WO2012021829A1 - Methods of treating bacterial infections - Google Patents

Abstract

The invention relates to a method of treating a bacterial infection in a human subject comprising administering intravenously to the subject a compound represented by the following structural formula (I): or a pharmaceutically acceptable salt thereof (Compound A). Compound A can administered once a day in an amount ranging from about 1-1.5 mg/kg of the body weight of the subject or twice a day from about 0.625-1 mg/kg of the body weight per dose of the subject.

Description

METHODS OF TREATING BACTERIAL INFECTIONS

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.

61/373,220, filed on August 12, 2010, U.S. Provisional Application No. 61/381,850, filed on September 10, 2010 and U.S. Provisional Application No. 61/381,848, filed on September 10, 2010. The entire teachings of the above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The rapid emergence of antimicrobial drug resistance has been recognized as an epidemic of global proportions. Many pathogenic bacteria, particularly in hospital-acquired infections, are multiply resistant to several classes of antibiotics, effectively narrowing therapeutic options. For example, colistin currently represents the last line of defense for treatment of multiple-drug resistant (MDR) gram- negative bacteria like extended-spectrum β-lactamase (ESBL) producing

Enterobacteriaceae and Acinetobacter baumannii. Even with the use of multiple antibiotics, patients with these infections have a high mortality rate.

In 2005, tigecycline (also known as TYGACIL®), an intravenous antibiotic with a broad spectrum of antimicrobial activity, including activity against drug- resistant bacteria such as methicillin-resistant Staphylococcus aureus was approved and launched. Tigecycline has provided physicians with an alternative to overcome the problems of resistance observed with the other antibiotics and to combat serious, resistant infections for all patients. However, the amount of tigecycline that can be given in a single intravenous dose is limited by the drug's side effects (e.g., nausea and vomiting) requiring multiple administrations per day by parenteral means to provide an acceptable tolerability profile and to maintain therapeutic effectiveness. Furthermore, multiple reports of breakthrough septicemias and frank failures with tigecycline treatment of patients with MDR Klebsiella pneumoniae or A. baumannii have been reported. As bacterial resistance to antibiotics has become an important public health problem, there is a continuing need to develop more effective antibiotics.

SUMMARY OF THE INVENTION

The invention relates to a method of treating a bacterial infection in a human subject comprising intravenously administering to the subject a compound represented by the following structural formula:

or a pharmaceutically acceptable salt thereof. The compound of the above structural formula or its salt will be referred to herein as Compound A. It has been found that in comparison to tigecycline, Compound A can be administered intravenously in doses which result in a higher exposure or Area Under the Curve (AUC) providing a more efficacious drug product.

In one embodiment, Compound A or a pharmaceutically acceptable salt thereof is administered intravenously once a day in an amount ranging from about 1- 1.5 mg/kg of the body weight of the subject. In one aspect, Compound A is administered by infusion over 30 to 120 minutes. In a more specific aspect, Compound A is administered by infusion over 30 to 60 minutes. In a most specific aspect, the concentration of the compound in the infusate administered in either of the above aspects is from about 0.2 mg/mL and 0.7 mg/mL.

In another embodiment, Compound A is administered intravenously twice a day from about 0.625-1.5 mg/kg of the body weight of the subject per dose. In one aspect, Compound A is administered intravenously twice a day from about 0.625 - 1 mg/kg of the body weight of a subject per dose. In another aspect, Compound A is administered by intravenous infusion over 30 to 120 minutes per administration. In a more specific aspect, Compound A is administered by intravenous infusion over 30 to 60 minute. In a most specific aspect, the concentration of the compound in the infusate administered in either of the above aspects is from about 0.2 mg/mL and 0.7 mg/mL.

In any one of the aspects or embodiments described herein, the infusion can be constant or intermittent. In a particular embodiment, the infusion is constant.

The invention also relates to a method of achieving an AUC of Compound A in a human subject that is at least 50% greater than the AUC achieved for Tygacil when the same subject is administered Tygacil at the recommended dose regimen. The method comprises administering intravenously to the subject a pharmaceutical composition comprising Compound A as the sole active agent once or twice a day in a therapeutically effective amount.

In one aspect, the AUC of Compound A in the subject is at least 75% greater than the AUC achieved for Tygacil following administration of Tygacil. In another aspect, Compound A is administered intravenously once a day in an amount equal to or greater than 1.5.mg/kg. In yet another aspect, Compound A is administered intravenously twice a day in an amount equal to or greater than 0.65 mg/kg per administration.

The invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of Compound A and formulated for intravenous administration, wherein administration of the composition to a human subject results in an AUC for Compound A that is at least 50% greater than the AUC of tigecycline when tigecycline is administered to the same subject in a pharmaceutical composition comprising an amount of tigecycline that is the same as the amount of compound on a per milligram basis of active ingredient and that is administered in the same dosing regimen as Compound A.

The invention also relates to a method of achieving a AUC/MIC ratio for

Compound A in a human subject suffering from an infection by a bacterial organism that is at least 20% greater than the AUC/MIC ratio for the bacterial organism in the same subject when the subject is administered Tygacil at the recommended dose, the method comprising administering intravenously to the subject a pharmaceutical composition comprising Compound A as the sole active agent once or twice a day in a therapeutically effective amount. In one aspect, the subject is suffering from a bacterial infection characterized by the presence of an organism with a MIC to compound of less than or equal to 2 μ^ηιΐ. As used herein, a MIC to compound of less than or equal to 2 μg/mL means the organism has a MIC of less than or equal to 2 μg/mL to Compound A.

For the purpose of comparing Tygacil to Compound A, the "same subject" can be administered Tygacil and Compound A at different times after a sufficient wash-out period between administrations.

The present invention further relates to a method of treating hospital- acquired or community-acquired bacterial pneumonia in a human subject comprising the step of administering intravenously to the subject a therapeutically effective amount of Compound A.

In one embodiment, the community-acquired bacterial pneumonia (CABP) is characterized by the presence of two or more CABP pathogens selected from:

Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, Streptococcus pyogenes, Legionella pneumophila,

Chlamydia pneumoniae, and Mycoplasma pneumoniae.

The invention also relates to a method of treating CABP in a human subject comprising the step of administering intravenously to the subject an effective amount of Compound A, wherein the community-acquired bacterial pneumonia is characterized by the presence of Legionella pneumophila.

The invention also relates to a method of treating hospital-acquired bacterial pneumonia in a human subject in need of treatment comprising the step of administering intravenously to the subject a therapeutically effective amount of Compound A, wherein the hospital-acquired bacterial pneumonia is characterized by the present of two or more of Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Escherichia coli and Klebsiella pneumonia.

The invention also relates to a method of causing a 2 log₁₀ reduction in the amount of a bacterial strain selected from MRSA and S. pneumoniae present in a human subject comprising the step of administering intravenously to the subject a pharmaceutical composition comprising a therapeutically effective amount of Compound A as the sole active ingredient.

In one embodiment, the bacterial strain is selected from the clonal lineage of methicillin-resistant Staphylococcus aureus USA 300. The invention also relates to a method of treating complicated urinary tract infections including pyelonephritis in a human subject comprising the step of administering intravenously to the subject a therapeutically effective amount of Compound A, wherein the urinary pathogens can be effectively treated. In certain embodiments, the urinary pathogens are selected from: Escherichia coli and Klebsiella pneumoniae, including strains producing extended -spectrum β- lactamases and/or carbapenem-resistant strains; other Enterobacteriaceae species {Proteus mirabilis, Proteus vulgaris, Citrobacter freundii, etc.) and Acinetobacter baumannii and other gram-negative species as detailed in Table 15, including by extraction other species of any of the genera tested.

In a particular embodiment, the urinary tract infection is characterized by two or more urinary pathogens selected from: Escherichia coli and Klebsiella pneumoniae, including strains producing extended -spectrum β-lactamases and/or carbapenem-resistant strains; other Enterobacteriaceae species {Proteus mirabilis, Proteus vulgaris, Citrobacter freundii, etc.); Acinetobacter baumannii; and other gram-negative species as detailed in Table 15, including by extraction other species of any of the genera tested; In an alternate embodiment, the invention relates to a method of treating complicated urinary tract infections including pyelonephritis in a human subject comprising the step of administering intravenously to the subject a therapeutically effective amount of Compound A, wherein the urinary pathogens can be effectively treated and the urinary tract infection is characterized by two or more urinary pathogens selected from Escherichia coli and Klebsiella pneumoniae, including strains producing extended -spectrum β-lactamases and/or carbapenem- resistant strains; other Enterobacteriaceae species {Proteus mirabilis, Proteus vulgaris, Citrobacter freundii, etc.); Acinetobacter baumannii; other gram-negative species as detailed in Table 15, including by extraction other species of any of the genera tested; enterococci, such as Enterococcus faecalis and Enterococcus faecium and including vancomycin-resistant isolates; staphylococci (including but not limited to MRSA and Staphylococcus epidermidis and other coagulase-negative species); and other gram-positive urinary pathogen species as detailed in Table 14, including by extraction other species of any of the genera tested. Gram positive bacteria are often the causative pathogen especially when an urinary catheter has been placed. The MIC ranges, MIC50 and MIC90 values for many gram-positive pathogens are shown in Table 14.

