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WO2013066469A2 - Inhibiteurs de kinases capables de renforcer la sensibilité d'agents pathogènes bactériens aux antibiotiques de la famille des b-lactamines - Google Patents

Inhibiteurs de kinases capables de renforcer la sensibilité d'agents pathogènes bactériens aux antibiotiques de la famille des b-lactamines Download PDF

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Publication number
WO2013066469A2
WO2013066469A2 PCT/US2012/050635 US2012050635W WO2013066469A2 WO 2013066469 A2 WO2013066469 A2 WO 2013066469A2 US 2012050635 W US2012050635 W US 2012050635W WO 2013066469 A2 WO2013066469 A2 WO 2013066469A2
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sulfonyl
group
kinase inhibitor
methoxy
substituted
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WO2013066469A3 (fr
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Lakshmi Rajagopal
Kellie Burnside HOWARD
Christopher WHIDBEY
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Seattle Childrens Hospital
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Seattle Childrens Hospital
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the present invention relates generally to compositions and methods for increasing the sensitivity of bacterial pathogens to antibiotics.
  • Staphylococcus aureus are gram positive bacteria that are frequently part of the bacterial flora found in the nose and on skin. About 20% of the human population is a long-term carrier of S. aureus. In addition, patients diagnosed with hyper IgE (Jobs) syndrome or chronic granulomatous disease (CGD) are predisposed to recurrent and life-threatening S. aureus infections. S.
  • aureus can cause a range of illnesses from minor skin infections, such as pimples, impetigo, boils, cellulitis folliculitis, carbuncles, scalded skin syndrome, and abscesses, to life- threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome (TSS), chest pain, bacteremia, and sepsis.
  • S. aureus is one of the five most common causes of nosocomial infections, with some 500,000 patients in American hospitals contracting a staphylococcal infection each year. Exotoxins, including the hemolysins, play an important role in the pathogenesis of S. aureus infections.
  • S. aureus infections often require treatment with antibiotics.
  • S. aureus has become resistant to many commonly used antibiotics.
  • Aminoglycoside antibiotics such as kanamycin, gentamicin, streptomycin, etc., were once widely effective against staphylococcal infections until strains evolved mechanisms to inhibit the aminoglycosides' action, which occurs via protonated amine and/or hydroxyl interactions with the ribosomal RNA of the bacterial 30S ribosomal subunit.
  • only about 2% of all S. aureus isolates are sensitive to penicillin.
  • Penicillinase-resistant ⁇ -lactam antibiotics such as methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, and flucloxacillin, are able to resist degradation by staphylococcal penicillinase and were developed to treat penicillin-resistant S. aureus. These antibiotics are commonly used as first-line treatment for S. aureus infections.
  • MRSA Methicillin-resistant Staphylococcus aureus
  • beta-lactam antibiotics methicillin, cloxacillin, dicloxacillin, nafcillin, oxacillin, etc.
  • cephalosporins Invasive infections caused by MRSA are escalating. Morbidity and mortality rates due to MRSA have surpassed those reported for HIV/ AIDS. The accelerating rate of morbidity and mortality due to S. aureus emphasize its importance as a human pathogen and the need for effective therapeutic strategies.
  • SCCmec Staphylococcal chromosome cassette mec
  • Bacterial cell wall synthesis is essential to growth and cell division (thus reproduction) and inhibition of Penicillin Binding Proteins leads to irregularities in cell wall structure leading to eventual cell death and lysis.
  • All ⁇ -lactam antibiotics (except for tabtoxinine-P-lactam, which inhibits glutamine synthetase) bind to Penicillin Binding Proteins, inhibiting the synthesis of the peptidoglycan layer of bacterial cell walls.
  • the ⁇ -lactam antibiotics have low binding affinity for PBP2a, which is important for cross-linking of cell wall peptidoglycan, therefore cell wall synthesis is able to proceed in their presence. This allows for resistance to all ⁇ -lactam antibiotics, and obviates their clinical use during MRSA infections.
  • the mecA gene is not found in methicillin sensitive S. aureus (MSSA) strains.
  • MRSA infections are commonly treated with ⁇ - ⁇ -lactam antibiotics, such as clindamycin and co-trimoxazole. Resistance to these antibiotics has also led to the use of broad-spectrum anti-Gram-positive antibiotics, such as linezolid, because of its availability as an oral drug.
  • First-line treatment for serious MRSA infections is currently the glycopeptide antibiotics, vancomycin and teicoplanin. There are number of problems with these antibiotics, such as the need for intravenous administration and toxicity. There are also concerns glycopeptide antibiotics do not penetrate very well into infected tissues.
  • VRSA Vancomycin-resistant S. aureus
  • VRSA Vancomycin-resistant S. aureus
  • the present embodiments relate to compositions and methods for increasing the sensitivity of bacterial pathogens to antibiotics.
  • a method for increasing the sensitivity of bacterial pathogens to ⁇ -lactam antibiotics by contacting the bacterial pathogen with one or more kinase inhibitors is provided.
  • the bacterial pathogen is MSRA.
  • the bacterial pathogen is Enterococcus faecalis.
  • compositions and methods for increasing the susceptibility of Gram negative pathogens to ⁇ -lactam antibiotics relate to compositions and methods for increasing the susceptibility or sensitivity of Gram positive pathogens to ⁇ -lactam antibiotics.
  • compositions and methods for increasing the susceptibility or sensitivity of Gram positive pathogens to ⁇ -lactam antibiotics relate to therapeutic formulas/cocktails, pharmaceutical composition, product combination, or kits for use against MRSA infections comprising kinase inhibitors and ⁇ -lactam antibiotics.
  • compositions and methods for inhibiting the growth of staphylococcus aureus relate to compositions and methods for increasing the susceptibility of Gram negative pathogens to ⁇ -lactam antibiotics.
  • the staphylococcus aureus is resistant to methicillin, other ⁇ -lactams, macrolides, lincosamides and aminoglicosides. In some embodiments, the staphylococcus aureus is sensitive to methicillin, other ⁇ -lactams, macrolides, lincosamides and/or aminoglicosides.
  • Several embodiments described herein relate to methods for increasing the sensitivity staphylococcus aureus to methicillin, other ⁇ -lactams, macrolides, lincosamides and aminoglicosides comprising administering an effective amount of a kinase inhibitor.
  • the staphylococcus aureus is resistant to methicillin, other ⁇ -lactams, macrolides, lincosamides and/or aminoglicosides.
  • the kinase inhibitors used with the pharmaceutical compositions, product combinations, kits, and methods described herein have following chemical structure:
  • j is 0 or 1 ;
  • p is 0 or 1 ;
  • m is 0 or 1 ;
  • k is 0 or 1 ;
  • u is 0 or 1 ;
  • Ri is selected from the group consisting of
  • V is selected from the group consisting of C, S and N,
  • R13 is selected from the group consisting of H and C1-C6 alkyl
  • R9 is selected from the group consisting of H, 0-CH3, 0-C2H5, 0-C3H7, 0-C4H9, O- C5H1 1 and 0-C6H13; wherein X is selected from the group consisting of C, S or N;
  • R3 is selected from the group consisting of H or C1-C6 alkyl
  • R4 is selected from the group consisting of a substituted or unsubstituted phenyl group, a substituted or unsubstituted naphthyl group,
  • RIO, Rl 1 and R12 are each independently selected from the group consisting of a CI to C6 alkyl or a substituted or unsubstituted phenyl group;
  • W and L are each independently selected from the group consisting of C, N and S; wherein R5 is selected from the group consisting of H, and
  • Z is selected from the group consisting of C, N and S;
  • R6 is selected from the group consisting of (0)s-R8, wherein s can be 0 or 1;
  • R8 is selected from the group consisting of H and a C1-C6 alkyl
  • R7 is selected from the group consisting of H
  • a pharmaceutically acceptable salt of the foregoing is used with the pharmaceutical compositions, product combinations, kits, and methods described herein.
  • the aforementioned kinase inhibitors can be used in any composition or method described herein (e.g., the aforementioned kinase inhibitors may be used in conjunction with one or more ⁇ -lactam antibiotics).
  • the kinase inhibitors used in the pharmaceutical compositions, product combinations, kits, and methods described herein have following chemical structure:
  • Cb is selected from the group consisting of an optionally substituted, unsaturated carbocyclic ring system, for example, phenyl or naphthyl groups and wherein the nitrogen atom of one or more piperidine rings is optionally substituted.
  • a pharmaceutically acceptable salt of the foregoing is used with the pharmaceutical compositions, product combinations, kits, and methods described herein.
  • the aforementioned kinase inhibitors can be used in any composition or method described herein (e.g., the aforementioned kinase inhibitors may be used in conjunction with one or more ⁇ -lactam antibiotics).
  • the kinase inhibitor used in the pharmaceutical compositions, product combinations, kits, and methods described herein can have following chemical structure:
  • Cb is selected from the group consisting of a substituted or unsubstituted, unsaturated ring system, for example, phenyl or naphthyl groups, and wherein Gl may be S or N.
  • Cb can be a fused ring system comprising two or more 5- or 6-membered rings, wherein each fused ring is substituted or unsubstituted and comprises 0 to 4 heteroatoms selected from the group consisting of nitrogen, oxygen, sulfur, phosphorus, chlorine, bromine, and iodine.
