial agents; where the combination has antimicrobial activity against microbes considered resistant to individual agents; and where the combination has antimicrobial activity against microbes considered resistant to individual agents at levels appropriate for systemic administration.

Depending upon the mode of administration, the antimicrobial agent combinations and concentrations described herein can be formulated and administered using techniques known in the art. In one aspect, the antimicrobial agents described herein can be administered locally to a subject via injection. In one aspect, the antimicrobial agents can be formulated into a single composition. Thus, in this aspect, the antimicrobial agents are administered concurrently to the subject. In other aspects, each antimicrobial agent can be in separate injectable solutions (e.g., a kit composed of different vials of each antimicrobial agent to be administered). Here, the antimicrobial agents are administered sequentially to the subject.

Preparations for injectable administration include sterile aqueous or non- aqueous solutions, suspensions, and emulsions. Examples of non-aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles, if needed for collateral use of the disclosed compositions and methods, include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles, if needed for collateral use of the disclosed compositions and methods, include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

In one aspect, the antimicrobial agents can be formulated as a topical composition. For example, the antimicrobial agents can be formulated as

pharmaceutically-acceptable creams, lotions, ointments, sprays, and other suitable topical formulations for localized administration. In other aspects, the antimicrobial agents can be applied to a wound dressing, where once again the administration of antimicrobial agents is localized. In another aspect, the antimicrobial agents can be formulated into

biocompatible and biodegradable packing materials or implants useful in orthopedic and dental procedures. For example, materials useful in packing joints can be formulated with the antimicrobial agents described herein in order to attain local release of the agents in the joint. Depending upon the composition of the packing material, the release pattern of the antimicrobial agents can be delayed or sustained as needed.

In other aspects, the antimicrobial agents can be formulated as instillation, solutions, irrigation solutions, or wash/soak solutions.

In a further aspect, the antimicrobial agents used herein are part of a delivery system. For example, delivery systems may provide needed solubilizing and/or separation and delivery components for the specific combination of antimicrobial agents. The delivery system can include, but are not limited to, multiple chambers, microbial filters, and sterile filtration units.

In another aspect, medical devices can be coated with the antimicrobial agents described herein. For example, medical implants (stents, catheters, artificial joints) can be coated with the antimicrobial agents described herein to prevent bacterial infection. In other aspects, surgical devices can be coated with the antimicrobial agents described herein to prevent infection during surgery.

In one aspect, described herein is a pharmaceutical composition comprising

(a) at least one cephalosporin in the amount of 0.001 mg/ml to 50 mg/ml and (b) at least one polymyxin in the amount of 0.0001 mg/ml to 5 mg/ml, (c) at least one penicillin in the amount of 0.001 mg/ml to 50 mg/ml, (d) at least one lipopeptide antibiotic, and optionally, (e) an aminoglycoside. In another aspect, the cephalosporin comprises cefazolin, cefuroxime, ceftazidime, or any combination thereof in the amount of 0.001 mg/ml to 20 mg/ml. In another aspect, the polymyxin comprises polymyxin B in the amount of 0.0001 mg/ml to 5 mg/ml. In another aspect, the composition further comprises amikacin in the amount of 0.001 mg/ml to 10 mg/ml. In another aspect, the composition further comprises daptomycin or ampicillin in the amount of 0.05 mg/ml to 50 mg/ml. In another aspect, the compositions can be formulated as injectable solutions.

In one aspect, described herein is a kit comprising (a) at least one

cephalosporin in the amount of 0.001 mg/ml to 50 mg/ml and (b) at least one polymyxin in the amount of 0.0001 mg/ml to 5 mg/ml, (c) at least one penicillin in the amount of 0.0001 mg/ml to 5 mg/ml. In another aspect, the kit also comprises (a) a lincosamide antibiotic in the amount of 0.001 mg/ml to 50 mg/ml and (b) at least one aminoglycoside in the amount of 0.001 mg/ml to 20 mg/ml. In another aspect, antibiotic combinations can be formulated as injectable solutions.