Anaerobic bacteria are most often seen in intra-abdominal infections and wounds (e.g., diabetic foot). The MIC ranges, MIC₅₀ and MIC₉₀ values of

Compound A and multiple comparators are shown in Table 16 for many anaerobic species. Compound A is overall the most potent agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the Area Under the Curve (AUC) and incidence of

Nausea/V omiting in subj ects administered the reported dose of Compound A or tigecycline.

FIG. 2 shows the results of testing Compound A in a murine model of lung infection challenged with either a multidrug-resistant Streptococcus pneumoniae or methicillin-resistant Staphylococcus aureus (MRSA).

FIG. 3 shows the results of testing Compound A in a model of

UTI/Pyelonephritis Model of infection.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

The compound or pharmaceutically acceptable salt thereof which is administered intravenously in the method described herein is represented by the following structural formula:

The compound of the above structural formula or its salts is referred to herein as Compound A. Suitable methods for making this compound are described in published International Application No. WO 2010/017470.

TYGACIL (tigecycline) is a tetracycline derivative (a glycylcycline) for intravenous infusion. The chemical name of tigecycline is (4S,4aS,5aR,12aS)-9-[2- (tert-butylamino)acetamido]-4,7bis(dimethylamino)-l,4,4a,5,5a,6,l l,12a-octan^ 3,10,12,12a-tetrahydroxy-l,l l-dioxo-2naphthacenecarboxamide. The empirical formula is C2 H39N₅O8 and the molecular weight is 585.65.

The following represents the chemical structure of tigecycline:

The recommended dose regimen for TYGACIL is an initial dose of 100 mg, followed by 50 mg every 12 hours. Intravenous infusions of TYGACIL should be administered over approximately 30 to 60 minutes every 12 hours. Adverse effects, such as a high incidence of nausea and vomiting have been reported at the recommended doses (-26% nausea, ~18%vomiting, and -12% diarrhea).

The recommended duration of treatment with TYGACIL for complicated skin and skin structure infections or for complicated intra-abdominal infections is 5 to 14 days. The recommended duration of treatment with TYGACIL for

community-acquired bacterial pneumonia is 7 to 14 days. The duration of therapy should be guided by the severity and site of the infection and the patient's clinical and bacteriological progress.

"AUC" as used herein is the area under the concentration-time curve at steady-state over 24 hours. The AUC is reported as Concentration x time (e.g., ng'h/L or μg·h/mL.

"MIC" as used herein is the Minimum Inhibitory Concentration or the lowest concentration to inhibit the growth of a bacterial isolate.

MIC90 is the concentration required to inhibit the growth of 90% of a collection of microorganisms.

MIC₅0 IS the concentration required to inhibit the growth of 50% of a collection of microorganisms. "MIC range" as used herein is the lowest and higher MIC concentrations observed in a collection of microorganisms.

As used herein, a MIC to compound of less than or equal to 2 μg/ml (<2 g/ml), means that the MIC for an organism treated with Compound A is less than or equal to a MIC of 2 μg/ml.

A PK/PD index is the quantitative relationship between a pharmacokinetic parameter (such as AUC, peak level, or percentage of time over MIC) and a microbiological parameter (such as MIC). An example used here includes

AUC/MIC.

As used herein, the recommended dose of tigecycline is an initial dose of 100 mg, followed by 50 mg every 12 hours by Intravenous (IV) infusions over approximately 30 to 60 minutes.

"Undersirable side effects" refer to a side effects following administration of drug which would require termination of treatment or prevent initiation of treatment due to the severity of the side effect. In one embodiment, the undesirable side effect can be nausea/vomiting.

PHARMACEUTICAL COMPOSITIONS

This application also relates to a pharmaceutical composition comprising a unit dose of Compound A or a pharmaceutically acceptable salt thereof, wherein the unit dose is from about 1-1.5 mg/kg subject body weight and a pharmaceutically acceptable carrier. In certain embodiments, the unit dose of Compound A is provided in dry powder form and reconstituted in a pharmaceutically acceptable carrier, such as a sterile aqueous formulation prior to administration to a subject. Such pharmaceutical compositions are administered by parenteral routes (e.g., intravenous or intramuscular).

"Pharmaceutically acceptable carrier" means non-therapeutic components that are of sufficient purity and quality for use in the formulation of a composition of the invention that, when appropriately administered to typically do not produce an adverse reaction, and that are used as a vehicle for a drug substance (i.e., Compound A).

Pharmaceutically acceptable salts of the compounds of the present invention are also included. For example, an acid salt can be obtained by reacting the compound with a suitable organic or inorganic acid, resulting in pharmaceutically acceptable anionic salt forms. Examples of anionic salts include (but are not limited to) the acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate,

methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate,

phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, and triethiodide salts.

Salts of the compound of the present invention can be prepared by reacting with a suitable base. Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceutically acceptable cation, which includes alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium.

DISEASES AND DISORDER

Compound A can be used to prevent or treat important mammalian and veterinary diseases. Such diseases include, but are not limited to, skin infections, GI infections, urinary tract infections, genito-urinary infections, respiratory tract infections, sinuses infections, middle ear infections, systemic infections, intraabdominal infections, pyelonephritis, pneumonia, bacterial vaginosis, streptococcal sore throat, chronic bacterial prostatitis, gynecological and pelvic infections, sexually transmitted bacterial diseases, ocular and otic infections, cholera, influenza, bronchitis, acne, psoriasis, rosacea, impetigo, malaria, sexually transmitted disease including syphilis and gonorrhea, Legionnaires' disease, Lyme disease, Rocky Mountain spotted fever, Q fever, typhus, bubonic plague, gas gangrene, hospital acquired infections, leptospirosis, whooping cough, anthrax and infections caused by the agents responsible for lymphogranuloma venereum, inclusion conjunctivitis, or psittacosis. Infections can be bacterial, fungal, parasitic and viral infections

(including those which are resistant to other tetracycline compounds). Specific diseases include diarrhea, urinary tract infections, infections of skin and skin structure including wounds, cellulitis, and abscesses, ear, nose and throat infections, pneumonias (CABP, hospital-acquired, healthcare-associated, chronic pneumonias), mastitis and the like. Chronic pneumonias mainly include those of Nocardia, Actinomyces and Blastomyces dermatitidis, as well as the granulomatous pneumonias (Mycobacterium tuberculosis and atypical mycobacteria, Histoplasma capsulatum and Coccidioides immitis). In addition, methods for treating neoplasms using tetracycline compounds of the invention are also included (van der Bozert et al., Cancer Res., 48: 6686-6690 (1988)).

In one embodiment, the infection can be caused by a bacterium (e.g. an anaerobic or aerobic bacterium).

In another embodiment, the infection is caused by a Gram-positive bacterium. In a specific aspect of this embodiment, the infection is caused by a Gram-positive bacterium selected from class Bacilli, including, but not limited to, Staphylococcus spp., Streptococcus spp., Enterococcus spp., Bacillus spp., Listeria spp.; phylum Actinobacteria, including, but not limited to, Propionibacterium spp., Corynebacterium spp., Nocardia spp., Actinobacteria spp. , and class Clostridia, including, but not limited to, Clostridium spp.

In another embodiment, the infection is caused by a Gram-negative bacterium. In one aspect of this embodiment, the infection is caused by a phylum Proteobacteria {e.g., Betaproteobacteria and Gammaproteobacteria), including Escherichia coli, Salmonella, Shigella, other Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella or alpha-proteobacteria such as Wolbachia. In another aspect, the infection is caused by a Gram-negative bacterium selected from cyanobacteria, spirochaetes, green sulfur or green non-sulfur bacteria. In a specific aspect of this embodiment, the infection is caused by a Gram-negative bacteria selected from Enterobacteriaceae (e.g., E. coli, Klebsiella pneumoniae including those containing extended-spectrum β-lactamases and/or carbapenemases), Bacteroidetes (e.g., Bacteroides fragilis), Vibrionaceae (Vibrio cholerae), Pasteur ellaceae (e.g.,

Haemophilus influenzae), Pseudomonadaceae (e.g., Pseudomonas aeruginosa), Neisseriaceae (e.g. Neisseria meningitidis), Rickettsiae, Moraxellaceae (e.g., Moraxella catarrhalis), any species of Proteeae, Acinetobacter spp., Helicobacter spp., and Campylobacter spp. In a particular embodiment, the infection is caused by Gram-negative bacterium selected from the group consisting of Enterobacteriaceae (e.g., E. coli, Klebsiella pneumoniae), Pseudomonas, sad Acinetobacter spp. In another embodiment, the infection is caused by an organism selected from the group consisting of K. pneumoniae, Salmonella, E. hirae, A. baumannii, M. catarrhalis, H. influenzae, P. aeruginosa, E. faecium, E. coli, S. aureus, and E. faecalis.

In one embodiment, the infection is caused by an organism that grows intracellularly as part of its infection process.

In another embodiment, the infection is caused by an organism selected from the group consisting of order Rickettsiales; phylum Chlamydiae; order

Chlamydiales; Legionella spp. (including L. pneumophila); class Mollicutes, including, but not limited to, Mycoplasma spp. (e.g. Mycoplasma pneumoniae); Mycobacterium spp. (e.g. Mycobacterium tuberculosis); and phylum Spirochaetales (e.g. Borrelia spp. and Treponema spp.).