  • a hydrogen on any carbon atom is substituted with at least one selected from mono-substituted, poly-substituted or unsubstituted, straight or branched chain variants of a C1-C12 alkyl, a C2-C12 alkenyl, a C2-C12 alkynyl, a C2-C12 alkoxy, a C1-C12 ether, a C2-C12 acylalkyl, a C7-C24 arylalkyl, a C1-C12 alkylsulfonyl, and a C5-C24 heteroarylalkyl; a C3-C12 cycloalkyl, a C3-C12 cycloalkenyl, a C3-C12 cycloalkoxy, a C 6 -Ci2 aryl, a C4-C12 heteroaryl, a C2-C12 heterocycloalkyl, a C4-C12 heterocycloalky
  • a pharmaceutically acceptable salt of the foregoing is used with the pharmaceutical compositions, product combinations, kits, and methods described herein.
  • the aforementioned kinase inhibitors can be used in any composition or method described herein (e.g., the aforementioned kinase inhibitors may be used in conjunction with one or more ⁇ - lactam antibiotics).
  • kinase inhibitors used in the pharmaceutical compositions, product combinations, kits, and methods described herein have the following chemical structure:
  • a pharmaceutically acceptable salt of the foregoing is used with the pharmaceutical compositions, product combinations, kits, and methods described herein.
  • the aforementioned kinase inhibitors can be used in any composition or method described herein (e.g., the aforementioned kinase inhibitors may be used in conjunction with one or more ⁇ -lactam antibiotics).
  • the kinase inhibitor used in the pharmaceutical compositions, product combinations, kits, and methods described herein can have the following chemical structure:
  • a pharmaceutically acceptable salt of the foregoing is used with the pharmaceutical compositions, product combinations, kits, and methods described herein.
  • the aforementioned kinase inhibitors can be used in any composition or method described herein (e.g., the aforementioned kinase inhibitors may be used in conjunction with one or more ⁇ -lactam antibiotics).
  • the kinase inhibitor used in the pharmaceutical compositions, product combinations, kits, and methods described herein can have the following chemical structure:
  • a pharmaceutically acceptable salt of the foregoing is used with the pharmaceutical compositions, product combinations, kits, and methods described herein.
  • the aforementioned kinase inhibitors can be used in any composition or method described herein (e.g., the aforementioned kinase inhibitors may be used in conjunction with one or more ⁇ -lactam antibiotics).
  • the kinase inhibitor used in the pharmaceutical compositions, product combinations, kits, and methods described herein can have the following chemical structure:
  • a pharmaceutically acceptable salt of the foregoing is used with the pharmaceutical compositions, product combinations, kits, and methods described herein.
  • the aforementioned kinase inhibitors can be used in any composition or method described herein (e.g., the aforementioned kinase inhibitors may be used in conjunction with one or more ⁇ -lactam antibiotics).
  • the kinase inhibitor used in the pharmaceutical compositions, product combinations, kits, and methods described herein can have the following chemical structure:
  • a pharmaceutically acceptable salt of the foregoing is used with the pharmaceutical compositions, product combinations, kits, and methods described herein.
  • the aforementioned kinase inhibitors can be used in any composition or method described herein (e.g., the aforementioned kinase inhibitors may be used in conjunction with one or more ⁇ -lactam antibiotics).
  • the kinase inhibitor used in the pharmaceutical compositions, product combinations, kits, and methods described herein can have the following chemical structure:
  • a pharmaceutically acceptable salt of the foregoing is used with the pharmaceutical compositions, product combinations, kits, and methods described herein.
  • the aforementioned kinase inhibitors can be used in any composition or method described herein (e.g., the aforementioned kinase inhibitors may be used in conjunction with one or more ⁇ -lactam antibiotics).
  • the kinase inhibitor used in the pharmaceutical compositions, product combinations, kits, and methods described herein can have the following chemical structure:
  • a pharmaceutically acceptable salt of the foregoing is used with the pharmaceutical compositions, product combinations, kits, and methods described herein.
  • the aforementioned kinase inhibitors can be used in any composition or method described herein (e.g., the aforementioned kinase inhibitors may be used in conjunction with one or more ⁇ -lactam antibiotics).
  • one or more kinase inhibitors that are used in the pharmaceutical compositions, product combinations, kits, and methods described herein are selected from the group consisting of 5-(azaperhydroepinylsulfonyl)-3-hydroxynaphthalene-2- carboxylic acid, (2-bromophenyl)(8-quinolylsulfonyl)amine, (2,5-dichlorophenyl)(8- quinolylsulfonyl)amine, 3-hydroxy-5-[(prop-2-enylamino)sulfonyl]naphthalene-2-carboxylic acid, 4-methyl- 1 -(naphthylsulfonyl)piperidine, 1 -((2E)-3-phenylprop-2-enyl)-4-(8- quinolylsulfonyl)piperazine, ethyl l-(8-quinolylsulfonyl)piperidine-4-
  • the aforementioned kinase inhibitors can be used in any composition or method described herein (e.g., the aforementioned kinase inhibitors may be used in conjunction with one or more ⁇ -lactam antibiotics).
  • two or more kinase inhibitors used in the pharmaceutical compositions, product combinations, kits, and methods described herein are selected from the group consisting of 5-(azaperhydroepinylsulfonyl)-3-hydroxynaphthalene-2-carboxylic acid, (2- bromophenyl)(8-quinolylsulfonyl)amine, (2,5-dichlorophenyl)(8-quinolylsulfonyl)amine, 3- hydroxy-5 -[(prop-2-enylamino)sulfonyl]naphthalene-2-carboxylic acid, 4-methyl- 1 - (naphthylsulfonyl)piperidine, l-((2E)-3-phenylprop-2-enyl)-4-(8-quinolylsulfonyl)piperazine, ethyl 1 -(8-quinolylsulfonyl)piperidine-4-car
  • the aforementioned kinase inhibitors can be used in any composition or method described herein (e.g., the aforementioned kinase inhibitors may be used in conjunction with one or more ⁇ -lactam antibiotics).
  • Figure 1 depicts an autoradiograph of Stkl and PBP3 incubated in kinase buffer containing [ ⁇ 32 ⁇ ] ATP resolved on an SDS-PAGE gel. Phosphorylation of PBP3 was observed only in the presence of Stkl (lane2).
  • Figure 2 depicts a graph showing the % inhibition in growth of WT MRSA
  • FIG. 3 depicts a graph showing the dose-dependent inhibition in growth of
  • WT MRSA LAC in the presence of the kinase inhibitor, ST085384.
  • the % inhibition in growth is calculated relative to WT MRSA LAC grown in the presence of 4 ⁇ g/ml Nafcillin.
  • Figure 4 depicts a graph showing the percent survival of mice intravenously infected with either WT MRSA LAC (represented by the open circles) or the isogenic Astkl mutant MSRA (represented by the triangles).
  • Figure 5B shows a graph of Blood Urea Nitrogen (BUN) levels (mg/dL) in blood collected from mice over a period of 24 hours following injection with vehicle alone (Omg/kg ST085405 (represented by the closed circles)), 1 Omg/kg ST085405 (represented by the closed squares), or lOOmg/kg ST085405 (represented by the closed triangles).
  • BUN Blood Urea Nitrogen
  • Figure 6B shows a graph of Blood Urea Nitrogen (BUN) levels (mg/dL) in blood collected from mice over a period of 24 hours following injection with vehicle alone (Omg/kg ST085384 (represented by the closed circles)), lOmg/kg ST085384 (represented by the closed squares), or lOOmg/kg ST085384 (represented by the closed triangles).
  • BUN Blood Urea Nitrogen
  • Figure 8A shows a graph of Glutamic Oxaloacetic Transaminase Activity
  • Figure 8B shows a graph of Alanine Transaminase Activity (ATA) (mU/ml) in blood collected from mice over a period of 24 hours following injection with vehicle alone (Omg/kg ST085405/ST085384 (represented by the closed circles)), lOmg/kg ST085405 (represented by the closed squares), lOOmg/kg ST085405 (represented by the closed upward- facing triangles), lOmg/kg ST085384 (represented by the closed downward-facing triangles), or lOOmg/kg ST085384 (represented by the closed diamonds).
  • ATA Alanine Transaminase Activity
  • Figure 9 shows a graph of kinase inhibitor concentration (ng/ml) in blood collected from mice over a period of 24 hours following injection at 0 hours with lOmg/kg ST085405 (represented by the closed circles), lOOmg/kg ST085405 (represented by the closed squares), lOmg/kg ST085384 (represented by the closed upward-facing triangles), or lOOmg/kg ST085384 (represented by the downward-facing triangles).
  • Figure 10 shows a graph of kinase inhibitor concentration (ng/ml) in blood collected from mice over a period of 24 hours following injection at 0 hours and again at 3 hours with lOO mg/kg ST085405 (represented by the closed circles) or lOOmg/kg ST085384 (represented by the closed squares).
  • the embodiments described herein relate to compositions and methods for treating S. aureus infections by increasing the sensitivity of S. aureus, in particular Methicillin- Resistant S. aureus, to antibiotics.
  • Most S. aureus strains both Methicillin- Sensitive Staphylococcus aureus (MSSA) and Methicillin-Resistant Staphylococcus aureus (MRSA), encode a single pair of signaling enzymes that are commonly found in eukaryotes, known as a serine/threonine kinase (Stkl (also known as PknB)) and a serine/threonine phosphatase (Stpl).