The antimicrobial agent combinations and concentrations described herein can kill a wide spectrum of bacteria quickly and effectively. Moreover, the antimicrobial agent combinations and concentrations described herein can prevent the growth of targeted bacteria in a subject. For example, the methods and compositions described herein are effective in killing concurrently both Gram-negative bacteria and Gram- positive bacteria. Examples of Gram-negative bacteria include, but not limited to, E. coli, Klebsiella, Enterobacter, H. influenzae, Proteus, Serratia, Pseudomonas species, or any combination thereof. Examples of Gram-positive bacteria include, but not limited to methicillin-resistant Staphylococcus aureus, a staphylococcal species, or species of Streptococci, Bacillus or Enterococci. In another aspect, the bacteria can include an anaerobe such as a Clostridium species, a Peptococcus species, a

Bacteroides species, or Mycobacteriaceae alone or in combination with Gram-positive and/or Gram-negative bacteria. As will be shown in the Examples below, the antimicrobial compositions and methods described herein are effective in killing the more resistant bacteria.

Not wishing to be bound by theory, the selection and combination of the antimicrobial agent combinations and concentrations described herein involve a combination of mechanisms of action that include, but are not limited to, the following: interference with microbial cell walls or membranes, interference with microbial DNA replication, inhibition of microbial protein synthesis, direct antimicrobial or cytotoxic effects, microbial enzyme inhibition, inhibition of microbial folic acid metabolism, interference with microbial folate metabolism, interference with microbial RNA, and interference with microbial metabolic pathways.

The antimicrobial agent combinations and concentrations described herein are effective in rapidly killing bacteria, which in turn can treat or prevent bacterial infection and the numerous symptoms associated with the infection including, but not limited to, inflammation, fever and diminished vision. Additionally, the ability to administer the antimicrobial agent combinations and concentrations described herein permit site-specific administration of the antimicrobial agents instead of systemic administration. This in turn permits the antimicrobial agents to effectively kill bacteria or prevent the growth of bacteria at a specific location in the subject.

Moreover, when it is desirable or practical to only administer one to two, three, four, or a limited number of, doses of the antimicrobial agents to the subject, the use of the antimicrobial agent combinations and concentrations described herein permit this.

In one aspect, the antimicrobial agent combinations and concentrations described herein are effective in killing targeted bacteria or preventing the growth of targeted bacteria at or near the eye of the subject. For example, the antimicrobial agents can be administered to the eyelash area or conjunctiva, in the tear film, anterior chamber, vitreous cavity, or the subconjunctival or subtenon space. In one aspect, the antimicrobial agents are injected into a chamber of the eye of the subject. In other aspects, the antimicrobial agents can be topically applied to one or more layers of the eye or to the surface of the eye.

In other aspects, antimicrobial agents described herein can be administered to the skin or skin structures topically or by subcutaneous injection, for example, when sterility measures have been breached, placing the subject at risk of serious infection.

In another aspect, antimicrobial agents described herein can be administered in limited areas or specific organs of the body, as in implants or localized release systems, or sprays for limited dose applications.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, and methods described and claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.

Antibacterial Activity and Efficacy of Clindamycin, Cefuroxime, Levofloxacin, Polymyxin B, Daptomycin, Amikacin, and/or Ampicillin Using a Suspension Time-Kill Procedure

Materials and Methods

Clindamycin, cefuroxime, levofloxacin hydrochloride, polymyxin B sulfate, amikacin (free base), and ampicillin were resuspended in water and diluted for use in the study.

Efficacy of these substances, alone and in combination, was tested against three strains of bacteria using the ASTM E2315 test method:

(1) Bacillus cereus 14579: This is a Gram-positive, rod-shaped, endospore- forming facultative aerobe bacterium. B. cereus is related to B. anthracis and B. thuringiensis and is commonly associated with food-borne illnesses as well as several local and systemic infections. This bacterium can be challenging to disinfect due to its ability to sporulate.

(2) Enterococcus faecalis (VRE) 51299: This is a Gram-positive, spherical- shaped strain of bacteria that has developed resistance to the antibiotic vancomycin. E. faecalis can cause local and systemic infections ranging from endocarditis to bacteremia to urinary tract infections; these can be difficult to treat because of this strain's acquired drug resistance. (3) Staphylococcus aureus (MRSA) 33592: This is a Gram-positive, spherical- shaped aerobic bacterium that is resistant to methicillin, a penicillin-derivative antibiotic. MRSA bacteria are resistant to drying and can survive on surfaces and fabrics for an extended period of time.

