Patent Application: US-201515322838-A

Abstract:
a composition comprising as components a polypeptide impiα and / or a polypeptide impiα - fusion and at least one antibiotic compound , in particular an aminoglycoside antibiotic , and / or at least one bactericidal compound , wherein the polypeptides , the at least one antibiotic and the at least one bactericidal compound is present in the composition in concentrations which exhibit in combination a synergistic effect against resistant bacteria .

Description:
the inventors discovered that applying impiα , having for example the amino acid sequences of seq id nos : 10 , 18 , 20 , 22 , 24 , 26 , 28 , 30 , 32 , 34 , 36 , 38 , 40 , 42 , 44 , 46 , 48 , 50 , 52 , 54 , 56 , 58 , 60 , 62 , 64 , 66 , 68 , 70 , 72 , 74 , 76 , 78 , 80 , 82 , 84 , and impiα - fusions , having for example the amino acid sequences of seq id nos : 6 , 8 , 12 , 86 , 88 , 90 , 92 , reduce and stop growth of resistant bacteria at any stage of the infection , especially when applied in combination with at least one further bactericidal compound , surprisingly when the composition of the invention was applied at early stages of the infection . this observation is surprising because impiα interfere with m4 protease activity , for example with thermolysin , pseudolysin , aureolysin , vibriolysin , bacillolysin and npr599 , which are shed only at high bacterial concentrations , e . g . during or after biofilm formation as is the case for pseudomonas aeruginosa . therefore one has to expect that m4 protease inhibitors only interfere with bacteria after biofilm growth , as several publications on non resistant bacteria suggest . the inventors discovered , however , that impiα and impiα fusions or its combination with bactericidal compounds delays , stops or even reverses growth of resistant bacteria in a solution containing just non adherent bacteria . in this experiment , care was taken by visual inspection that biofilm formation and hence protease shedding had not yet started . more precisely it was observed that a delay of bacterial growth starts much earlier during the so called “ late log ” ( late logarithmic ) phase , when bacterial growth kinetics still follows a logarithmic function of time . the inventors also discovered that impiα and impiα fusions or its combination with at least one bactericidal compound effectively prohibits biofilm formation , even for resistant bacterial strains . furthermore the inventors discovered that combining impiα or impiα - fusions and antibiotics always inhibits growth of resistant bacteria synergistically , regardless of the antibiotics concentration and the stage of the infections . even at sub inhibitory antibiotic doses impiα or impiα - fusions interfere with bacterial growth . the inventors observed , for example , that the synthesis of the p . aeruginosa siderophore pyoverdine , known to be involved in the synthesis of virulence factors , is inhibited synergistically by impiα and the antibiotic gentamycin . thus , subject matter of the invention are compositions containing impiα or impiα - fusions and at least one further bactericidal compound , and the use of any impiα against partially or completely resistant or even multiresistant strains of bacteria , such as multiresistant staphylococcus aureus ( mrsa ) alone or in combination with antibiotics or other bactericidal compounds to treat patients or protect devices , especially implants . this use of impiα or impiα - fusions is particularly advantageous since it is bactericidal even without applying antibiotics in parallel , or even with very low doses of antibiotics . impiα or impiα - fusions may be used to affect bacteria being planktonic , isolated sessile or forming biofilms , at any stage of an infection including early stages . thus , bacteria resistant against antibiotics will still be affected by impiα . moreover , impiα can act synergistically with antibiotics in areas of the patient &# 39 ; s body where antibiotics concentrations are low due to , e . g ., rapid dilution or low drug influx related to low diffusion rate or mechanisms inducing active outflow of the drug . so by use of impiα and impiα fusions and combinations with bactericidal compounds according to the invention these areas cannot become areas where , due to sub inhibitory concentrations of the antibiotic , bacteria could respond to the challenge by developing resistance . further contemplated is the use of impiα or impiα - fusions with the impiα element exhibiting additional modifications , such as chemical modifications in the side chain or at the n and / or c terminal for improving biological or chemical properties such as bioavailability , stability , and effectivity . the modification may also provide for a detectable label , for example a chemiluminescent structural element , one or more radioactive isotopes in one or more side chains of an amino acid in the polypeptide , an enzyme which is able to generate a colour reaction and the like . a cystein , for example , may be added for linking a water soluble polymer such as polyethylene glycol , or other amino acids like lysine , cysteine , histidine , arginine , asparaginic acid , glutamic acid , serine , threonine , or tyrosin could also be used for coupling polymers to the peptide . another example is the insertion of tripeptide sequences nxt or nxs or fragments thereof with x designating any amino