  • MSSA Methicillin- Sensitive Staphylococcus aureus
  • MRSA Methicillin-Resistant Staphylococcus aureus
  • stkl or stpl does not affect antibiotic resistance in MSSA (Newman strain). However, as shown in Table 1, resistance to ⁇ -lactam antibiotics is attenuated by mutation of stkl, but not stpl, in the MRSA strains USA400 (MW-2) and USA300 (LAC). Mutation of the stkl gene also increases sensitivity to ⁇ -lactam antibiotics in the N315, COL, and USA300 MRSA strains. In some instances, stkl mutant MRSA strains exhibit 4-32 fold increases in ⁇ - lactam sensitivity compared to wild-type (WT) MRSA strains. While MRSA Astkl mutants are more sensitive than WT MRSA to ⁇ -lactam antibiotics, MRSA Astkl mutants are similar to WT MRSA in virulence (see Fig. 4).
  • both methicillin-sensitive and methicillin- resistant strains of S. aureus encode four major penicillin binding proteins (PBP) known as PBP1, PBP2, PBP3 and PBP4 with approximate molecular masses of 85, 81, 75 and 45 kDa, respectively.
  • PBP3 major penicillin binding protein
  • the major penicillin binding protein, PBP3 has a C-terminal transpeptidase domain (from amino acids 350-677, which is also the penicillin binding domain) and an N- terminal domain of unknown function. Loss of PBP3 function does not appear to affect virulence, as MRSA mutants lacking pbp3 were not identified in screens for attenuated virulence in S. aureus.
  • kinase activity of Stkl is important for antibiotic resistance as complementation with the kinase domain, and not the extracellular PASTA domain, restored WT levels of antibiotic resistance to Astk mutants.
  • Stkl phosphorylates PBP3 in vitro. See Fig. 1.
  • inhibition of Stkl activity by the kinase inhibitor ST085384 increases susceptibility of WT (Stkl expressing) MRSA to ⁇ -lactam antibiotics. Therefore, Stkl phosphorylation activity is an important modulator of MRSA's resistance to ⁇ -lactams and compositions that disrupt phosphorylation of Stkl targets can be used for the treatment of MRSA infections.
  • Phosphopeptide enrichment and mass spectrometry identified a threonine phosphopeptide corresponding to PBP3 that was expressed in WT MRSA but not in the Astkl mutant MRSA, indicating that PBP3 is a Stkl -specific target.
  • a transposon (TN5EZ) insertion in monocistronic pbp3 increased sensitivity of MRSA to a wide range of ⁇ -lactam antibiotics, similar to the stkl mutant. Therefore, disruption of PBP3 activity represents a novel mechanism for sensitizing drug resistant pathogens to ⁇ -lactam antibiotics.
  • Several embodiments described herein relate to methods and compositions for increasing the sensitivity of MRSA to ⁇ -lactams by disrupting PBP3 activity. Some embodiments relate to a method of increasing the sensitivity of MRSA to ⁇ -lactams by reducing phosphorylation of amino acid 105 of PBP3.
  • PBP3 activity may be disrupted by a phosphatase.
  • PBP3 activity may be disrupted by a kinase inhibitor.
  • the kinase inhibitor prevents phophorylation of PBP3 by Stpl .
  • addition of a kinase inhibitor reduces PBP3 activity by at least about or any number in between about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%
  • addition of a kinase inhibitor completely blocks PBP3 activity.
  • MRSA methicillin resistant Staphylococcus aureus
  • small molecule libraries are screened to identify compounds that increase the sensitivity of MRSA to ⁇ -lactams in vitro. For example, a natural compound library, the Timtec ActiTarg-K library, was screened and the kinase inhibitor ST085384 was identified as increasing the sensitivity of MRSA to a wide spectrum of ⁇ -lactams in vitro.
  • kinase inhibitor reduces Stkl kinase activity by at least about or any number in between about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 6
  • Some embodiments relate to methods for treating or inhibiting an antibiotic- resistant pathogenic bacterial infection by co -administration of a kinase inhibitor, for example a serine/threonine kinase inhibitor, and an antibiotic. More specifically, some embodiments relate to methods of treating or inhibiting a MRSA infection by administering an amount of a kinase inhibitor to the body of a patient sufficient to sensitize the MRSA to the antibiotic.
  • the antibiotic is a ⁇ -lactam antibiotic.
  • the kinase inhibitor used in the methods of treating or inhibiting a bacterial infection, for example a MRSA infection, by administering an amount of a kinase inhibitor to the body of a patient sufficient to sensitize the MRSA to the antibiotic has the following chemical Formula I:
  • Cb can be an optionally substituted, unsaturated carbocyclic ring system and wherein the nitrogen atom of one or more piperidine rings is optionally substituted.
  • Cb can be a phenyl group.
  • Cb can be a naphthyl group.
  • the kinase inhibitor used in the methods of treating or inhibiting a bacterial infection, for example a MRSA infection, by administering an amount of a kinase inhibitor to the body of a patient sufficient to sensitize the MRSA to the antibiotic has the following chemical Formula II:
  • Cb can be a substituted or unsubstituted, unsaturated ring system and wherein Gl is S or N.
  • Cb can be a fused ring system comprising two or more 5- or 6- membered rings, wherein each fused ring is substituted or unsubstituted and comprises 0 to 4 heteroatoms selected from the group consisting of nitrogen, oxygen, sulfur, phosphorus, chlorine, bromine, and iodine.
  • a hydrogen on any carbon atom of Formula II is substituted with at least one selected from mono-substituted, poly-substituted or unsubstituted, straight or branched chain variants of a alkyl, a C2-C12 alkenyl, a C2-C12 alkynyl, a C2-C12 alkoxy, a C1-C12 ether, a C2-C12 acylalkyl, a C7-C24 arylalkyl, a C1-C12 alkylsulfonyl, and a C5-C24 heteroarylalkyl; a C3-C12 cycloalkyl, a C3-C12 cycloalkenyl, a C3-C12 cycloalkoxy, a C 6 -Ci2 aryl, a C4-C12 heteroaryl, a C2-C12 heterocycloalkyl, a C4-C12 heterocycloalkyl,
  • Some embodiments disclosed herein relate to methods of ameliorating and/or treating a bacterial infection that can include administering to a subject suffering from the bacterial infection an effective amount of one or more compounds of Formula (I) and/or Formula (II), or a pharmaceutically acceptable salt of the foregoing, or a pharmaceutical composition that includes one or more compounds of Formula (I) and/or Formula (II), or a pharmaceutically acceptable salt of the foregoing.
  • Other embodiments described herein relate to using one or more compounds of Formula (I) and/or Formula (II), or a pharmaceutically acceptable salt of the foregoing, in the manufacture of a medicament for ameliorating and/or treating a bacterial infection.
  • Still other embodiments described herein relate to compounds of Formula (I) and/or Formula (II), or a pharmaceutically acceptable salt of the foregoing, that can be used for ameliorating and/or treating a bacterial infection.
  • methods of ameliorating and/or treating a bacterial infection can include contacting a cell infected with the bacterial infection with an effective amount of one or more compounds of Formula (I) and/or Formula (II), or a pharmaceutically acceptable salt of the foregoing, or a pharmaceutical composition that includes one or more compounds of Formula (I) and/or Formula (II), or a pharmaceutically acceptable salt of the foregoing.
  • Some embodiments disclosed herein relate to methods of inhibiting the replication of a bacteria that can include contacting a cell infection with the bacteria with an effective amount of one or more compounds of Formula (I) and/or Formula (II), or a pharmaceutically acceptable salt of the foregoing, or a pharmaceutical composition that includes one or more compounds of Formula (I) and/or Formula (II), or a pharmaceutically acceptable salt of the foregoing.
  • the bacterial infection can be a S. aureus infection.
  • the kinase inhibitor used in the methods of treating or inhibiting a MRSA infection by administering an amount of a kinase inhibitor to the body of a patient sufficient to sensitize the MRSA to the antibiotic has the following chemical structure:
  • the kinase inhibitor used in the methods of treating or inhibiting a MRSA infection by administering an amount of a kinase inhibitor to the body of a patient sufficient to sensitize the MRSA to the antibiotic has the following chemical structure:
  • the kinase inhibitor used in the methods of treating or inhibiting a MRSA infection by administering an amount of a kinase inhibitor to the body of a patient sufficient to sensitize the MRSA to the antibiotic has the following chemical structure:
  • the kinase inhibitor used in the methods of treating or inhibiting a MRSA infection by administering an amount of a kinase inhibitor to the body of a patient sufficient to sensitize the MRSA to the antibiotic has the following chemical structure:
  • the kinase inhibitor used in the methods of treating or inhibiting a MRSA infection by administering an amount of a kinase inhibitor to the body of a patient sufficient to sensitize the MRSA to the antibiotic has the following chemical structure:
  • the kinase inhibitor used in the methods of treating or inhibiting a MRSA infection by administering an amount of a kinase inhibitor to the body of a patient sufficient to sensitize the MRSA to the antibiotic has the following chemical structure:
  • the kinase inhibitor used in the methods of treating or inhibiting a MRSA infection by administering an amount of a kinase inhibitor to the body of a patient sufficient to sensitize the MRSA to the antibiotic has the following chemical structure:
  • one or more kinase inhibitors are used in the methods of treating or inhibiting a MRSA infection by administering an amount of a kinase inhibitor, for example a serine/threonine kinase inhibitor, to the body of a patient sufficient to sensitize the MRSA to the antibiotic and said kinase inhibitors are selected from the group consisting of 5- (azaperhydroepinylsulfonyl)-3-hydroxynaphthalene-2-carboxylic acid, (2-bromophenyl)(8- quinolylsulfonyl)amine, (2,5-dichlorophenyl)(8-quinolylsulfonyl)amine, 3-hydroxy-5-[(prop-2- enylamino)sulfonyl]naphthalene-2-carboxylic acid, 4-methyl- l-(naphthylsulfonyl)piperidine, 1- ((2E)-3
  • substituted means any substitution of a hydrogen atom with a functional group.