ASTM E2315 is a quantitative test method designed to assess changes in the population of a microorganism in an antimicrobial liquid suspension. This method can be conducted using contact times ranging from 10 seconds to 24 hours. The test method uses non-antimicrobial agents as controls to establish baselines for microbial reductions.

The following criteria were used to establish scientific defensibility of the

ASTM E2315 study:

(1) The average number of viable bacteria recovered from time zero samples must be approximately 1 x 10⁶ cells/mL or greater.

(2) Ordinary consistency between replicates must be observed for the time zero samples.

(3) Positive/growth controls must demonstrate growth of the appropriate test microorganism.

(4) Negative/purity controls must demonstrate no growth of the test organism. Summary of the Experimental Procedure

Test microorganisms were prepared in liquid culture medium. Suspensions of test microorganisms were standardized as needed by dilution in a buffered saline solution. Test and control substances were dispensed in identical volumes to sterile vessels.

Independently, test and control substances were inoculated with each test microorganism, then mixed and incubated. Control substances were immediately harvested and used to represent the concentration present at the start of the test.

At the conclusion of the contact time, a volume of liquid test solution was harvested and chemically neutralized. Test microorganisms were spun down and the cell pellet was resuspended in phosphate buffered saline (PBS) prior to inoculation of the test systems. 10 mL volumes of Dey/Engley neutralizer broth supplemented with 5% magnesium chloride and 35% polysorbate 80 were used for chemical neutralization. After chemical neutralization, test systems were filtered using a filtration manifold to recover surviving microorganisms.

Dilutions of neutralized test solution were assayed using appropriate growth media to determine surviving microorganism levels at each respective contact time. Reductions of microorganisms were calculated by comparing initial microbial concentrations to final microbial concentrations. The limit of detection for the assay was 10 CFU/mL.

Testing parameters used in the current study are described in Table 1.

Table 1 : Testing Parameters

Test substance volume 4-5 mL

Replicates Single

Control substance volume 4-5 mL

Control substance PBS

Culture growth media Tryptic soy broth

Culture growth time 18 hours

Culture dilution media N/A

Inoculum volume 0.020-0.025 mL

Inoculum concentration 1.0 x 10⁶ CFU/mL

Contact temperature Ambient (25 °C + 2 °C)

Contact time 2 Hours

Volume harvested 0.100 mL

Plating media Tryptic soy agar

Enumeration plate incubation temperature 36 °C + 1 °C

Enumeration plate incubation time 24-48 hours

Concentrations of antibiotics and identities of microorganisms used in the experimental trials are presented in Table 2.

Table 2: Experimental Microorganisms and Concentrations of Antibiotics

Test Number Test Microorganism Antibiotic (s) and Concentration

1 Daptomycin 1 mg/mL

2 Cefuroxime 10 mg/mL

E. faecalis 51299 Levofloxacin 5 mg/mL

Ampicillin 1 mg/mL

Polymyxin B 1 mg/mL

3 Daptomycin 1 mg/mL

S. aureus 33592

4 Daptomycin 1 mg/mL Cefuroxime 10 mg/mL

Amikacin 2 mg/mL

Polymyxin B 1 mg/mL

Calculations

Percent reduction of bacterial population was calculated according to the following equation, where A = number of viable test microorganisms in the test substance after the contact time and B = number of viable test microorganisms in the control substance immediately after inoculation. Logio reduction was also calculated.

% Reduction =

B

Log ^reduction = Log (— )

Results

Results of the study are presented in Table 3.