In another embodiment, the infection is caused by a Category A, B, or C Biodefense organisms as described at http://www.bt.cdc.gov/agent/agentlist- category.asp, the entire teachings of which are incorporated herein by reference. Examples of Category A organisms include, but are not limited to, Bacillus anthracis (anthrax), Yersinia pestis (plague), Clostridium botulinum (botulism) or Francisella tularensis (tularemia). In another embodiment the infection is a Bacillus anthracis infection. "Bacillus anthracis infection" includes any state, diseases, or disorders caused or which result from exposure or alleged exposure to Bacillus anthracis or another member of the Bacillus cereus group of bacteria.

Additional infections that can be treated using compounds of the invention or a pharmaceutically acceptable salt thereof include, but are not limited to, anthrax, botulism, bubonic plague, and tularemia.

In another embodiment, the infection is caused by a Category B Biodefense organism as described at http://www.bt.cdc.gov/agent/agentlist-category.asp, the entire teachings of which are incorporated herein by reference. Examples of Category B organisms include, but are not limited to, Brucella spp, Clostridium perfringens, Salmonella spp., Escherichia coli 0157:H7, Shigella spp., Burkholderia mallei, Burkholderia pseudomallei, Chlamydia psittaci, Coxiella burnetii,

Staphylococcal enterotoxin B, Rickettsia prowazekii, Vibrio cholerae, and

Cryptosporidium parvum.

Additional infections that can be treated using compounds of the invention or a pharmaceutically acceptable salt thereof include, but are not limited to,

Brucellosis, Clostridium perfringens, food-borne illnesses, Glanders, Melioidosis, Psittacosis, Q fever, viral encephalitis, and water-borne illnesses. These are defined as Category B pathogens.

Category C pathogens include, but are not limited to, emerging pathogens that could be engineered for mass dissemination in the future because of availability; ease of production and dissemination; and potential for high morbidity and mortality rates and major health impact (e.g., multidrug-resistant Mycobacterium

tuberculosis).

In yet another embodiment, the infection can be caused by one or more than one organism described above. Examples of such infections include, but are not limited to, intra-abdominal infections (often a mixture of a gram-negative species like E. coli and an anaerobe like B. fragilis), diabetic foot (various combinations of Streptococcus, Serratia, Staphylococcus and Enterococcus spp., anaerobes (S.E. Dowd, et al., PloS One 2008;3:e3326, the entire teachings of which are incorporated herein by reference) and respiratory disease (especially in patients that have chronic infections like cystic fibrosis - e.g., S. aureus plus P. aeruginosa or H. influenzae, atypical pathogens), wounds and abscesses (various gram-negative and gram- positive bacteria, notably MSSA/MRSA, coagulase-negative staphylococci, enterococci, Acinetobacter, P. aeruginosa, E. coli, B. fragilis), and bloodstream infections (13% were polymicrobial (H. Wisplinghoff, et al., Clin. Infect. Dis. 2004; 39:311-317, the entire teachings of which are incorporated herein by reference)).

In one embodiment, the infection is caused by an organism resistant to one or more antibiotics.

In another embodiment, the infection is caused by an organism resistant to tetracycline or any member of first and second generation of tetracycline antibiotics (e.g., doxycycline or minocycline). In another embodiment, the infection is caused by an organism resistant to methicillin.

In another embodiment, the infection is caused by an organism resistant to vancomycin.

In another embodiment, the infection is caused by an organism resistant to a quinolone or fluoroquinolone.

In another embodiment, the infection is caused by an organism resistant to tigecycline or any other tetracycline derivative. In a particular embodiment, the infection is caused by an organism resistant or nonsusceptible to tigecycline.

In another embodiment, the infection is caused by an organism resistant to a β-lactam or cephalosporin antibiotic or an organism resistant to penems or carbapenems.

In another embodiment, the infection is caused by an organism resistant to an antimicrobial peptide or a biosimilar therapeutic treatment. Antimicrobial peptides (also called host defense peptides) are an evolutionarily conserved component of the innate immune response and are found among all classes of life. In this case, antimicrobial peptide refers to any naturally occurring molecule or any

semi/synthetic molecule that are analogs of anionic peptides, linear cationic ex- helical peptides, cationic peptides enriched for specific amino acids (i.e., rich in proline, arginine, phenylalanine, glycine, tryptophan), and anionic and cationic peptides that contain cysteine and form disulfide bonds.

In another embodiment, the infection is caused by an organism resistant to macrolides, lincosamides, streptogramin antibiotics, oxazolidinones, tetracycline and/or pleuromutilins.

In another embodiment, the infection is caused by an organism resistant or nonsusceptible to PTK0796 (7-dimethylamino, 9-(2,2-dimethyl-propyl)- aminomethylcycline) .

In another embodiment, the infection is caused by a multidrug-resistant pathogen (having intermediate or full resistance to any two or more antibiotics). ADMINISTRATION

As used herein, the term "subject" means a mammal in need of treatment or prevention, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses, sheep, goats and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like). Typically, the subject is a human in need of the specified treatment.

Same subject as used herein is the same human person. For comparison of Tygacil to Compound A, the "same subject" can be administered Tygacil and Compound A at different times after a sufficient wash-out period between administrations.

As used herein, the term "treating" or 'treatment" refers to obtaining desired pharmacological and/or physiological effect. The effect can include achieving, partially or substantially, one or more of the following results: partially or totally reducing the extent of the disease, disorder or syndrome; ameliorating or improving a clinical symptom or indicator associated with the disorder; delaying, inhibiting or decreasing the likelihood of the progression of the disease, disorder or syndrome.

" As used herein, "preventing" or "prevention" refers to reducing the likelihood of the onset or development of disease, disorder or syndrome.

"Therapeutically effective amount" means that amount of Compound A that elicits the desired biological response, for example, treatment of a bacterial infection in a subject. In a specific embodiment, a therapeutically effective amount is an amount which in comparison to the recommended dosing regimen of tigecycline results in a higher AUC than tigecycline without undesirable side effects. In one embodiment, the effective amount of Compound A is from about 1- 1.5 mg/kg subject body weight once a day. In another embodiment, the effective amount of a compound of the invention is from about 0.625-1 mg/kg subject body weight twice a day.

As used herein, "population of human subjects" means the entire population tested and for which there is AUCo-t_au(ss) data. In certain aspects, the entire population must be healthy human subjects and must comprise at least six members. In alternate aspects, the entire population must be human subjects who are being treated for a bacterial infection and must comprise at least six members.

Once a day, as used herein, refers to one administration per every 24 hour period. Twice a day, as used herein, refers to two administrations per every 24 hour period. A suitable interval between the two administrations includes any time period which maintains a therapeutically effective plasma level of Compound A. Such an interval can be, for example, about 12 hours.

In a specific embodiment, Compound A was prepared for administration as follows: 52.5 mg of Compound A (free base equivalent) in the form of a lyophilized powder (59.4 mg of salt form and 420 mg mannitol) is reconstituted with a volume of 10 mL of sterile water for injection. The reconstituted sterile liquid is further diluted for infusion in D5W (and/or normal saline).

EXPERIMENTAL METHODS

The activity of Compound A and tigecycline were compared with respect to in vivo efficacy in animal models, drug exposure and tolerability in Phase 1 clinical trials and PK/PD relationships. An improved tolerability profile was seen for Compound A with respect to the incidence and severity of gastrointestinal tolerability. Such an improvement can allow once daily dosing of Compound A and higher AUCs than tigecycline.

Experiment 1: Phase 1 Single Ascending Dose (SAD) Study

Overview: The SAD study investigated single doses of 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, and 3.0 mg/kg, administered by intravenous infusion over 30 minutes.

Objectives: Study objectives were exploration of safety, tolerability, plasma PK and urinary excretion of single ascending doses of Compound A administered intravenously.

Methodology: Dose escalations were performed sequentially. 56 healthy adult volunteers 18-50 years old were recruited. Each of the 7 Dose Groups (DGs) consisted of 8 subjects (6 randomized to receive Compound A and 2 matching placebo). All subjects provided plasma and urine samples for an assessment of the pharmacokinetics.

Results: Plasma PK was dose-proportional and linear with respect to AUC, C_max, and 0-8 hour urine concentrations. All estimated T½ were between 12-24 hours, with DG 3 mg/kg having the largest estimated T½ while DG 0.10 mg/kg had the smallest. Table 1

Table 2 Mean ± SD Urine Concentrations of Compound A (ng/mL) for SAD Cohorts 1 to 7

*AU pre-infusion values were below the limit of detection

No serious adverse events were reported. Gastrointestinal side effects were observed at >2 mg/kg. No clinically significant safety lab values or significant changes in ECG readings were observed in any dose group.

Conclusions: PK, safety and tolerability data indicate Compound A may be of utility at dose regimens up to and including 2 mg/kg/day and are consistent with the potential utility of once-daily doses in the treatment of important bacterial infections.