  • the term "functional group” has its common definition, and refers to chemical moieties, for example chemical moieties selected from the group consisting of a halogen atom, C1-C20 alkyl, substituted C1-C20 alkyl, perhalogenated alkyl, cyloalkyl, substituted cycloalkyl, aryl, substituted aryl, benzyl, heteroaryl, substituted heteroaryl, cyano, and nitro.
  • salt refers to a salt of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound.
  • the salt is an acid addition salt of the compound.
  • Pharmaceutical salts can be obtained by reacting a compound with inorganic acids such as hydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid and phosphoric acid.
  • compositions can also be obtained by reacting a compound with an organic acid such as aliphatic or aromatic carboxylic or sulfonic acids, for example formic, acetic, succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic, ethanesulfonic, p-toluensulfonic, salicylic or naphthalenesulfonic acid.
  • organic acid such as aliphatic or aromatic carboxylic or sulfonic acids
  • Pharmaceutical salts can also be obtained by reacting a compound with a base to form a salt such as an ammonium salt, an alkali metal salt, such as a sodium or a potassium salt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamine, cyclohexylamine, triethanolamine, ethylenediamine, and salts with amino acids such as arginine and lysine.
  • a salt such as an ammonium salt, an alkali metal salt, such as a sodium or a potassium salt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamine, cyclohexylamine
  • kinase inhibitors described above may be administered alone or in combination with other therapeutic agents, such as antibiotic, anti-inflammatory or antiseptic agents such as anti-bacterial agents, anti-fungicides, anti-viral agents, and anti-parasitic agents.
  • a pharmaceutical composition comprises one or more kinase inhibitors and one or more antibiotic or antiseptic agent(s). Examples are penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, and fluoroquinolones.
  • Antiseptic agents include iodine, silver, copper, clorhexidine, polyhexanide and other biguanides, chitosan, acetic acid, and hydrogen peroxide. These agents may be incorporated as part of the same pharmaceutical composition or may be administered separately.
  • the pharmaceutical compositions may also contain anti-inflammatory drugs such as steroids and macro lactam derivatives.
  • ⁇ -Lactam antibiotics are bactericidal, and act by inhibiting the synthesis of the peptidoglycan layer of bacterial cell walls.
  • the peptidoglycan layer is important for cell wall structural integrity, especially in Gram-positive organisms.
  • ⁇ -lactam antibiotics include, but are not limited to: benzathine penicillin, benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V), procaine penicillin, methicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin, flucloxacillin, temocillin, amoxicillin, ampicillin, co-amoxiclav, azlocillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, cephalosporins, cephalexin, cephalothin, cefazolin, cefaclor, cefuroxime, cefamandole, cephamycins, cefotetan, cefoxitin, ceftriaxone, cefotaxime, cefpodoxime, cefixime, ceftazidime, cefepime, cefpirome, imipenem, meropenem, cefix
  • Gram-positive bacteria are stained dark blue or violet by Gram staining, which is in contrast to Gram-negative bacteria, which do not retain the crystal violet stain. Gram-positive organisms retain the crystal violet stain because of the high amount of peptidoglycan in their cell walls.
  • Susceptible organisms generally include those gram positive and gram negative, aerobic and anaerobic organisms whose growth can be inhibited by the embodiments described herein.
  • Susceptible organisms include, but are not limited to, Staphylococcus, Lactobacillus, Streptococcus, Streptococcus agalactiae, Sarcina, S. pneumoniae, S. pyogenes, S.
  • mutans Escherichia, Enterobacter, Klebsiella, Pseudomonas, Pseudomonas aeruginosa, Acinetobacter, Proteus, Campylobacter, Citrobacter, Nisseria, Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacteroides, Peptococcus, Clostridium, Salmonella, Shigella, Serratia, Haemophilus, Brucella, Mycobacterium tuberculosis and other organisms.
  • susceptible organisms may include fungi, protozoa and viruses.
  • the term "patient” refers to the recipient of a therapeutic treatment and includes all organisms within the kingdom animalia.
  • the animal is within the family of mammals, such as humans, bovine, ovine, porcine, feline, buffalo, canine, goat, equine, donkey, deer and primates.
  • the animal is human.
  • Treatment covers preventing growth of harmful infectious bacterial, fungal, parasitic, and viral infections, in an animal, particularly a human, and includes: (a) preventing the infection from occurring or developing in the subject which may be predisposed to the infection but has not yet been diagnosed as having it; (b) inhibiting the infection, i.e.
  • Treatment may be specifically directed towards treating patients with wounds caused by trauma and/or surgery.
  • treating can comprise using formulations of kinase inhibitor and antibiotic during surgical procedures.
  • an effective amount of a compound of the present embodiments is used, optionally in combination with a pharmaceutically acceptable carrier.
  • the composition may be dry, or it may be a solution.
  • Treatment may be reactive, for combating an existing infection, or prophylactic, for preventing infection in an organism susceptible to infection.
  • compositions of this invention are used to treat infections by drug -resistant strains of bacteria, for example MRS A (methicillin resistant S.
  • MRSE methicillin resistant S. epidermidis
  • PRSP penicillin resistant S. pneumoniae
  • VIRSA vancomycin intermittently resistant Staphylococcus aureus
  • VRE vancomycin resistant Enterococci
  • the kinase inhibitors described herein may be provided prior to, simultaneously with, or subsequent to a beta-lactam antibiotic ("co-administration").
  • the kinase inhibitor and antibiotic may be administered separately by different routes, if desired.
  • the term "co-administered” is used to denote simultaneous or sequential administration. Preferably, such co-administration produces a synergistic effect.
  • the terms “synergy” and “synergistic effect” indicate that the effect produced when two or more drugs are co-administered is greater than would be predicted based on the effect produced when the compounds are administered individually. In general, a synergistic effect is most clearly demonstrated at sub-optimal concentrations of the compounds (i.e., sub-therapeutic dosages). A lower dosage minimizes the potential of side effects, thereby providing an increased margin of safety. Synergy can be in terms of lower cytotoxicity, increased antimicrobial effect, or some other beneficial effect of the combination compared with the individual components.
  • one or more kinase inhibitors are co-administered with an antibiotic selected from the group consisting of cephalosporin, penicillin, monobactam or carbapenem. In one embodiment, one or more kinase inhibitors are co-administered with a ⁇ - lactam antibiotic.
  • one or more kinase inhibitors are co-administered with an antibiotic selected from the group consisting of benzathine penicillin, benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V), procaine penicillin, methicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin, flucloxacillin, temocillin, amoxicillin, ampicillin, co- amoxiclav, azlocillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, cephalosporins, cephalexin, cephalothin, cefazolin, cefaclor, cefuroxime, cefamandole, cephamycins, cefotetan, cefoxitin, ceftriaxone, cefotaxime, cefpodoxime, cefixime, ceftazidime, cefepime, ce
  • Combination when used in reference to the administration of two or more compounds, mean that the compounds are administered to a subject concurrently.
  • Concurrent administration includes administration at the same time, in the same formulation or separately, and sequential administration in any order or at different points in time so as to provide the desired therapeutic effect.
  • the kinase inhibitor and antibiotic will be administered by the same route and in a single composition, so as to ensure that they are given simultaneously to the subject. In some embodiments, the kinase inhibitor and antibiotic will be administered by different routes and in separate compositions, for example to improve stability and/or efficacy.
  • a method of treating or inhibiting a bacterial infection in an animal comprising administering to the animal, for example a human, an effective amount of a kinase inhibitor or a combination of kinase inhibitors (as described herein) in combination with an effective amount of an antibiotic or a combination of antibiotics.
  • a method of treating or inhibiting a bacterial infection in an animal comprising co-administration of an effective amount of a kinase inhibitor or a combination of kinase inhibitors and an effective amount of an antibiotic or a combination of antibiotics to the animal.
  • a method of preventing or inhibiting bacterial infection in an animal comprising administering to the animal, for example a human, amounts of a kinase inhibitor or a combination of kinase inhibitors (as described herein) and an antibiotic or a combination of antibiotics that are effective to prevent infection or reduce the likelihood of infection.
  • the administration of an effective amount of a kinase inhibitor or a combination of kinase inhibitors in combination with an effective amount of an antibiotic or combination of antibiotics to a subject reduces the likelihood of infection by at least about or any number in between about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
  • a method of reducing the amount of antibiotic required to reduce, inhibit, or prevent bacterial proliferation by co-administration of a kinase inhibitor for example a serine/threonine kinase inhibitor, or a combination of kinase inhibitors.
  • a kinase inhibitor for example a serine/threonine kinase inhibitor, or a combination of kinase inhibitors.
  • Several embodiments relate to a method of reducing the amount of antibiotic required to reduce, inhibit, or prevent symptoms associated with bacterial infection, reduce the likelihood of bacterial infection, or prevent bacterial infection by administering an antibiotic or combination of antibiotics in combination with a kinase inhibitor or combination of kinase inhibitors.
  • the amount of antibiotic in an effective dose is reduced by about or any number in between about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or more.