The E. faecalis strain used was more susceptible to a mixture of cefuroxime, levofloxacin, ampicillin, and polymyxin B (Test 2) than to daptomycin alone (Test 1). Further, the MRSA strain used in these tests was more susceptible to a mixture of daptomycin, cefuroxime, amikacin, and polymyxin B (Test 4) than to daptomycin alone (Test 3). The mixed antibacterial agents displayed kill rates that approached bactericidal (99.26% for E. faecalis in Test 4 and 99.89% for S. aureus in Test 4), which is especially useful since both the strain of E. faecalis and the strain of S. aureus used in these tests have known resistances to common antimicrobial agents (respectively, vancomycin and methicillin). In both instances, a synergistic effect of a four-antibiotic combination has been demonstrated.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the compounds, compositions and methods described herein.

Various modifications and variations can be made to the compounds, compositions and methods described herein. Other aspects of the compounds, compositions and methods described herein will be apparent from consideration of the specification and practice of the compounds, compositions and methods disclosed herein. It is intended that the specification and examples be considered as exemplary.

Claims

What is claimed:

1. A method for rapidly killing bacteria in a subject or preventing the growth of bacteria in a subject, comprising administering to the subject the agents comprising at least one cephalosporin and at least one polymyxin in combination with (a) at least one fluoroquinolone and at least one penicillin or (b) at least one aminoglycoside and at least one lipopeptides antibiotic.

2. The method of claim 1 wherein the cephalosporin comprises cefazolin,

cefuroxime, ceftazidime, cephalexin, cephaloridine, cefamandole, cefsulodin, cefonicid, cefoperazine, cefoprozil, ceftriaxone, or any combination thereof.

3. The method of claim 1 wherein the cephalosporin comprises cefazolin,

cefuroxime, ceftazidime, or any combination thereof.

4. The method of claim 1 wherein the cephalosporin has a concentration from 0.01 mg/ml to 200 mg/ml.

5. The method of claim Iwherein the cephalosporin has a concentration from 0.2 mg/ml to 50 mg/ml.

6. The method in claim 1 wherein the polymyxin comprises polymyxin B,

colistin, or a combination thereof.

7. The method of claim 1 wherein the polymyxin has a concentration from 0.1 mcg/ml to 1 ,000 mcg/ml.

8. The method of claim 1 wherein the polymyxin has a concentration from

0.0001 mg/ml to 50 mg/ml.

9. The method of claim 1 wherein the fluoroquinolone comprises levofloxacin, norfloxacin, ofloxacin, ciprofloxacin, perfloxacin, lomefloxacin, fleroxacin, sparfloxacin, grepafloxacin, trovafloxacin, clinafloxacin, gemifloxacin, enoxacin, sitafloxacin, nadifloxacin, tosulfloxacin, cinnoxacin, rosoxacin, miloxacin, moxifloxacin, gatifloxacin, cinnoxacin, enoxacin, fleroxacin, lomafloxacin, lomefloxacin, miloxacin, nalidixic acid, nadifloxacin, oxolinic acid, pefloxacin, pirimidic acid, pipemidic acid, rosoxacin, rufloxacin, temafloxacin, tosufloxacin, trovafloxacin, besifloxacin, or any combination thereof.

10. The method of claim 1 wherein the fluoroquinolone has a concentration from 0.0001 mcg/ml to 100 mg/ml.

11. The method of claim 1 wherein the penicillin comprises ampicillin,

amoxicillin, oxacillin, carbenicillin, ticarcillin, piperacillin or any combination thereof.

12. The method of claim 1 wherein the penicillin has a concentration of 0.0001 mcg/ml to 100 mg/ml.

13. The method of claim 1 wherein the lipopeptides antibiotic comprises

daptomycin.

14. The method of claim 1 wherein the lipopeptide antibiotic has a concentration from 0.0001 mg/ml to 100 mg/ml.

15. The method of claim 1 wherein the aminoglycoside comprises gentamicin, amikacin, tobramycin, debekacin, kanamycin, neomycin, netilmicin, paromomycin, sisomycin, spectinomycin, streptomycin, or any combination thereof.

16. The method of claim 1 wherein the aminoglycoside has a concentration from 0.001 mcg/ml to 1,000 mcg/ml.

17. The method of claim 1 wherein the aminoglycoside has a concentration from 0.0001 mg/ml to 100 mg/ml.

18. The method of claim 1 wherein the subject is administered cefuroxime, polymyxin B, levofloxacin, and ampicillin.

19. The method of claim 18 wherein the cefuroxime has a concentration of from 0.2 mg/ml to 50 mg/ml, polymyxin B has a concentration from 0.0001 mg/ml to 5 mg/ml, levofloxacin has a concentration from 1 mg/ml to 20 mg/ml, and ampicillin has a concentration of 0.1 mcg/ml to 10 mg/ml.