Experiment 2: Comparison of Nausea/Emesis and AUC of Compound

A and Tigecycline

The AUC data and incidence of nausea/emesis resulting from the SAD study of Compound A were compared to corresponding data available from Phase 1 trials of tigecycline. A comparison of AUC and nausea/emesis is set forth in Table 3. Table 3. Com arison of incidence of nausea and emesis in SAD Phase 1 trials

Predicted dose based on 80 kg average body weight

bAUC₀-24 or AUCo-inf-; AUC data for compound A is from population PK analyses

Compound A demonstrated a lower incidence and severity of nausea and emesis as compared to tigecycline, despite reaching higher levels of exposure.

Experiment 3: Population Pharmacokinetic (PK) Analyses of SAD Study Methods: Population pharmacokinetic (PK) analyses were done with ADAPT 5 after the completion of each SAD cohort using plasma and urinary data. The best model was chosen using standard model discrimination criteria. Simulations were then performed with the model to obtain clinical endpoints associated with various dosing regimens, including AUC/MIC (area under the concentration-time curve (AUC)/ minimal inhibitory concentration (MIC)), T>MIC (% time drug

concentration exceeds MIC at steady-state) and Cmax/MIC. MAD regimens were proposed using these endpoints.

Results: Seven cycles of modeling and simulation were conducted with data from 42 subjects. Compound A PK was described by a 4-compartment model with linear elimination. Mean parameters (% intersubject variability) were Vc = 10.8 L (20.6%), CLnr = 11.5 L/h (19.5%), Vpl = 16.1 L (23.8%), CLdl = 44.3 L/h (8.69%), Vp2 = 132 L (20.2%), CLd2 = 6.95 L/h (40.8%), Vp3 = 103 L (25.1%), CLd3 = 26.9 L/h (30.8%) & CLr = 2.34 L/h (18.4%). Residual variability for plasma and urine data was 12.7% and 21.9%, respectively. When only AUC/MIC was considered, model-based simulations suggested that a minimum of 1.0 mg/kg QD for 10 days would be efficacious for organisms with an MIC90 = 2 μg/mL. Conclusion: The PK of Compound A was well-described by a 4-compartment model. Experiment 4: Phase 1 Multiple Ascending Dose (MAD) Study

Overview: 4 Dosing Groups

1- 0.50 mg/kg q24h (0.50 mg/kg of body weight given once a day by IV infusion over 30 minutes and for 10 days; based on 80 kg subject would have been given a 40 mg total daily dose)

2- 1.5 mg/kg q24h (1.5 mg/kg given once a day by IV infusion over 30 minutes and for 10 days; based on 80 kg subject would have been given a 120 mg total daily dose)

3- 1.5 mg/kg q24h, 60" infusion (1.5 mg/kg given once a day by IV infusion for 60 minutes and for 10 days; based on 80 kg subject would have been given a 120 mg total daily dose)

4- 1.0 mg/kg ql2h 60" infusion (1.0 mg/kg given twice a day by IV infusion for 60 minutes and for 10 days; based on 80 kg subject would have been given a 160 mg total daily dose)

Objectives: Study objectives were exploration of safety, tolerability, and PK of multiple ascending doses of Compound A.

Methodology: 32 healthy adult volunteers, 6 randomized to receive Compound A and 2 matching placebo per dose group, received 30 minute (30') infusions of 0.50 mg/kg q24h and 1.50 mg/kg q24h or 60 minute (60') infusions of 1.5 mg/kg q24h and 1.0 mg/kg ql2h.

Results: When evaluated by compartmental analysis, the AUCo-tau(ss) increased proportionally with dose, reaching steady state values of 8671 ng-h/mL when given once daily at 1.5 mg/kg and 13,335 ng-h/mL (over 24 hrs) when give 1.0 mg/kg twice daily. Clearance and volume of distribution at steady state averaged 13.9 L/h and 319.6 L, respectively, for subjects receiving 1.5 mg/ml q24h. Based on PK population modeling, steady-state should be attained within 7 days. The mean half- life of Compound A in plasma (all cohorts confounded) was 47.7 h and the median value was 35.3 h. Renal clearance accounted for approximately 15.5 ± 2.37% of total clearance of Compound A.

Based upon these results, the invention provides a method of achieving an arithmetic mean AUCo-tau(ss) of greater than 7,050 ng Compound A-h/mL over 24 hrs in a population of human subjects by administering intravenously to each member of the population the same dose of Compound A, wherein the dose is selected from 1.5 mg/kg body weight once a day in a 30 minute infusion; 1.5 mg/kg body weight once a day in a 60 minute infusion; and 1.0 mg/kg body weight twice a day, each administration in a 60 minute infusion; and wherein the dose is administered over at least 7 consecutive days. In a more specific aspect of this embodiment, the method achieves an arithmetic mean AUCo-tau(ss) of greater than 8,500 ng Compound A-h/mL over 24 hrs in the population.

In a related embodiment, the invention provides a method of achieving an arithmetic mean AUCo-t_au(ss) of greater than 10,000 ng Compound A-h/mL over 24 hrs in a population of human subjects by administering intravenously to each member of the population Compound A at a dose 1.0 mg/kg body weight twice a day, each administration in a 60 minute infusion; and wherein the dose is administered over at least 7 consecutive days. In a more specific aspect of this embodiment, the method achieves an arithmetic mean AUCo-tau(ss) of greater than 12,500 ng Compound A-h/mL over 24 hrs in the population.

In another related embodiment, the invention provides a pharmaceutical composition comprising Compound A and a pharmaceutically acceptable carrier, wherein the composition is formulated for intravenous administration to a human, wherein when administered to a population of human subjects over 7 consecutive days, wherein each member of the population is administered the same dosage selected from 1.5 mg Compound A /kg body weight once a day in a 30 minute infusion; 1.5 mg Compound A /kg body weight once a day in a 60 minute infusion; and 1.0 mg Compound A /kg body weight twice a day, each administration in a 60 minute infusion; produces an arithmetic mean AUCo-tau(ss) of greater than 7,050 ng Compound A- h/mL over 24 hrs. In one aspect of this embodiment, the

pharmaceutical composition produces an arithmetic mean AUCo_-tau(ss) of greater than 8,500 ng Compound A-h mL over 24 hrs.

In another related embodiment, the invention provides a pharmaceutical composition comprising Compound A and a pharmaceutically acceptable carrier, wherein the composition is formulated for intravenous administration to a human, wherein when administered to a population of human subjects over 7 consecutive days, wherein each member of the population is administered 1.0 mg Compound A /kg body weight twice a day, each administration in a 60 minute infusion; produces an arithmetic mean AUCo-tau(ss) of greater than 10,000 ng Compound A-h/mL over 24 hrs. In a more specific aspect of this embodiment, the pharmaceutical composition produces an arithmetic mean AUCo-t_au(ss) of greater than 12,500 ng Compound A-h/mL over 24 hrs in the population.

In any of the above embodiments and aspects thereof, ACUo-t_aU(ss) over 24 hours can be determined by compartmental or non-compartmental analyses. As long as one of those analyses yields results within the parameters set forth above, the method or composition is deemed to be a part of the present invention.

Table 4: Summary Statistics and Pharmacokinetic Parameter Values for Compound A MAD Study Using Population PK Modeling

Arithmetic Mean (%CV)

Median (Range) for T_max

Parameter 30 min infusion I 60 min infusion

D10 = Cmax after 10 days of Compound A dosing. For DG4, AUC values measured for 12 hours. Table 5. Mean ± SD Urine Concentrations of Compound A (ng/mL) for MAD Cohorts 1 to 4 on Days 1-4

aInfused over 30 minutes

bInfused over 60 minutes

°A11 Day 1 pre-infusion values were below the limit of detection

Table 6. Mean ± SD Urine Concentrations of Compound A (ng/mL) for MAD Cohorts 1 to 4 on Days 10-13

aInfused over 30 minutes

bInfused over 60 minutes

For urinary tract infections, it is desirable to have Compound A urine concentrations in excess of the MIC90 of key pathogens that cause cUTIs (e.g., E. coli, K. pneumoniae, E. faecalis, E. faecium, MRS A, MSSA; see Tables 14-16 for MIC data). Compound A has an excellent spectrum for empiric treatment of cUTIs caused by either gram-negative or gram-positive bacteria, with MIC₅0/MIC90 values of 0.25/0.5 and 0.5/1 μg/ml against 176 Escherichia coli and 219 Klebsiella pneumoniae isolates, respectively, and MIC90S of 0.12 μg ml vs. vancomycin- susceptible (n=102) or -resistant (n=88) enterococci or MRSA (n=137). From the data presented in Tables 5 and 6, the urine levels are in excess of 2 μg/ml in the 0-8 hour samples for all dose groups. Subjects receiving 1.5 mg/kg q24hrs (30 or 60 min infusions) or 1.0 mg/kg ql2hrs had urine concentrations in excess of 2 μg/ml for all sampling times through Day 10.

No serious adverse events (AEs) were reported. The most common adverse events by system organ class were gastrointestinal, administration site, and vascular disorders. No clinically significant safety lab values or significant changes in ECG readings were observed.

Conclusions: PK, safety and tolerability data indicate Compound A has significant exposure at dose regimens up to and including 2 mg/kg/day and are consistent with the potential utility of once-daily doses for the treatment of serious bacterial infections, including those caused by multidrug-resistant gram-negative pathogens. The urine concentrations of Compound A are >2 μg/ml throughout 10 days of therapy, thereby providing sufficient levels to treat infections caused by key urinary tract patho gens .