  • a biological sample is obtained from a subject and the infective agent is identified and assayed for ⁇ -lactam antibiotic resistance. If a ⁇ -lactam antibiotic resistant infective agent is detected, the subject is treated with an effective amount of one or more kinase inhibitors as described herein and one or more ⁇ -lactam antibiotics. During the course of treatment, one or more biological samples may be obtained and assayed to monitor the levels of the infective agent and/or resistance of the infective agent to ⁇ -lactam antibiotics. Treatment may be concluded when a ⁇ -lactam antibiotic resistant infective agent is no longer detected in a biological sample obtained from the subject. In some embodiments, the amount of kinase inhibitor and/or antibiotic administered to the subject may be reduced once the levels of the infective agent fall below a threshold.
  • the term "effective amount” means an amount of compound, which upon administration, is capable of reducing or preventing proliferation of the bacteria, reducing or preventing symptoms associated with the bacterial infection, reducing the likelihood of bacterial infection, or preventing bacterial infection.
  • the subject is treated with an amount of a therapeutic composition of the invention sufficient to reduce a symptom of a disease or disorder by at least about or any number in between about 25%, 50%, 90% or 100%.
  • the actual amount of compound administered, the route of administration, and the frequency of administration will depend upon the particular disease or type of bacteria, virus or fungal infection, as well as other factors such as the size, age, sex, weight, health status, metabolic activity and ethnic origin of the individual being treated and is determined by routine analysis. Suitable dosages can readily be determined by one skilled in the art.
  • a suitable dosage is about 0.5 to 250 mg/kg body weight or more, e.g., about 1 to 10 mg/kg body weight, 10 to 20 mg/kg body weight, 20 to 30 mg/kg body weight, 30 to 40 mg/kg body weight, 40 to 50 mg/kg body weight, 50 to 60 mg/kg body weight, 60 to 70 mg/kg body weight, 70 to 80 mg/kg body weight, 80 to 90 mg/kg body weight, 90 to 100 mg/kg body weight, 100 to 110 mg/kg body weight, 110 to 120 mg/kg body weight, 120 to 130 mg/kg body weight, 130 to 140 mg/kg body weight, 140 to 150 mg/kg body weight, 150 to 160 mg/kg body weight, 160 to 170 mg/kg body weight, 170 to 180 mg/kg body weight, 180 to 190 mg/kg body weight, 190 to 200 mg/kg body weight, 200 to 210 mg/kg body weight, 210 to 220 mg/kg body weight, 220 to 230 mg/kg body weight, 230 to 240 mg/kg body weight,
  • a treatment regime may require administration over extended periods of time, e.g., for several hours, days or for from two to four weeks or more.
  • the amount per administered dose or the total amount administered will depend on such factors as the nature and severity of the infection, the age and general health of the recipient subject, the tolerance of the recipient subject to the kinase inhibitor or antibiotic and the type of the bacterial infection.
  • Dosage levels and time course of administration of the kinase inhibitor (as described herein) and/or antibiotic may be varied so as to obtain an amount of the kinase inhibitor and/or antibiotic which is effective to achieve a prophylactic or therapeutic effect for a particular subject, pharmaceutical formulation, and mode of administration, without being toxic to the subject.
  • Therapeutically effective blood levels of the kinase inhibitor and/or antibiotic may be achieved in one or more administrations, applications or dosage regimens. Determination of the dosing needed to achieve a therapeutically effective blood level for a given kinase inhibitor and/or antibiotic is within the ability of one of ordinary skill in the pharmaceutical arts.
  • Administration of the dose of kinase inhibitor and/or antibiotic can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals. Supplemental doses may be administered so as to maintain effective blood serum levels of kinase inhibitor and/or antibiotic during the entire treatment period.
  • an effective amount of a kinase inhibitor is administered as a single dose. In other embodiments, an effective amount of a kinase inhibitor is administered as a series of doses. Doses may be administered, for example, every 30 minutes, every hour, every 1.5 hours, every 2 hours, every 3 hours, every 3.5 hours, every 4 hours, every 5 hours, every 6 hours, every 7 hours, every 8 hours, every 9 hours, every 10 hours, every 11 hours, every 12 hours, every 13 hours, every 14 hours, every 15 hours, every 16 hours, every 17 hours, every 18 hours, every 19 hours, every 20 hours, every 21 hours, every 22 hours, every 23 hours, or every 24 hours.
  • kinase inhibitor is administered continuously, for example by IV drip.
  • an effective amount of kinase inhibitor is administered concomitantly with an antibiotic, in other embodiments, a kinase inhibitor and an antibiotic are administered alternately.
  • a loading dose amount of kinase inhibitor is administered for the first administration dose of the treatment.
  • the loading dose amount is higher than the dose amount administered for subsequent administrations in the treatment.
  • the loading dose amount is about double in quantity, of the amount in subsequent administrations in the treatment.
  • the first dose of kinase inhibitor administered at dosage of about 120 mg/kg and subsequent doses of kinase inhibitor are administered at a dosage of about 60 mg/kg.
  • the first dose of kinase inhibitor administered at dosage of about 240 mg/kg and subsequent doses of kinase inhibitor are administered at a dosage of about 120 mg/kg.
  • the first dose of kinase inhibitor administered at a dosage of about 480 mg/kg and subsequent doses of kinase inhibitor are administered at a dosage of about 240 mg/kg.
  • the first dose of kinase inhibitor administered at a dosage of about 680 mg/kg and subsequent doses of kinase inhibitor are administered at a dosage of about 340 mg/kg.
  • the first dose of kinase inhibitor administered at a dosage of about 880 mg/kg and subsequent doses of kinase inhibitor are administered at a dosage of about 440 mg/kg.
  • Further embodiments include any of the above-mentioned embodiments and where the loading dose concept in used for an antibiotic, e.g., the first dose of antibiotic administered is double in quantity to the subsequent doses.
  • the amount of kinase inhibitor (as described herein) and/or antibiotic may be reduced during the course of treatment. For example, once an effective blood serum level of kinase inhibitor and/or antibiotic is reached or maintained for a desired period of time, the amount of kinase inhibitor and/or antibiotic administered in subsequent doses may be decreased. In some embodiments, the amount of kinase inhibitor and/or antibiotic administered in subsequent doses may be reduced by about 5%, 10%, 15%, 20%>, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.
  • the kinase inhibitor may be selected from the compounds listed in Table 2, or a pharmaceutically acceptable salt thereof.
  • a compound inhibitor may be selected from the group consisting of ST081981 [N-benzyl-8-methoxy-N-phenylquinoline-5-sulfonamide], ST085384 [N-(l-benzylpiperidin-4- yl)-l -(naphthalene- l-ylsulfonyl)piperidine-3-carboxamide], ST085399 [N-(4-methylphenyl)-2- oxo- 1 ,2-dihydrobenzo[cd]indole-6-sulfonamide], ST085404 [2-oxo-N-(2-oxonaphtho[2, 1 - d][l,3]oxathiol-5-yl)-l,2-dihydrobenzo[cd]indole-6-s
  • kinase inhibitor for example, ST081981 [N-benzyl-8-methoxy-N- phenylquinoline-5 -sulfonamide], ST085384 [N-(l -benzylpiperidin-4-yl)- 1 -(naphthalene- 1 - ylsulfonyl)piperidine-3-carboxamide], ST085399 [N-(4-methylphenyl)-2-oxo-l,2- dihydrobenzo[cd]indole-6-sulfonamide], ST085404 [2-oxo-N-(2-oxonaphtho[2,l- d][l,3]oxathiol-5-yl)-l,2-dihydrobenzo[cd]indole-6-sulfonamide], ST085409 [4-ethoxy-N-(3- methylpyri
  • the pharmaceutical formulation further comprises an antibiotic, for example a ⁇ -lactam antibiotic.
  • the compounds of the present embodiments may be formulated into compositions together with pharmaceutically acceptable carriers for local or systemic administration.
  • the pharmaceutical formulations may be administered orally (including buccal, sublingual, inhalation), nasally, rectally, vaginally, intravenously, intradermally, subcutaneously and topically. Also administration from implants is possible.
  • Compounds may be formulated into compositions suitable for administration for example with suitable carriers, diluents, thickeners, adjuvants, etc., as are routine in the formulation art.
  • Pharmaceutical formulations may also include additional active ingredients. Dosage forms include solutions, powders, tables, capsules, gel capsules, suppositories, topical ointments and creams and aerosols for inhalation.
  • the kinase inhibitor described herein may be dissolved or suspended in saline, water, polyethylene glycol, propylene glycol, PBS, Tween-80, Cremaphor, ethanol, oil (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum, various buffers or various combinations thereof.
  • kinase inhibitor is dissolved or suspended in Cremophor:Ethanol:PBS.
  • Pharmaceutical formulations may also include ions and a defined pH.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, troches, tablets or SECs (soft elastic capsules or caplets). Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, carrier substances of binders may be desirably added to such formulations.
  • the use of such formulations has the effect of delivering the nucleic acid to the alimentary canal for exposure to the mucosa thereof.
  • the formulation can consist of material effective in protecting the compound from pH extremes of the stomach, or in releasing the compound over time, to optimize the delivery thereof to a particular mucosal site.
  • Enteric coatings for acid-resistant tablets, capsules and caplets are known and typically include acetate phthalate, propylene glycol and sorbitan monoleate.
  • compositions may be formulated in a conventional manner using additional pharmaceutically acceptable carriers or excipients as appropriate.
  • the composition may be prepared by conventional means with additional carriers or excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrates (e.g., starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art.
  • the preparations may be also contain flavoring, coloring and/or sweetening agents as appropriate.
  • compositions which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided soled carriers or both, and then, if necessary, shaping the product.