acid except p , which may be recognized by a cellular enzyme adding glycosylation elements . suitable , clinically acceptable , water soluble polymers include polyethylenglycol ( peg ) and polysialic acid ( psa ). impiα - fusions according to the invention comprise impiα and at least one polypeptide having a physiological function , in particular impi13 , an antibody or antibody fragment , scaffolds such as lipocalin , ankyrin , fibronectin , transferrin , tetranectin , adnectin , albumin , uteroglobin , or protein a , functional peptides such as transferrin , peptides useful for diagnostic applications , such as green fluorescent protein ( gfp ), or peptide tags enabling immobilization on technical surfaces , such as hexahistidine , or glutathione - s - transferase ( gst ). there are three super families ( cytosolic , mitochondrial , and mapeg ) of gsts : while classes from the cytosolic super family of gsts possess more than 40 % sequence homology , those from other classes may have less than 25 %. cytosolic gsts are divided into 13 classes based upon their structure : alpha , beta , delta , epsilon , zeta , theta , mu , nu , pi , sigma , tau , phi , and omega . mitochondrial gsts are in class kappa . the mapeg super family of microsomal gsts consists of subgroups designated i - iv , between which amino acid sequences share less than 20 % identity . human cytosolic gsts belong to the alpha , zeta , theta , mu , pi , sigma , and omega classes , while six isozymes belonging to classes i , ii , and iv of the mapeg super family are known to exist : an impiα fusion may also comprise a linker of 1 - 100 amino acids between impiα and the polypeptide . another subject matter of the invention are nucleic acids , especially single stranded rna , coding for impiα or impiα - fusion , which are administered into a patient and taken up by cells into their cytoplasm , where the cellular protein expression machinery expresses the impiα or impiα - fusion from the nucleic template . preferred are nucleic acids coding for an impiα - fusion , wherein the fused element comprises a signal peptide inducing secretion of the assembled and posttranslationally modified impiα - fusion protein . once secreted , the impiα - fusion protein acts in a manner similar to an impiα or impiα - fusion protein directly applied to the patient . nucleic acids according to the invention may be modified to resist degradation and improve delivery . useful modifications include lna ( locked nucleic acids ) or pna ( peptide nucleic acids ), and phosphodiester or phosphorothioate modified backbones . specific formulations for nucleic acid administration in a pharmaceutical composition include liposomes . the use of the polypeptide or fusion polypeptide comprising impiα according to the invention includes treating patients , such as humans or animals infected by microorganisms capable of secreting bacterial toxins of the m4 or metzincin family of metalloproteinases , in particular thermolysine , aureolysin , bacillolysin , pseudolysin , vibriolysin , msp peptidase , mpl peptidase , or anthrax npr599 . in another aspect of the invention , the simultaneous use of antibiotics or other bactericidal compounds , and impiα or impiα - fusions is provided at any time of infection , including early stages . simultaneous application may comprise dosing schemes with a delay between application of antibiotics and m4 protease inhibitors , a different application frequency or different and individually evolving dosings . these drug application schemes may prove beneficial for the patient or facilitate the application . bactericidal compounds amenable for use according to the invention include all antibiotics , such as listed in http :// en . wikipedia . org / wiki / list_of_antibiotics , for example . they further include antibodies like the anti - pseudomonas - pcrv antibody fab ′ fragment ( kb001 , kalos therapeutics , inc . ), and a fully human igg1 antibody highly specific for s . aureus exotoxin ( kbsa 301 , kenta biotech ltd .) subject matter of the invention are also the use of impiα or impiα fusions or a nucleic acid comprising a section coding for impiα or impiα - fusions in a suitable pharmaceutical composition and the use thereof to treat bacterial infections , especially in combination with antibiotics in a single pharmaceutical composition so that they are always applied simultaneously to the patient . another embodiment of the invention comprises the use of antibiotics or other bactericidal compounds in one pharmaceutical composition and of impiα or impiα - fusions or a nucleic acid comprising a section coding for impi or impiα or impiα - fusions in a separate one . the separate entities can be advantageous for treating patients since the doses relative to each other can be varied independently over time , as well as the individual frequency of administration . a delayed application of the two compositions may be beneficial to prohibit side effects to occur or to limit their strength . separate entities exhibit the further advantage that different formulations can be chosen , which may even be required for particular molecule combinations . subject matter of the invention are also particular dosing schemes , such as combining maximum doses for both , impiα and bactericidal compounds . another dosing scheme may include reduction of the applied dose of the bactericidal compound for