Experiment 5: Comparison of Nausea/Emesis and AUC of Compound

A and Tigecycline

A comparison of the AUCs from the MAD trial for Compound A is set forth in Table 7.

Table 7. Comparison of AUCs from MAD trials with Compound A to ublished AUCs of ti ec cline.

24h-AUC at steady state based on compartmental PK analysis; AUC₀_24ii from Tygacil

Package Insert, March 2009 FIG. 1 is a graphical representation of the results of Nausea/Vomiting and the AUCs for Compound A at the indicated dose and tigecycline at the indicated dose. The comparison of AUC and incidence of nausea/vomiting show that

Compound A can be dosed to achieve higher doses and systemic exposure levels before generating a similar rate of gastrointestinal adverse events associated with tigecycline. In FIG. 1, N=Nausea, V=Vomiting and W= Withdrawn and adverse events for the 1.5 mg/kg g24h at both 30 min and 60 min are combined to make a total of N=12 subjects. Experiment 6: Population Pharmacokinetic (PK) Analyses of MAD Study

Methods: Population PK analyses were done with ADAPT 5 (maximum likelihood expectation maximization) using plasma and urinary data collected for each group. Standard model discrimination criteria were used to determine the best model. Two-, three- and four-compartment models were tested. Results were then compared to those obtained from single dose (SD) data.

Results: The PK of Compound A was described by a 4-compartment model with linear elimination. Mean parameters (% intersubject variability) were Vc = 12.2 L (10.9%), CLnr = 11.5 L/h (23.0%), Vpl = 16.6 L (2.28%), CLdl = 29.9 L/h

(21.2%), Vp2 = 188 L (15.4%), CLd2 = 4.90 L/h (46.7%), Vp3 = 103 L (9.56%), CLd3 = 21.2 L/h (29.4%) & CLr = 2.05 L/h (15.7%). Steady-state volume of distribution was 320 L and mean half-life was around 48 hours. Residual variability for plasma & urinary data was 14.0% & 21.5%, respectively. Results were similar to those reported following SD administration & daily doses > 1.5 mg/kg were predicted to be effective for organisms with MIC < 2 g/ml.

Conclusion: The multiple dose PK of Compound A was well-described by a 4- compartment model. Results are in line with those previously determined using SAD data and indicate dose linearity. Experiment 7: Pharmacokinetics (PK) of Compound A in Mouse, Rat, Dog, Monkey and Chimpanzee

Methods: Disposition of single IV doses of Compound A have been determined in species relevant for safety assessment (mice=10; rats =3; beagle dogs=3; cynomolgus monkeys=2; and chimpanzees=3) and for projection of human pharmacokinetics. Animals were fasted overnight (minimum of 12 hours) and given a single IV dose followed by a sampling scheme for 24-48 hours. Urine collection was done in chimpanzee. Plasma, urine and dosing solution concentrations of Compound A were evaluated by sensitive and selective HPLC Turbolonspray ionization or HPLC/MS-MS assay using appropriate standard curves with LLOQs of 1-25 ng/mL. PK parameters were calculated by noncompartmental analysis using WinNonlin.

The in vitro protein binding of 1 μΜ Compound A in plasma collected with EDTA from mice, rats, beagle dogs, and cynomolgus monkeys was determined in triplicate using ultrafiltration and LC/MS/MS analysis.

Results: Peak plasma concentrations after a single 1 mg/kg IV dose ranged from approximately 0.5 μg/mL to 1.3 μg/mL, and declined in a multi-phasic manner with estimated elimination half-lives ranging from 4 to 19 hrs in mice, rats, dogs, monkeys, and chimpanzees. The PK of Compound A was species-dependent, with no notable gender differences (Table 8). Systemic clearances (Cls) of low pharmacological doses in mice, rats, dogs, and chimpanzees were low relative to hepatic blood flow, appearing somewhat higher in monkey. Cls in dogs appeared saturable at higher dosage, resulting in disproportionately higher plasma

concentrations. The mechanism for this saturable clearance is unknown, but was not seen in doses evaluated in rats. Cls after a single dose in chimpanzees, a useful surrogate for the human pharmacokinetics of many drugs, was 0.421 L/hr/kg.

Compound A was widely distributed in extravascular tissue, with a volume of distribution ranging from 3.2 L/kg to 15 L kg, demonstrating not only extensive tissue distribution but also tissue binding. When Compound A plasma protein binding was evaluated using ultrafiltration at 1 μΜ, the percent free ranged from 10.8-59.3% (Table 9). Biliary excretion is likely the major route for elimination of the dose since -20% of the IV dose was recovered in the urine of dogs and chimpanzees. The data is as follows: Table 8. Mean Pharmacokinetic Parameters of Compound A in Animals Given

Table 9. Protein Binding (% free) of 1 uM Compound A in Plasma (EDTA) from Mouse, Rat, Beagle Dog and Cynomolgus Monkey

Human Plasma Protein Binding

The in vitro protein binding of 1 μΜ Compound A in plasma collected with EDTA from humans was determined in triplicate using ultrafiltration and LC- MS/MS analysis. Compound A was added to 1.0 mL of plasma which was then incubated for 10 minutes at 37° C at 1800 rpm for 12 minutes and approximately 0.10-0.12 mL of filtrate was collected. Aliquots of the filtrate and plasma were analyzed by HPLC-MS/MS and the peak area ratios of Compound A were used to calculate the percent unbound for each sample. Plasma protein binding was also determined over a concentration range of 0.1-2.5 μg/mL using human plasma collected with heparin and equilibrium (Teflon microdialysis chambers) of test compound in phosphate-buffered saline against phosphate-buffered saline at 37° C for 22 hours.

The unbound (free) fraction of 1 μΜ (-0.5 μg/mL) Compound A was 1 1.5

±0.4% in human plasma by ultrafiltration. However, when the unbound fraction was measured by equilibrium dialysis in plasma collected with heparin, concentration- dependent protein binding was observed and is shown below: Table 10. Protein Binding of Compound A to Human Plasma

Conclusions: PK has been adequately determined in animal models for safety assessment. The binding of Compound A to human plasma appears to be concentration-dependent.

Experiment 8: Efficacy of Compound A in a Murine Neutropenic Thigh Model

CD-I female mice were rendered neutropenic by pretreatment with cyclophosphamide and then challenged with different bacterial strains in the thigh. Treatment was IV with a single dose of Compound A. Treatment was IV 1.5 hours post-challenge, with bacterial burden reduction recorded 24 hours post-challenge. The results are shown in Table 11 along with MIC values.

Table 11. Efficacy of Compound A in the Neutropenic Thigh Model

QD=once daily As can be seen in Table 11, Compound A demonstrated good activity, requiring less compound than comparators to achieve a 2 log reduction in bacterial counts of both susceptible (Smith) and resistant (MRS A SA161 tet(M)⁺) strains of S. aureus. Compound A showed activity similar to tigecycline against the S, aureus tet(K)⁺ strain (which contains a tetracycline-specific efflux pump), MRSA USA300, and S. pyogenes.

Experiment 9: Efficacy of Compound A in Murine Models of Systemic

Infections

The efficacy of Compound A to tigecycline was also compared in conventional systemic murine infection models. CD-I female mice were infected

intraperitoneally for septicemia models; compound was dosed 1 hr post-challenge, and survival was recorded 48 post-challenge. Results are presented using the PD₅₀ values, the dose that protects 50% of the mice from death, for comparison (Table 12).

Efficacy of Compound A and Comparators in Murine Systemic

aMean and standard deviation of PD50 from three experiments.

NT = not tested

CI = confidence interval

As shown in Table 12, Compound A was highly effective in septicemia models, demonstrating PD50S of <1 mg/kg against S. aureus, including tetracycline- resistant strains, MRSA and S. pyogenes. The PD₅₀s against E. coli were 1.2-4.4 mg/kg. The PD50 values for Compound A against S. aureus strains were comparable to those for tigecycline (MICs to strains used in the systemic models are shown in Table 11). In addition, Compound A PD₅₀ values were comparable to tigecycline against Escherichia coli isolates for infections with an E. coli strain expressing an extended spectrum β-lactamase (ESBL), EC133, and one that does not, E. coli ATCC 25922.

Experiment 10: Minimum Inhibitory Concentration (MIC) Assay for Strains Used in Murine Mouse Models

MICs were determined according to the Clinical and Laboratory Standards

Institute (CLSI) guidances (e.g., CLSI. Performance standards for antimicrobial susceptibility testing; nineteenth information supplement. CLSI document M100- S19, CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA, 2009). Briefly, frozen bacterial strains were thawed and subcultured onto Mueller Hinton Broth (MHB) or other appropriate media (Streptococcus requires blood and Haemophilus requires hemin and NAD). Following incubation overnight, the strains were subcultured onto Mueller Hinton Agar and again incubated overnight. Colonies were observed for appropriate colony morphology and lack of contamination. Isolated colonies were selected to prepare a starting inoculum equivalent to a 0.5 McFarland standard. The starting inoculum was diluted 1 :125 (this is the working inoculum) using MHB for further use. Test compounds were prepared by dilution in sterile water to a final concentration of 5.128 mg/mL.