  • Formulations of the present embodiments suitable for oral administration may be presented as discrete units such as capsules, cachets or tables each containing predetermined amounts of the active ingredients; as powders or granules; as solutions or suspensions in an aqueous liquid or a non-aqueous liquid; or as oil-in-water emulsions or water-in-oil liquid emulsions.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredients therein.
  • compositions can be configured in various ways and in a variety of dosage forms to modify a dissolution rate of the kinase inhibitor and/or antibiotic.
  • the dissolution rate of the pharmaceutical formulation determines how quickly an active component, i.e. a kinase inhibitor or antibiotic, becomes available for absorption into the blood stream and therefore controls the bioavailability of the active component.
  • Dissolution rate is dependent on the size and the composition of the dosage form.
  • the dissolution rate of the pharmaceutical formulation can be changed by altering components of the formulation.
  • Disintegrants such as starch or corn starch, or crosslinked PVPs, can be used to increase solubility when desired.
  • Solubilizers can also be used to increase the solubility of the pharmaceutical formulations.
  • alternative binders such as hydroxypropylmethyl cellulose (HPMC), hydroxypropyl cellulose (HPC), methyl cellulose (MC), PVP, gums, xanthine, and the like, can be used to increase the dissolution rate of the kinase inhibitor and/or antibiotic.
  • HPMC hydroxypropylmethyl cellulose
  • HPC hydroxypropyl cellulose
  • HPC hydroxypropyl cellulose
  • MC methyl cellulose
  • PVP methyl cellulose
  • gums xanthine, and the like
  • the pharmaceutical formulation is a sustained-release pharmaceutical formulation.
  • a "sustained-release” formulation is a type of controlled-release formulation, wherein ingredients have been added to a pharmaceutical composition such that the dissolution profile is extended over a longer period of time than that of an immediate release formulation comprising a similar pharmaceutical composition.
  • Sustained-release pharmaceutical formulations can contain a variety of excipients, such as retardant excipients (also referred to as release modifiers) and/or fillers that are selected and incorporated into the formulation in such a way as to slow the dissolution rate of the formulation (and thereby slow the dissolution and/or release of the kinase inhibitor and/or antibiotic) under in vivo conditions as compared to an otherwise comparable immediate-release formulation.
  • a “comparable" immediate-release formulation is one that is substantially identical to the sustained-release formulation, except that that it is configured to provide immediate-release of a kinase inhibitor and/or antibiotic dose under substantially identical conditions.
  • a sustained-release pharmaceutical formulation comprises one or more retardant excipients.
  • retardant excipient is used herein in its ordinary sense and thus includes an excipient that is configured (e.g., incorporated into the formulation) in such a way as to control a dissolution profile of the drug, e.g., slow the dissolution of the kinase inhibitor and/or antibiotic in a standard dissolution test, as compared to an otherwise comparable pharmaceutical formulation that does not contain the retardant excipient.
  • Examples of pharmaceutically acceptable retardant excipients include hydroxypropyl methylcellulose (HPMC), hydroxyethylcellulose, hydroxypropylcellulose (HPC), methylcellulose, ethylcellulose, cellulose acetate butyrate, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, microcrystalline cellulose, corn starch, polyethylene oxide, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), cross-linked PVP, polyvinyl acetate phthalate, polyethylene glycol, zein, poly-DL-lactide-co-glycolide, dicalcium phosphate, calcium sulfate, and mixtures thereof.
  • the retardant excipient comprises a sustained-release polymer, e.g., at least one of hydroxypropyl methylcellulose (HPMC), hydroxyethylcellulose, hydroxypropylcellulose (HPC), methylcellulose, ethylcellulose, cellulose acetate butyrate, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, microcrystalline cellulose, corn starch, polyethylene oxide, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), cross-linked PVP, polyvinyl acetate phthalate, polyethylene glycol, zein, poly-DL-lactide-co-glycolide, and mixtures thereof.
  • Retardant excipients may be referred to herein as release modifiers.
  • the dissolution rate of the sustained-release pharmaceutical formulation is such that about 25% of the kinase inhibitor and/or antibiotic in the dosage form is dissolved within the first hour, about 60% of the kinase inhibitor and/or antibiotic is dissolved within the first 6 hours, about 80% of the kinase inhibitor and/or antibiotic is dissolved within the first 9 hours, and substantially all of the kinase inhibitor and/or antibiotic is dissolved within the first 12 hours.
  • the dissolution rate of the sustained-release pharmaceutical formulation is such that about 35% of the kinase inhibitor and/or antibiotic in the dosage form is dissolved within the first hour, about 85% of the kinase inhibitor and/or antibiotic is dissolved within the first 6 hours, and substantially all of the kinase inhibitor and/or antibiotic is dissolved within the first 9 hours.
  • the dissolution rate of the sustained-release pharmaceutical formulation in the dosage form is such that about 45% of the kinase inhibitor and/or antibiotic is dissolved within the first hour, and substantially all of the kinase inhibitor and/or antibiotic is dissolved within the first 6 hours.
  • a dosage form comprising an immediate- release pharmaceutical formulation of kinase inhibitor and/or antibiotic and a sustained-release pharmaceutical formulation of kinase inhibitor and/or antibiotic.
  • the immediate -release pharmaceutical formulation comprises a loading dose of kinase inhibitor and/or antibiotic.
  • a peak blood plasma level of kinase inhibitor and/or antibiotic is achieved within 2-4 hours after administration and then the blood plasma level of kinase inhibitor and/or antibiotic begin to fall through a protracted, substantially linear decrease from the peak plasma level for the reminder of the period, maintaining therapeutic level of the kinase inhibitor and/or antibiotic for about 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24 hours or more.
  • Some embodiments relate to injectable or intravenously administered formulations of one or more kinase inhibitors described herein and a pharmaceutically acceptable carrier (e.g., an aqueous carrier).
  • the injectable formulations further comprise one or more antibiotics.
  • the injection formulation may comprise one or more components selected from a free base, a lyoprotectant, an anti-oxidant, a pH adjustment compound, and a carrier.
  • lyoprotectant include, for example, sugars such as sucrose.
  • antioxidants include, but are not limited, to sodium bisulfite.
  • pH adjustment compounds include, but are not limited to, sodium bicarbonate, hydrochloric acid and sodium hydroxide.
  • a pharmaceutical formulation comprising one or more kinase inhibitors as described herein may also be in the form of a liposome, in which the kinase inhibitor is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids, which exist in aggregated forms as micelles, insoluble monolayers and liquid crystals.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is can be found in for example U.S. Patent No. 4,235,871.
  • a pharmaceutical formulation comprising one or more kinase inhibitors as described herein may also be in the form of biodegradable microspheres.
  • Aliphatic polyesters such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly(carprolactone) (PCL), and polyanhydrides have been widely used as biodegradable polymers in the production of microshperes. Preparations of such microspheres can be found in U.S. Patent No. 5,851 ,451.
  • a pharmaceutical formulation comprising one or more kinase inhibitors as described herein may also be in the form of polymer gels, where polymers such as starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone are used for thickening of the solution containing the kinase inhibitor.
  • the polymers may also comprise gelatin or collagen.
  • a pharmaceutical formulation as described above may be administered to a subject prophylactically to prevent or reduce the likelihood of contracting a bacterial infection or therapeutically to treat a bacterial infection, for example a MRSA infection.
  • a first dose of a pharmaceutical formulation as described above may be administered to a subject at the time of infection, immediately after infection, within 30 minutes of infection, within 1 hour of infection, within 1 to 2 hours of infection, within 2 to 3 hours of infection, within 1 to 2 hours of infection, within 2 to 3 hours of infection, within 1 to 2 hours of infection, within 2 to 3 hours of infection, within 3 to 4 hours of infection, within 4 to 5 hours of infection, within 5 to 6 hours of infection, within 6 to 7 hours of infection, within 7 to 8 hours of infection, within 8 to 9 hours of infection, within 9 to 10 hours of infection, within 10 to 11 hours of infection, within 11 to 12 hours of infection, within 12 to 24 hours of infection, within 24 to 36 hours of infection, within 24 to 48 hours of infection, within 48 to 72 hours of infection, or within 72 to
  • a subject may be treated with a pharmaceutical formulation as described above for a period of about or for any period between about 1-3 hours, 3-6 hours, 6-12 hours, 12-24 hours, 24-48 hours, 48-72 hours, 72-96 hours, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more.
  • a subject may be treated with a pharmaceutical formulation as described above until symptoms of infection subside.
  • a subject may be treated with a pharmaceutical formulation as described above until one or more biological samples obtained from the subject are free of the infectious agent.
  • An additional embodiment is directed to a kit comprising a packaging containing one or more doses of a pharmaceutical formulation as described above together with written instructions directing the administration of the pharmaceutical formulation.
  • the first dose comprises a loading dose of a kinase inhibitor and/or antibiotic.
  • the individual doses of the pharmaceutical formulation can be in any dosage form, e.g. tablets, capsules, solutions, creams, etc. and packaged within any of the standard types of pharmaceutical packaging materials, e.g. bottles, blister-packs, IV bags, syringes, etc., that may themselves be contained within an outer packaging material such as a paper/cardboard box.
  • kits further comprises one or more of culture media, culture plates, PCR primers, test strips, and stains for identifying the infective agent.