some time , even down to sub inhibitory doses , where the combination of impiα and the bactericidal compound is still active . the benefit of such temporary dose regimen would be to encounter development of resistance against the compounds . impiα or impiα - fusions may be combined with ingredients to form a pharmaceutical composition . the pharmaceutical composition may include water and salts at physiological concentrations , solubilizing or dispersing agents , or anti - oxidant , or particles forming micelles , such as liposomes . this pharmaceutical composition may be filled in a glass or plastic vials , or in a syringe . the pharmaceutical composition may also contain additives supporting drying or freeze - drying of the pharmaceutical composition , for example cyclodextrins or saccharides , in particular disaccharides . impiα or impiα - fusions or nucleic acids encoding for impiα or impiα - fusions and combinations with bactericidal compounds may be administered parenterally , orally , or topically using suitable pharmaceutical compositions , or attached to a patch or wound debridement from where the medication elutes into a wound of the patient . impiα or impiα - fusions or nucleic acids encoding for impiα or impiα - fusions and combinations with bactericidal compounds may be administered in biodegradable containers suitable for implantation into patients , or a reservoir attached or included in a device may contain impiα or impiα - fusions actively or passively deployed so that the device is situated in an area with known high load of target bacteria . a method for tracking edna ( extracellular dna ) was derived and compiled partly from similar procedures and conditions found in the literature and modified by the inventors . the reference strain p . aeruginosa ( dsm no . 50071 ; mic 8 mg / liter ), partially resistant to gentamycin , was studied in parallel with a clinical isolate of p . aeruginosa pao1 ( dsm no . 19880 ) to evaluate edna accumulation over time in the presence and absence of impi . first the strains were cultured in nb medium ( nutrient broth no . 4 ) overnight at 37 ° c . and grown to stationary phase . from these bacteria , 5 μl were used to inoculate 96 - well black flat bottom plates ( greiner ) containing nb medium ( 200 μl ). the nb medium contained either double concentrated nb medium ( 2 ×; 100 μl ) diluted with 100 μl tbs - buffer ( negative control ) or medium diluted with impi , which was previously solved in tbs ( 100 μl ; positive control ). the final concentration of impi per well was 35 μm for experiments with p . aeruginosa dsm 50071 and dsm 19880 respectively . to stain extracellular dna and membrane - compromised ( dead ) bacteria in aggregates of p . aeruginosa , 1 μl of 1 mm stock solution of bobo - 3 stain ( life technologies ) was added to 5 ml cultures at the start of growth experiments ( incubation in the dark ). bobo - 3 is a membrane - impermeable fluorescent dye ( λ ex 570 , λ em 602 ) that binds to dna and therefore specifically stains extracellular dna which images were taken over 48 h by a high - definition area scan ( 99 × 99 points ) on a well of a 96 - well microplate using a synergy h4 plate reader ( biotek ). gentamycin mic ( minimal inhibitory concentration ) values were determined using a standard two fold microtiter broth dilution protocol with nutrient broth as medium . midexponential phase cultures of p . aeruginosa reference strain dsm no . : 50071 and two antibiotic - resistant clinical isolates ( p . aeruginosa vb7623 and vb7444 ), which were isolated from the tracheal secrete of a patient , were tested . antimicrobial susceptibility testing of the clinical isolates was performed by the university clinic tubingen . typically gentamicin concentrations between 8 μg / ml and 0 . 0625 μg / ml were chosen to estimated bacterial growth and determine the mic values . further , the od ( optical density ) at 600 nm in the presence and absence of the insect metalloprotease inhibitor impi was investigated . for assessing influences of impi on bacterial growth final concentrations of impi between 20 and 75 μm were used . bacterial growth was monitored at 37 ° c . over 48 h . all mic values were done as triplicates . the subsequent table lists the sequences printed in the ensuing sequence protocol . the leading number denotes the seq id no for the nucleotide sequence , the subsequent even number missing number would denote the seq id no of the respective peptide sequence . 95 : impi like ( solenopsis , peptide only , no nucleotide sequence provided ) aloush , v ., et al ., multidrug - 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160 el solh , a . a . and a . alhajhusain , update on the treatment of pseudomonas aeruginosa pneumonia . the journal of antimicrobial chemotherapy , 2009 . 64 ( 2 ): p . 229 - 38 . strateva , t . and d . yordanov , pseudomonas aeruginosa — a phenomenon of bacterial resistance . journal of medical microbiology , 2009 . 58 ( pt 9 ): p . 1133 - 48 . wedde m , weise c , kopacek p , franke p , vilcinskas a . purification and characterization of an inducible metalloprotease inhibitor from the hemolymph of greater wax moth larvae , galleria mellonella . eur j biochem 1998 ; 255 : 534 - 43 zimlichman , e ., et al ., “ health care - associated infections : a meta - analysis of costs and financial impact on the us health care system .” jama intern med , 2013 .