Antibiotics (stored frozen, thawed and used within 3 hours of thawing) and compounds were further diluted to the desired working concentrations.

The assays were run as follows. Fifty of MHB was added to wells 2 - 12 of a 96-well plate. One hundred μΐ, of appropriately diluted antibiotics was added to well 1. Fifty μΐ, of antibiotics was removed from well 1 and added to well 2 and the contents of well 2 mixed by pipetting up and down five times. Fifty μΐ_^ of the mixture in well 2 was removed and added to well 3 and mixed as above. Serial dilutions were continued in the same manner through well 12. Fifty iL was removed from well 12 so that all contained 50 ih. Fifty iL of the working inoculum was then added to all test wells. A growth control well was prepared by adding 50 i of working inoculum and 50 μΐ, of MHB to an empty well. The plates were then incubated at 37 °C overnight, removed from the incubator and each well was read on a plate reading mirror. The lowest concentration (MIC) of test

compound that inhibited the growth of the bacteria was recorded.

Example:

(cloudiness)

Interpretation: MIC = 2 μg/mL

Protocol for Determining Inoculum Concentration (Viable Count)

Fifty 50μ1 of the inoculum was pipetted into well 1. Ninety μΐ of sterile

0.9% NaCl was pipetted into wells 2-6 of a 96-well microtiter plate. Ten μΐ, from was removed from well 1 and added it to well 2 followed by mixing. Ten μΤ was removed from well two and mixed with the contents of well 3 and so on creating serial dilutions through well 6. Ten μΐ, was removed from each well and spotted onto an appropriate agar plate. The plate was placed into an incubator overnight.

The colonies in spots that contain distinct colonies were counted. Viable count was calculated by multiplying the number of colonies by the dilution factor.

Bacterial Strains

The following bacterial strains, listed below, can be examined in minimum inhibitory concentration (MIC) assays.

STRAIN

ORGANISM DESIGNATION KEY PROPERTIES

SA100

Staphylococcus aureus ATCC 13709, MSSA, Smith strain

SA101 ATCC 29213, CLSI quality control strain,

Staphylococcus aureus

MSSA

SA191 HA-MRSA, tetracycline-resistant, lung

Staphylococcus aureus

infection model isolate

Staphylococcus aureus SA161 FIA-MRSA, tetracycline-resistant, tet(M)

Staphylococcus aureus SA158 Tetracycline-resistant tet(K)

ATCC 12228, CLSI quality control strain,

Staphylococcus epidermidis SE164

tetracycline-resistant STRAIN

ORGANISM DESIGNATION KEY PROPERTIES

Enterococcus faecalis EF103 ATCC 29212, tet-I/R, control strain

Tetracycline-resistant, tet(M)

Enterococcus faecalis EF159

Enterococcus faecalis EF327 Wound isolate (US) tet(M)

Enterococcus faecium EF404 Blood isolate (US) tet(M)

Streptococcus pneumoniae SP106 ATCC 49619, CLSI quality control strain

Streptococcus pneumoniae SP160 Tetracycline-resistant, tet(M)

Streptococcus pyogenes SP312 2009 clinical isolate, tet(M)

S. pyogenes for efficacy models; tetS;

Streptococcus pyogenes SP193

sensitive to sulfonamides

Haemophilus influenzae HI262 Tetracycline-resistant, ampicillin-resistant

Moraxella catarrhalis MC205 ATCC 8176, CLSI quality control strain

Escherichia coli EC 107 ATCC 25922, CLSI quality control strain

Escherichia coli EC155 Tetracycline-resistant, tet(A)

Escherichia coli EC878 MG1655 tolCr.kan

Escherichia coli EC880 IpxA

Escherichia coli EC882 impA

MDR uropathogenic; serotype

Escherichia coli EC200 017:K52:H18; UMN 026; trimeth/sulfa-R;

BAA-1 161

Enterohacter cloacae EC108 ATCC 13047, wt

Enterobacter cloacae EC603 Urine isolate (Spain)

Klebsiella pneumoniae KP109 ATCC 13883, wt

Klebsiella pneumoniae KP153 Tetracycline-resistant, tet(A), MDR, ESBL⁺

Klebsiella pneumoniae KP457 2009 ESBL⁺, CTX-M, OXA

Proteus mirabilis PM112 ATCC 35659

Proteus mirabilis PM385 Urine ESBL⁺ isolate

Pseudomonas aeruginosa PA111 ATCC 27853, wt, control strain

Wt, parent of PA 170- 173

Pseudomonas aeruginosa PA169

PA 170 AmexX; MexXY-(missing a

Pseudomonas aeruginosa PA173 functional efflux pump)

Pseudomonas aeruginosa PA555 ATCC BAA-47, wild type strain PAOl

Pseudomonas aeruginosa PA556 Multiple-Mex efflux pump knockout strain

Pseudomonas aeruginosa PA689 Blood isolate (US)

Acinetobacter baumannii ABl lO ATCC 19606, wt

Acinetobacter baumannii AB250 Cystic fibrosis isolate, MDR

Stenotrophomonas

SM256 Cystic fibrosis isolate, MDR maltophilia

Burkholderia cenocepacia BC240 Cystic fibrosis isolate, MDR

*MDR, multidrug-resistant; MRS A, rnethicillin-resistant S. aureus; MSSA, methicillin- sensitive S. aureus; HA-MRSA, hospital-associated MRS A; tet(K), major gram-positive tetracycline efflux mechanism; tet(M), major gram-positive tetracycline ribosome-protection mechanism; ESBL⁺, extended spectrum β-lactamase Results

Values of minimum inhibition concentration (MIC) for Compound A and comparators are provided in Table 13 and Table 13 A.

Table 13. MICs of Compound A and Comparators for Strains used in Murine Mouse Models

NT=Not Tested

Table 13A. MICs of Compound A and Comparators for Strains used in

Murine Mouse Models

NT=Not Tested; N/A=not intrinsically active

aMIC to meropenem

Additional Values of minimum inhibition concentration (MIC) for

Compound A and comparators are provided in Tables 14-16.

Table 14. The susceptibility of Compound A and comparators against Gram- Positive Pathogens

MIC range

MIC /MIC

50 90

( δ πιΐ)

Linezoli Daptomyci Vancomyci Levofloxaci

Compd. A Tigecycline

Organism³ N d n n n

0.12-

0.12-0.25 0.25 2-4 0.5-1 0.5-2 0.12->32

MSSA 56

0.25/0.25 0.12/0.2 4/4 1/1 1/1 1/1

5

MIC range

MIC /MIC

50 90

Linezoli Daptomyci Vancomyci Levofloxaci

Compd. A Tigecycline

Organism³ N d n n n

0.12/0.1

2

0.03-

0.03-0.12 0.12 1-8 0.5-16 >64 32->32

VRE 88

0.06/0.12 0.12/0.1 2/4 2/8 >64/>64 >32/>32

2

MSSA=methicillin-susceptible S. aureus; MRSA=methicillin-resistant S. aureus;

MRSA PVL⁺= MRSA isolates confirmed to contain Panton- Valentine leukocidin;

MSSE=methicillin-susceptible Staphylococcus epidermidis; MRSE=methicillin-resistant S.

epidermidis; VSE=vancomycin-susceptible E. faecalis and E. faecium; VRE=vancomycin-resistant E.

faecalis and E. faecium. Results

The MIC₉₀ of Cpd A < MIC₉₀ of TGC for: MRSA, MRSAPVL⁺, Streptococcus anginosus, Streptococcus intermedius, Streptococcus mitis, Streptococcus sanguis,

Streptococcus pyogenes, and Streptococcus agalactiae

Table 15. The susceptibility of Compound A and comparators against Gram- Negative Pathogens

MIC range ^ /ml)

MIC /MIC (ng/ml)

rd

Organism N Compound A Tigecycline Carbapenem FQ 3 Gen Ceph Gentamicin Pip/ Tazo

0.016-

<0.016-4 0.03->8 <0.25->32 0.03->64 <0.25->32 . <0.5->128

Escherichia coli 176 >32

0.2₅/0.₅ 0.25/1 0.25/32 8/>64 2/>32 4/>64

0.25/<l

ESpL⁺ 0.03-4 0.06->8 0.06-8 0.03->32 0.5->64 <0.25->32 <0.5->128

97

E, coli 0.2₅/0.₅ 0.25/2 0.25/<l >4/32 >32/>64 4/>32 8/>64

0.063-

Klebsiella 0.13-16 0.13-16 <0.016->32 0.03->64 <0.25->32 0.5->64

219 >32

pneumoniae 0.5/1 0.5/2 2/>32 32/64 4/>32 16/>64

0.5/16

ESpL⁺ Klebsiella 0.12-16 0.25-16 0.03->32 0.03->32 0.25->64 <0.25->32 2->128

90

pneumoniae 0.₅/l 1/4 <l/32 >4/>32 >32/>64 >8/>32 >64/>64

Carbapenem- >128-

0.13-16 0.25-16 4->32 4->32 32->32 2->32

resistant 19 >128

0.5/1 1/1 32/>32 >32/>32 >32/>32 16/>32

K. pneumoniae >128/>128

0.03-2 0.06-4 <1-<1 <0.25->4 <0.5->32 <0.25->32

Klebsiella oxytoca 41 <0.5->64^a

0.25/1 0.5/2 <1/<1 <0.25/>4 <0.5/>32 0.5/>32 2/8

0.5-8 1-16 2->32

Proteus mirabilis 68 0.02-32^b <0.02-64 0.5->64 <0.13-8^C

2/4 4/8 4/32 0.063/>2 <0.02/4 2/16 0.25/2 MIC range ^g/ml)