  • An additional embodiment is directed to a kit comprising a packaging containing one or more doses of a first pharmaceutical formulation comprising a kinase inhibitor described herein or a pharmaceutically acceptable salt thereof, and one or more doses of a second pharmaceutical formulation comprising an antibiotic, each together with written instructions directing the co-administration of the first pharmaceutical formulation and the second pharmaceutical formulation for the treatment of bacterial infection.
  • the first dose of the first pharmaceutical formulation comprises a loading dose of a kinase inhibitor.
  • the first dose of the second pharmaceutical formulation comprises a loading dose of an antibiotic.
  • the individual doses of the pharmaceutical formulations can independently be in any dosage form, e.g. tablets, capsules, solutions, creams, etc. and packaged within any of the standard types of pharmaceutical packaging materials, e.g. bottles, blister-packs, IV bags, syringes, etc., that may themselves be contained within an outer packaging material such as a paper/cardboard box.
  • the kit further comprises one or more of culture media, culture plates, PCR primers, test strips, and stains for identifying the infective agent.
  • the serine/threonine kinase Stkl affects resistance of MRS A to ⁇ -lactam antibiotics
  • Stkl substrates in MRS A were identified by deriving Astkl and Astpl mutants from MRSA LAC and MW-2 using methods as described in Burnside et al., PLoS ONE 5, el 1071 (2010).
  • Antibiotic susceptibility testing was performed using the standards established by the Clinical and Laboratory Standards Institute, CLSI (2008) Performance standards for antimicrobial susceptibility testing; 18th informational supplement.
  • the WT MSSA Newman strain showed increased susceptibility to ⁇ -lactams compared to WT MRSA. See Table 1.
  • MW-2 and LAC isolates deficient in Stpl showed a resistance to ⁇ -lactam antibiotics that was similar to the WT (Table 1), suggesting that Stkl regulation of antibiotic resistance may be independent of Stpl .
  • qRT-PCR revealed that transcription of mecA in Astkl (and Astpl) mutants of MW-2 and LAC was similar to the isogenic WT.
  • PEN Penicillin
  • AMP Ampicillin
  • CAZ Caftazidime
  • FEB Cefepime
  • IMI Imipenem
  • ETP Ertapenem
  • FOX Cefoxitin disc.
  • Minimal inhibitory concentration (MIC) was determined using Etest strips (AB Biodisk) or growth in liquid broth.
  • a TN5EZ insertion located in the transpeptidase domain (at amino acid 470) of the monocistronic pbp3 gene from the RN4220 transposon insertion library was transduced into MRSA MW-2.
  • Antibiotic susceptibility testing was performed as described in Example 1.
  • the MW-2 pbp3 transposon mutant showed a significant increase in sensitivity to ⁇ -lactams, similar to the stkl mutant (see MW-2pbp3::TN5EZ in Table 1).
  • a natural compound library consisting of kinase inhibitors was screened to determine whether inhibition of kinase activity increases the sensitivity of WT MRSA to ⁇ -lactam antibiotics.
  • a subset of Timtec's kinase library known as ActiTarg-K, which includes 280 drug-like, low molecular weight compounds that have the ability to inhibit protein kinases was selected.
  • WT MRSA (LAC) was grown in the presence of sub-lethal concentrations of Nafcillin (4 ⁇ g/ml) in the presence of 40 ⁇ g/ml of the respective kinase inhibitor in 96 well plates at 37°C overnight.
  • the toxicity of the kinase inhibitor ST085405 was evaluated in mice by injecting increasing doses of ST085405 dissolved in vehicle.
  • amounts of ST085405 leading to lOmg/kg and lOOmg/kg doses for a 20g mouse were dissolved in 500ul of a 1 :1 mixture of Tween-80 and Ethanol and stored at 4°C overnight.
  • Five mice were then injected intraperitoneally with either 500ul of vehicle (Tween-80 :Ethanol (1 : 1)) or 500ul of the lOmg/kg or lOOmg/kg doses of ST085405 and observed for signs of distress.
  • Cremophor:Ethanol:PBS 4 mice were injected with 50ul, lOOul, 150ul or 200ul of Cremophor:Ethanol:PBS vehicle and evaluated for signs of toxicity and survival. All 4 vehicle- injected mice survived and were healthy.
  • mice were divided into 3 groups.
  • ⁇ 50ul of blood was obtained from each of 10 mice via a submandibular bleed at 15 minutes, 45 minutes, 3 hours, 6 hours and 24 hours post injection.
  • Blood from 5 mice per group was pooled into 1.5ml Eppendorf tubes with or without ⁇ 10ul of heparin for a total of two tubes per dose group.
  • Blood with heparin was used for mass spectrometry (MS) analysis and blood without heparin was clotted and the serum was used for toxicology testing.
  • MS mass spectrometry
  • mice from each group were euthanized and the livers, kidneys and spleens were collected and placed in 1ml of 4% formalin for histopathology to be analyzed for signs of toxicity. Blood was also obtained from each mouse at 24 hours prior to euthanasia.
  • BUN blood urea nitrogen
  • alanine transaminase activity (ATA) and glutamic oxaloacetic transaminase activity (GOTA) were measured in the samples of pooled blood collected from the control, 10 mg/ml ST085405, and 100 mg/ml ST085405 groups at the various time points.
  • the dose of ST085405 administered did not correlate with elevated GOTA (Figure 8A) or ATA ( Figure 8B), indicating that liver function was not affected by administration of the kinase inhibitor.
  • the toxicity of the kinase inhibitor ST085384 was evaluated in mice by injecting intraperitoneally increasing doses of ST085384 dissolved in vehicle. Amounts of ST085384 leading tolO mg/kg and 100 mg/kg doses for a 20g mouse were suspended in 100 ul vehicle (Cremophor:Ethanol:PBS in a ratio of 1 : 1 :4) and extensively vortexed prior to being loaded into needles for injection.
  • the mice were monitored for 9 days for survival.
  • ⁇ 50ul-100ul of blood was obtained from each of 5 mice via a submandibular bleed at 15 minutes, 45 minutes, 3 hours, 6 hours and 24 hours post injection.
  • Blood from 2 mice per group was pooled into 1.5 ml Eppendorf tubes without heparin and blood from 3 mice per group was pooled into 1.5 ml Eppendorf tubes with ⁇ 10ul of heparin for a total of two tubes per dose group.
  • Blood with heparin was used for mass spectrometry (MS) analysis and blood without heparin was clotted and the serum was used for toxicology testing.
  • MS mass spectrometry
  • mice from each group were euthanized and the livers, kidneys and spleens were collected and placed in 1ml of 4% formalin for histopathology to be analyzed for signs of toxicity. Blood was also obtained from each mouse at 24 hours and day 9 prior to euthanasia.
  • alanine transaminase activity (ATA) and glutamic oxaloacetic transaminase activity (GOTA) were measured in the samples of pooled blood collected from the control, 10 mg/ml ST085384, and 100 mg/ml ST085384 groups at the various time points.
  • the dose of ST085384 administered did not correlate with elevated GOTA (Figure 8A) or ATA ( Figure 8B), indicating that liver function was not affected by administration of the kinase inhibitor.
  • Amounts of ST085405 or ST085384 leading to a lOOmg/kg dose for a 20g mouse were suspended in 200ul vehicle (Cremophor:Ethanol:PBS in a ratio of 1 : 1 :4) and extensively vortexed prior to being loaded into needles for injection.
  • mice were divided into 3 groups.
  • ⁇ 20ul-50ul of blood was obtained from each of 2 mice via a submandibular bleed at 15 minutes, 45 minutes, 3 hours, 6 hours and 24 hours post injection.
  • Blood from 1 mouse per group was collected into a 1.5 ml Eppendorf tube without heparin and blood from 1 mouse per group was collected into a 1.5 ml Eppendorf tube with ⁇ 10ul of heparin for a total of two tubes per dose group.
  • Blood with heparin was used for mass spectrometry (MS) analysis and blood without heparin was clotted and the serum was used for toxicology testing.
  • MS mass spectrometry
  • mice from each group were euthanized and the livers, kidneys and spleens were collected and placed in 1ml of 4% formalin for histopathology to be analyzed for signs of toxicity. Blood was also obtained from each mouse at 24 hours and day 9 prior to euthanasia.
  • PBP3 requires Stkl (i.e., phosphorylated PBP3 is absent in the Astkl mutant) and that Stkl phosphorylates PBP3 in vitro.
  • Stkl i.e., phosphorylated PBP3 is absent in the Astkl mutant
  • Stkl phosphorylates PBP3 in vitro.
  • the link between Stkl and PBP3 in conferring resistance to ⁇ -lactam antibiotics in MRSA is evaluated by examining antibiotic resistance in MRSA strains that a) lack PBP3 (Apbp3), b) lack both PBP3 and Stkl (Apbp3Astkl) or c) encode the non- phosphorylatable PBP3 T105A substitution.
  • MRSA (MW-2 and LAC) and MSSA (Newman) strains containing Apbp3 and Apbp3Astkl mutations are constructed as described in Burnside et al, PLoS ONE 5, el 1071 (2010).
  • MRSA and MSSA strains encoding the non-phosphorylated PBP3 T105A substitution are generated by amplifying > 1 Kb of DNA Flanking the 5' and 3' ends of amino acid 105 from WT MRSA using primers that contain the T ⁇ A amino acid substitution. The PCR products are cloned into the temperature sensitive vector pHY304, sequenced to confirm the presence of the substitution, and then transformed into Apbp3 MW-2, LAC and Newman strains.