20. The method of claim 1 wherein the subject is administered cefuroxime, polymyxin B, daptomycin, and amikacin.

21. The method of claim 20 wherein the cefuroxime has a concentration of from 0.2 mg/ml to 50 mg/ml, polymyxin B has a concentration from 0.0001 mg/ml to 5 mg/ml, daptomycin has a concentration from 0.1 mg/ml to 10 mg/ml, and amikacin has a concentration of 0.1 mg/ml to 20 mg/ml.

22. The method of claim 1 wherein the subject is further administered

corticosteroid agents and congeners thereof comprising betamethasone, dexamethasone, fludrocortisone, hydrocortisone, tixorcortol, prednisolone, methylprednisolone mometasone, amcinonide, budesonide, desonide, fluocinonide, halcinonide, fluocortolone, flunisolide, fluocorotolone, fluticasone, fluprednidene, beclomethasone, budesonide, clobetasone, prednicarbate, fluticasone, or any combination thereof.

23. The method in any one of claims 1-22 wherein the targeted bacteria comprises Gram-negative bacteria.

24. The method of claim 23 wherein the Gram-negative bacteria comprises E. coli, Klebsiella, Enterobacter, H. influenzae, Proteus, Serratia, Pseudomonas species, or any combination thereof.

25. The method in any one of claims 1-22 wherein the bacteria comprises Gram- positive bacteria.

26. The method of claim 25 wherein the Gram-positive bacteria comprises

methicillin-resistant Staphylococcus aureus, a staphylococcal species, or a streptococcal species, Bacillus species such as B. cereus, or Enterococcus species such as E. faecalis.

27. The method in any one of claims 1-22 wherein the bacteria comprises Gram- negative bacteria and Gram-positive bacteria.

28. The method in any one of claims 1-22 wherein the bacteria comprises an anaerobe comprising a Clostridium species, a Peptococcus species, a

Bacteroides species, or Mycobacteriaceae alone or in combination with Gram- positive and/or Gram-negative bacteria.

29. The method in any one of claims 1-22 wherein the bacteria is at or near the eye of the subject.

30. The method in any one of claims 1-22 wherein the bacteria is present at the eyelash area or conjunctiva, in the tear film, anterior chamber, vitreous cavity, or the subconjunctival or subtenon space.

31. The method in any one of claims 1-22 wherein the agents are injected into a chamber of the eye of the subject.

32. The method in any one of claims 1-22 wherein the agents are topically applied to one or more layers of the eye or to the surface of the eye.

33. The method in any one of claims 1-22 wherein the agents are administered to the skin or skin structures topically or by subcutaneous injection.

34. The method in any one of claims 1-22 wherein the agents are administered in limited areas or specific organs of the body.

35. The method in any one of claims 1-22 wherein each agent is administered concurrently to the subject.

36. The method in any one of claims 1-22 wherein each agent is administered sequentially to the subject.

37. The method in any one of claims 1-22 wherein from 10 to 100% of the

targeted bacteria in the subject are killed within 1 to 16 hours after administration of the agents to the subject.

38. The method in any one of claims 1-22 wherein from 10 to 100% of the

targeted bacteria in the subject are killed within 6 hours after administration of each component to the subject.

39. A pharmaceutical composition comprising at least one cephalosporin and at least one polymyxin in combination with (a) at least one fluoroquinolone and at least one penicillin or (b) at least one aminoglycoside and at least one lipopep tides antibiotic. The composition of claim 39 wherein the composition comprises an injectable composition.

A coated medical device, an antibiotic pack, a wound dressing, or a delayed or extended release delivery system comprising the composition of claim 39.

A kit comprising at least one cephalosporin and at least one polymyxin in combination with (a) at least one fluoroquinolone and at least one penicillin or (b) at least one aminoglycoside and at least one lipopeptides antibiotic.