MIC₅₀/MIC₉₀ (fig/ml)

rd

Organism N Compound A Tigecycline Carbapenem FQ 3 Gen Ceph Gentamicin Pip/ Tazo

Providencia 0.12-8 0.06-16 0.25-16 0.02->2 <0.02->16

50 0.5->32 <0.02->16 stuartii 1/2 1/2 2/4 >2/>2 0.25/16 . 4/>32 4/>128

0.5-2 0.5-4 <\.<\ <0.25-l 0.25->64 <0.25->8 <0.5-4

Proteus vulgaris 29

1 /2 2/4 <1/<1 <0.25/l <0.25/>64 l/>8 <0.5/2

Morganella 0.5-2 0.25-8

30 <1-<1 <0.25->4 <0.5-16 0.5->8 <0.5-2 morganii 1/2 2/4 <1/<1 <0.25/4 <0.5/4 l/>8 <0.5/l

Serratia 0.5-2 0.5-2

30 <1-<1 <0.25->4 <0.5->64 0.5-8 1-32 marcescens 1/1 1/2 <1/<1 <0.25/2 <0.5/<0.5 0.5/1 2/8

Citrobacter 0.12-2 0.12-8 0.12->32 0.008->2 0.06->16 0.25->32

50 0.25->128 freundii 0.5/2 0.5/2 1/8 0.06/>2 4/>16 5/>32 16/>128

Enterobacter 0.03-4 0.06-8 0.06-32 0.008->32

134 0.03->64^d <0.25->32 0.5->128^e cloacae 0.5/2 0.5/4 0.5/4 0.25/>4 >16/>64 0.5/>8 >64/>128

Enterobacter 0.25-2 0.25-4 <l-2 <0.25-0.5 <0.5->64 <0.25-l <0.5->64

30

aerogenes 0.25/0.25 0.5/0.5 <1/<1 <0.25/<0.25 <0.5/16 <0.25/0.5 2/16 _.

0.12-0.5 0.12-1 <l-8 <0.25->4 <0.5-<0.5 <0.25->8 1-64

Salmonella spp. 30

0.25/0.25 0.25/0.5 <1/<1 <0.25/<0.25 <0.5/<0.5 0.5/1 2/4

0.06-1 0.12-1 <1-<1 <0.25-l <0.5-2 <0.25->8 <0.5-4

Shigella spp. 30

0.12/0.5 0.25/0.5 <1/<1 <0.25/0.5 <0.5/<0.5 1/1 2/2

Slenotrophomonas 0.12-4 0.25-8 8->8 <0.25->4 l->64 <0.25->8 8->64

29

maltophilia 0.25/1 0.5/2 >8/>8 <0.25/>4 8/>64 >8/>8 32/>64

Acinetobacter 0.03-0.25 0.06-0.5 <l->8 <0.25-2 <0.5->64 <0.25-8 <0.5-16

29

Iwoffli 0.12/0.25 0.12/0.5 <l/4 <0.25/<0.25 1/16 <0.25/l <0.5/8

Acinetobacter <0.016-4 <0.016-8 0.12->32 0.02->32

89 0.12->16^f 0.5->32^h 1->128^S baumannii 0.5/2 1/4 l/>32 8/>16 >16/>16 32/>32 >128/>128

Pseudomonas l->64 1->16 0.12->32 0.06->2 1->16 0.12->32 1->128

88 h

aeruginosa 8/16 16/>16 1/16 025/>2 >16/>16 2/16 8/>128'

Haemophilus 0.06-0.25 0.13-1 <0.016-0.13 <0.016-0.6

15

influenzae 0.13/0.2 0.5/0.5 ND <0.016/0.03 <0.016/<0.016 ND ND

<0.016- <0.016-

Moraxella 0.03-0.25

15 0.063 0.063 ND ND ND ND catarrhalis <63/0.063

<0.016/0.063 0.03/0.063

Legionella 0.016-2

70 ND ND ND ND ND ND pneumophila 1/2

Shaded Organisms indicate that the MIC₉₀ of Compound A < MIC₉₀ of tigecycline

Carbapenem = meropenem, ertapenem, or imipenem; FQ = Fluoroquinolone levofloxacin or ciprofloxacin; 3rd Gen Ceph = third generation cephalosporin (either cefotaxime or ceftazidime); Pip/Tazo = Piperacillin/tazobactam (only the piperacillin MIC in the presence of 4

of tazobactam is shown); ES L⁺ = extended spectrum p-lactamase producing isolates

a30 isolates tested against Pip/Tazo; ^b55 tested with ciprofloxacin; 13 with levofloxacin;

c d

55 isolates tested with Pip/Tazo ; 13 not tested; 97 isolates tested, 68 isolates tested with cefotaxime and 29 isolates tested with ceftazidime;

e81 isolates tested; ^f29 isolates tested with cefotaxime and 60 isolates not tested; ^g29 isolates tested with PTZ and 60 not tested;

h36 isolates tested with tobramycin and 52 isolates tested with gentamicin; '52 isolates with Pip/Tazo and 36 isolates not tested; ^JND = Not done Results

MIC90 of Cmpd. A < MIC₉₀ of tigecycline for: Escherichia coli, ESpL⁺ E. coli, Klebsiella pneumoniae, ESpL⁺ . pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris, Morganella morganii, Serratia marcescens, Enterobacter cloacae, Enterobacter aerogenes, Stenotrophomonas maltophilia, Acinetobacter baumannii, Acinetobacter hvqffii, Salmonella spp., Haemophilus influenzae, Moraxella catarrhalis, and Pseudomonas aeruginosa

Table 16. The susceptibility of Compound A and comparators against Anaerobes

3 F. mortiferum and 2 F. varium

Results:

The MIC₉₀ of Cmpd. A < MIC90 of TGC for: Bacteroides fragilis, Bacteroides fiagilis (that produce a cefinase), Bacteroides vulgatus, Bacteroides

thetaiotaomicron, Bacteroides ovatus,, Prevotella bivia, Prevotella disiens,

Prevotella intermedia, Clostridium perfringens, Anaerococcus spp., Parabacteroides distasonis, and Peptoniphilus asaccharolyticus.

Experiment 11: Activity of Compound A in Murine Models of Lung Infection

Compound A was evaluated in murine lung infection models. After cyclophosphamide treatment that rendered the mice neutropenic, mice were intranasally challenged with either a MRSA isolate containing a tet(M) mechanism or with a multidrug-resistant S. pneumoniae tet(M) strain. Compound A or comparators were dosed 2 and 12 hours post-challenge by the indicated route (FIG. 2). Twenty-four hours post-initiation of treatment, mice were euthanized, lungs removed, and the bacterial burden was determined. Compound A reduced S.

pneumoniae by >2.5 logs when administered IV at 3-12 mg/kg, BID while linezolid reduced the bacterial burden by 2 logs when given as 30 mg/kg, orally (PO). Oral doxycycline was ineffective as the isolate contained tet(M), a mechanism that provides tetracycline resistance at the level of the ribosome.

When mice were challenged with MRSA, 10 mg/kg of Compound A administered intravenously was as effective as 30 mg/kg linezolid given orally. Both compounds were more effective than vancomycin given as an IV dose at 50 mg/kg.

MICs of Compound A (Cpd A) and doxycycline against S. pneumoniae SP160 tet(M) was <0.016 and 8 μg/ml, respectively. MICs of Compound A (Cpd A) and doxycycline against S. pneumoniae SP160 tet(M) was <0.016 and 8 μg/ml, respectively.

Experiment 12: Activity of Compound A in UTI/pyelonephritis Models of Infection

Compound A is also effective in reducing the bacterial burden in kidneys from mice that have been infected with a tetracycline-resistant uropathogenic strain of E. coli or an ESpL-producing strain of K. pneumoniae (FIG. 3). Treatment with Compound A or comparators was IV at the dose indicated, 12 and 24 hours post- challenge. Compound A at 5 or 10 mg/kg BID, reduced the bacterial burden by 4 logs and was comparable to tigecycline at 10 mg/kg, BID and to levofloxacin at 2 mg/kg, BID in treatment of a tetracycline-resistant (tetR) E. coli strain. In treating the ES PL-producing isolate of K. pneumoniae 10-20 mg/kg of Compound A was found to be equivalent to the gold standard, meropenem at 30 mg/kg BID.

MICs of Compound A (Cpd A) and doxycycline against S. pneumoniae

SP160 tet(M) was <0.016 and 8 μg/ml, respectively. MICs of Compound A (Cpd A) and meropenem against K. pneumoniae KP453 were 0.5 and 0.031 μg/ml, respectively.