  • the Apbp3 and double Apbp3Astkl mutant strains are expected to exhibit increased sensitivity to ⁇ -lactam antibiotics compared to the WT, similar to the increased sensitivity observed in Astkl mutants, indicating a link between Stkl and PBP3. Additionally, MRS A strains encoding the non-phosphorylatable PBP3 T105A substitution mutation are expected to exhibit antibiotic susceptibility similar to that of the Astkl and Apbp3 strains, indicating that phosphorylation of PBP3 is important for conferring resistance to the ⁇ -lactam antibiotics.
  • the mouse sepsis/kidney abscess model is used to evaluate virulence potential of MRSA MW-2 and LAC Astkl, Apbp3, and AstklApbp3 mutant strains.
  • the virulence potential of the MRSA MW-2 and LAC Apbp2a mutant strains is evaluated as a control.
  • Controls also include PBS mock-infected mice. Log rank tests are used to evaluate statistical significance of the morbidity data. Kidneys and other organs are harvested to enumerate bacterial CFU and histopathology is performed on infected renal tissue.
  • MSRA Apbp3 and Apbp2a mutant strains will exhibit similar virulence as the WT MRSA strains. If MSRA Apbp3 or Apbp2a exhibit attenuated virulence, the virulence of the complemented strains will be tested to determine if complementation restores virulence to WT levels.
  • mice peritonitis model of infection is used to determine if the absence of Stkl or PBP3 activity will render MRSA sensitive to ⁇ -lactam antibiotics in vivo.
  • peritoneal fluid (PF) and blood is obtained from 3 mice in each group including controls to evaluate bacterial CFU/mL.
  • PF and blood is obtained from 3 mice of each group including controls to evaluate CFU/mL of bacteria and concentration of the antibiotic as described.
  • Astkl MRS A will succumb to the infection and that the ED50 for nafcillin against WT MRS A infections will be >100mg/kg.
  • the ED50 for mice infected with Astkl, Apbp3 and the control Apbp2a MRS A and treated with nafcillin is expected to be significantly lower than lOOmg/kg, which is similar to the ED50 for mice infected with MSSA Newman and treated with nafcillin.
  • the number of bacterial CFU/mL in PF after nafcillin treatment is expected to be significantly lower in mice infected with Astkl, Apbp3 and control Apbp2a compared to WT MRS A.
  • Inhibitor doses include 1 mg/kg, 50 mg/kg, lOO mg/kg, 200 mg/kg and 250 mg/kg.
  • Controls include untreated groups of mice (i.e. lacking the inhibitor but containing equivalent amounts of the solvent in PBS) and mice inoculated only with PBS.
  • mice All groups of mice are continuously monitored for the initial 2 hr period and then every 8 hours for signs of morbidity (i.e., defective motor activity, muscle spasms, convulsions, writhing, sedation, hypnosis, ruffling of fur, not eating/drinking, lack of spontaneous movement, fatigue, labored breathing, lethargy or mice with significant weight loss, >10%) for a period of 48 -72 hours.
  • Moribund animals are euthanized using C02 asphyxiation as soon as they are detected. Morbidity data is analyzed and interpreted as percent survival of mice in each group over the three day period of observation. The dose at which 50% of the treated mice die (LD50) is determined. At the end of the experiment, all surviving animals are euthanized and necropsies are performed to determine if there are any gross pathological effects of kinase inhibitor administration.
  • LD50 dose at which 50% of the treated mice die
  • mice will not exhibit signs of morbidity.
  • the LD50 values of the tested kinase inhibitors are anticipated to be lower than the LD50 of staurosporine @ 6.5mg/kg. If the inhibitors do not exhibit toxicity at the above tested doses, the dosages of the inhibitors will be increased to identify the LD50 values.
  • LCMS liquid chromatography/mass spectrometry
  • the proteins are then precipitated from the plasma samples using acetonitrile and the supematants are analyzed for the presence of the queried inhibitors, ST081981, ST085384, ST085404, ST085405, ST085409, ST085397, and ST085399, and the internal standard using reverse-phase LC/MS coupled to a quadrupole mass spectrometer.
  • Standard curves are generated using varying amounts of ST081981, ST085384, ST085404, ST085405, ST085409, ST085397, and ST085399.
  • the amounts of the inhibitors are normalized to the levels of the internal standard and quantified using the standard curve. The experiment is repeated for reproducibility. The half life of the inhibitor (tl/2) and elimination or clearance from the plasma is estimated.
  • the mouse sepsis/kidney abscess model is used to evaluate virulence potential of MRSA treated with an effective amount of ST081981, ST085384, ST085404, ST085405, ST085409, ST085397, or ST085399.
  • mice are examined every 12 hours for signs of morbidity for two weeks. Controls also include MRSA infected mice that are concurrently or subsequently administered PBS. Log rank tests are used to evaluate statistical significance of the morbidity data. Kidneys and other organs are harvested to enumerate bacterial CFU and histopathology is performed on infected renal tissue.
  • mice peritonitis model of infection is used to determine if administration of kinase inhibitors will render MRSA sensitive to ⁇ -lactam antibiotics in vivo.
  • Control mice are injected with 0.5mL PBS+ 5% porcine gastric mucin.
  • peritoneal fluid (PF) and blood is obtained from 3 mice in each group including controls to evaluate bacterial CFU/mL.
  • PF and blood is obtained from 3 mice of each group including controls to evaluate CFU/mL of bacteria and concentration of the antibiotic kinase inhibitor.
  • ED50 effective dose
  • a human patient suffering from MRSA infection is identified.
  • a dosage of, for example, 2 g IV nafcillin is co-administered to the patient every 4 to 6 hours with an effective amount of ST085384 [N-(l-benzylpiperidin-4-yl)-l -(naphthalene- 1- ylsulfonyl)piperidine-3-carboxamide].
  • the dosage can be adjusted so that it is enough to be effective in reducing inflammation.
  • a human patient suffering from MRSA infection is identified.
  • a dosage of, for example, 0.5 g IV nafcillin is co-administered to the patient every 4 to 6 hours with an effective amount of ST085384 [N-(l-benzylpiperidin-4-yl)-l -(naphthalene- 1- ylsulfonyl)piperidine-3-carboxamide].
  • ST085384 N-(l-benzylpiperidin-4-yl)-l -(naphthalene- 1- ylsulfonyl)piperidine-3-carboxamide.
  • Patient is observed for reduction in signs of infection.
  • a human patient at risk for MRS A infection is identified.
  • An effective amount of ST085384 [N-(l-benzylpiperidin-4-yl)-l -(naphthalene- l-ylsulfonyl)piperidine-3- carboxamide] is co-administered with 500 mg ampicillin administered to the patient orally every 5-6 hours.
  • a human patient suffering from MRSA infection is identified.
  • a dosage of, for example, .5 g IV nafcillin is co-administered to the patient every 4 to 6 hours with an effective amount of ST085384 [N-(l-benzylpiperidin-4-yl)-l -(naphthalene- 1- ylsulfonyl)piperidine-3-carboxamide] and ST085404 [2-oxo-N-(2-oxonaphtho[2,l- d][l,3]oxathiol-5-yl)-l,2-dihydrobenzo[cd]indole-6-sulfonamide].
  • ST085384 N-(l-benzylpiperidin-4-yl)-l -(naphthalene- 1- ylsulfonyl)piperidine-3-carboxamide
  • ST085404 [2-oxo-N-(2-oxonaphtho

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Abstract

Staphylococcus aureus est une bactérie à gram positif constituant actuellement la principale cause d'infections invasives chez l'être humain. Si l'antibiothérapie est actuellement utilisée pour traiter les infections à S. aureus, l'apparition de souches résistantes aux antibiotiques telles que celles qui résistent à la méthicilline épuisent rapidement les options thérapeutiques. L'invention concerne donc des méthodes et des compositions destinées à renforcer la sensibilité d'agents pathogènes bactériens aux antibiotiques de la famille des β-lactamines. L'invention concerne également des gènes impliqués dans la modulation de la résistance aux antibiotiques qui constituent des cibles inédites pour les traitements destinés à inhiber les agents pathogènes bactériens résistants aux antibiotiques. L'invention concerne également des inhibiteurs de kinases démontrant une activité supérieure en termes de sensibilisation des agents pathogènes bactériens aux antibiotiques de la famille des β-lactamines.
PCT/US2012/050635 2011-08-15 2012-08-13 Inhibiteurs de kinases capables de renforcer la sensibilité d'agents pathogènes bactériens aux antibiotiques de la famille des b-lactamines Ceased WO2013066469A2 (fr)

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WO2015075166A1 (fr) * 2013-11-22 2015-05-28 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes et compositions pharmaceutiques pour traiter une infection bactérienne
EP4326696A4 (fr) * 2021-04-22 2025-06-25 Kayothera Inc. Composés hétérocycliques et leurs utilisations

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US20060070135A1 (en) * 1998-07-24 2006-03-30 Michel Tremblay Compositions and methods for inhibiting the enzymatic activity of PTP-1B
DE102005035741A1 (de) * 2005-07-29 2007-02-08 Merck Patent Gmbh Quadratsäurederivate
ES2497766T3 (es) * 2005-12-29 2014-09-23 Mitsui Norin Co., Ltd Composiciones y procedimientos de sensibilizar a oxacilina Staphylococcus aureus resistente a meticilina

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015075166A1 (fr) * 2013-11-22 2015-05-28 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes et compositions pharmaceutiques pour traiter une infection bactérienne
EP4326696A4 (fr) * 2021-04-22 2025-06-25 Kayothera Inc. Composés hétérocycliques et leurs utilisations

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