Experiment 13: Activity of Compound A Against L. pneumophila Isolates

Methods: The in vitro activity of Compound A was compared to tetracycline and erythromycin against a total of 70 L. pneumophila isolates (serogroup 1 (n=20), 2 (n=10), 3 (n=10), 4 (n=10), 5 (n=10) and 6 (n=10)) by standard agar dilution using buffered yeast extract agar containing BCYE growth supplement (BYE). A pretest to determine if Compound A's activity was impacted artificially by BCYE supplement or iron was done by testing ATCC isolates of Staphylococcus aureus and Escherichia coli on BYE, BYE without ferric pyrophosphate (modB YE) and cation-adjusted Mueller-Hinton agar (MH).

Strains and Growth Conditions

Recent Legionella pneumophila strains were isolated from the respiratory tract from 1992 to 2010 and identified by standard methods described by Murray et al. (1). Isolates from six serogroups were tested for a total number of 70 L. pneumophila. Buffered Yeast extract (BYE) (with original

Legionella BCYE Growth supplement) was used as the medium to test Legionella strains.

(1) Murray et al, Manual of Clinical Microbiology, 9^th ed., 2007, A.S.M., Chap. 53; 835- 849.

• Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213 were tested in a pilot study comparing the activities of antibiotics in Mueller Hinton Broth (MH), standard BYE, and modified BYE ("Mod BYE";

lacking ferric pyrophosphate).

Determination of Minimal Inhibitory Concentrations (MICs)

• MICs were determined using the CLSI agar dilution method (2,3) with

replicate plating of the organisms onto a series of agar plates of increasing concentrations of compound from 0.004 mg/L to 64 μg/ML.

(2) Performance standards for antimicrobial susceptibility testing; 18^th Information Supplement; Ml 00-518, Clinical and Laboratory Standards Institute (CLSI), Wayne, PA, January 2008.

(3) Method for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard 17^th edition, M7-A7, Clinical and Laboratory Standards Institute (CLSI), Wayne, PA, 2006. RESULTS

Table 17. Pilot Media Study: Susceptibility of E. coli ATCC 25922 QC Strain

_T , , . η · Media

Incubation l ime , ,

Tested Compd A Tetracycline Erythromycin

24 hours MH 0.12 1 >64

Mod BYE 0.5 0.5 >64 BYE 2 16 >64

MH ND ND ND

48 hours Mod BYE 1 1 >64

BYE 16 >64 >64

Expected MIC range MH 0.06-0.12* 0.5-2** U

ND= Not Done; U= Unavailable

Table 18. Susceptibility of Legionella pneumophila

pneumophila Antibiotic Range 50% 90% (no. tested)

All serogroups Compd. A 0.016-2 1 2

Tetracycline 0.5-8 4 8 Erythromycin 0.06-1 0.25 0.5 sero group 1 Compd. A 0.016-2 0.5 2

Tetracycline 0.5-8 4 8 Erythromycin 0.06-1 0.25 0.5 sero group 2 Compd. A 0.12-2 1 2

Tetracycline 1-8 4 8 Erythromycin 0.06-0.5 0.25 0.25 serogroup 3 Compd. A 0.5-2 0.5 2

Tetracycline 1-8 2 8 Erythromycin 0.12-0.5 0.25 0.5 serogroup 4 Compd. A 0.25-2 1 2

Tetracycline 2-8 8 8 Erythromycin 0.12-0.5 0.5 0.5 serogroup 5 Compd. A 0.25-1 0.5 1

Tetracycline 2-8 4 8 Erythromycin 0.06-1 0.25 0.5 serogroup 6 Compd. A 0.06-1 0.5 1

Tetracycline 2-8 4 8 Erythromycin 0.12-0.25 0.12 0.25

Only BYE supported L. pneumophila growth. Pilot tests indicated that BYE resulted in a 16- to 64-fold increase in MICs relative to MH for S. aureus

ATCC29213 and E. coli ATCC25922, suggesting that, similar to other tetracycline class antibiotics, the MIC values of Compound A obtained in BYE for L. pneumophila were artificially elevated due to media effects. Regardless, the MIC50/90 values of Compound A, tetracycline and erythromycin against all L.

pneumophila strains were 1/2, 4/8 and 0.25/0.5 μg/ML, respectively. Against L. pneumophila serogroup 1, usually the most frequently recovered serogroup, the MIC50/90 of Compound A, tetracycline, and erythromycin was 0.5/2, 4/8, and 0.25/0.5 μg/mL.

Conclusions: Compound A had excellent activity against L. pneumophila, especially as its activity was artificially suppressed in BYE agar.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS claimed is:

A method of treating a bacterial infection in a human subject comprising administering intravenously to the human subject once a day from about 1- 1.5 mg/kg subject body weight of Compound A.

A method of treating a bacterial infection in a human subject comprising administering intravenously to the human subject twice a day from about 0.625-1 mg/kg subject body weight of Compound A.

The method of claim 1 or 2, wherein Compound A is administered by infusion over 30 to 120 minutes per administration.

The method of claim 3, wherein the infusion is a constant infusion.

The method of claim 3 or 4, wherein the compound is administered by infusion over 30 to 60 minutes per administration.

The method of any one of claims 3-5, wherein the compound is

administered at a concentration of from about 0.2 mg/mL to 0.7 mg/mL.

A method of achieving an AUC of Compound A in a human subject that is at least 50% greater than the AUC achieved for tigecycline when the same subject is administered tigecycline at the recommended dosing regimen, the method comprising administering intravenously to the human subject a pharmaceutical composition comprising Compound A as the sole active agent once or twice a day in a therapeutically effective amount.

The method of claim 7, wherein the AUC of Compound A achieved in the human subject is at least 75% greater than the AUC for tigecycline that is achieved when the same subject is administered tigecycline at the recommended dose regimen.

The method of claim 7 or 8, wherein Compound A is administered once a day in an amount equal to or greater than 1.5 mg/kg.

The method of claim 7 or 8, wherein Compound A is administered twice a day in an amount equal to or greater than 0.65 mg/kg per administration.

A pharmaceutical composition comprising a therapeutically effective amount of Compound A, wherein intravenous administration of the composition to a human subject results in an AUC of Compound A that is at least 50% greater than the AUC of tigecycline when tigecycline is administered intravenously to the same subject in a pharmaceutical composition comprising an amount of tigecycline that is the same as the amount of Compound A on a milligram basis of active ingredient and that is administered in the same dosing regimen as Compound A.

A method of achieving an AUC/MIC ratio in a human subject suffering from an infection by a bacterial organism that is at least 20% greater than the AUC/MIC ratio for the bacterial organism in the same subject when the same subject is administered tigecycline at the recommended dose regimen, the method comprising administering intravenously to the subject a

pharmaceutical composition comprising Compound A as the sole active agent once or twice a day in a therapeutically effective amount.

The method of claim 12, wherein the subject is suffering from a bacterial infection.

The method of claim 13, wherein the subject is suffering from a bacterial infection characterized by the presence of an organism with a MIC to compound for Compound A of less than or equal to 2 μg/mL. A method of treating community-acquired bacterial pneumonia in a human subject in need of treatment comprising the step of administering

intravenously to the subject a therapeutically effective amount of Compound A, wherein the community-acquired bacterial pneumonia is characterized by the presence of two or more of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, Streptococcus pyogenes, Legionella pneumophila, Chlamydia pneumoniae, and

Mycoplasma pneumoniae.

A method of treating hospital-acquired bacterial pneumonia in a human subject in need of treatment comprising the step of administering

intravenously to the subject a therapeutically effective amount of Compound A, wherein the hospital-acquired bacterial pneumonia is characterized by the present of two or more of Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae.

17. A method of treating community-acquired bacterial pneumonia in a human subject comprising the step of administering intravenously to the subject a therapeutically effective amount of Compound A, wherein the community- acquired bacterial pneumonia is characterized by the presence of Legionella pneumophila.

A method of causing a 2 log₁₀ reduction in the amount of a bacterial strain selected from MRS A and S. pyogenes present in a human subject comprising the step of administering intravenously to the subject a pharmaceutical composition comprising a therapeutically effective amount of Compound A as the sole active ingredient. The method of claim 18, wherein the bacterial strain is selected from the clonal lineage of MRS A US A300.

A method of treating complicated urinary tract infections in a human subject comprising the step of administering intravenously to the subject a therapeutically effective amount of Compound A, wherein the UTI is characterized by the presence of two or more pathogens selected Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Citrobacter freundii, Acinetobacter baumannii; Enter ococcus faecalis, Enterococcus faecium, MRSA and Staphylococcus epidermidis.

The method of claim 20, wherein one or more of the bacterial strains is multidrug-resistant.

The method of claim 21, wherein the bacterial strain produces an extended- spectrum β-lactamase and/or is carbapenem-resistant.

A method of treating complicated acute or chronic bacterial skin and skin structure infections in a human subject comprising the step of administering intravenously to the subject a therapeutically effective amount of Compound A, wherein the infection is characterized by the presence of two or more skin pathogens.

The method of claim 23, wherein one or more of the bacterial strains is multidrug-resistant.

25. The method of claim 24, wherein the bacterial strain produces an extended- spectrum β-lactamase and/or is carbapenem-resistant.