Patent Application: US-200913131563-A

Abstract:
the present invention relates generally to the field of antibacterials and to the treatment , inhibition and prevention of disorders caused by bacteria . more particularly , this 5 invention concerns a method for inhibiting gram - positive bacteria with a composition comprising at least one non - phospholpid lipid vesicle .

Description:
as used herein , the terms “ liposome ” and “ lipid vesicle ” are used interchangeably to designate a small sphere made of lipid shells enclosing a central cavity mostly composed of an aqueous volume . the lipids are in the form of bimolecular layers , or lamellae , in an onion - like structure . non - phospholipid lipid vesicle ( nplv ) refers to vesicles that are spherical structures made of material having high lipid content . these lipids are preferably comprised of non - phospholipids that can be selected from the group comprising the compounds set forth below in table 2 . the terms “ unilamellar ”, “ paucilamellar ”, “ multilamellar ”, as used herein , refer to the number of peripheral bilayers surrounding the central cavity of the liposome , in particular the nplv of the invention . a unilamellar nplv consists of one peripheral bilayer surrounding the central cavity whereas a multilamellar nplv consists of more than 2 peripheral bilayers . paucilamellar nplv , which can be considered as a sub - class of the multilamellar nplv , consists of 2 to 8 peripheral bilayers . the molecular bilayers of nplvs have a physical structure similar to classical phospholipid bilayers . for example , it has been shown that x - ray diffraction of c 16 ( peg ) 2 ether vesicles showed a simple and principal reflection , representing the thickness of a hydrated , double layer ( 5 , 8 - 6 , 1 nanometers ) of amphiphile , with smaller spacing at higher cholesterol levels — fully analogous to phospholipid bilayers . the spacing of 6 . 1 nanometers corresponds to the maximum extension of two amphiphile molecules plus a layer of bound water ( mitchell , et al . 1983 ; adam et al . 1984 ). lantzsch et al . ( 1996 ) used fluorescent transfer techniques to determine the surfaces of surfactant type c 12 ( peg ) n in 1 - palmitoyl - 2 - oleoyl phosphatidylcholine / c 12 ( peg ) 1 - 8 liposomes . for n = 1 - 3 , the expansion of surface is equivalent to a liquid - crystalline hydrocarbon phase per molecule of c 12 ( peg ) n . for n = 4 - 8 , the surface area per molecule of surfactant increased gradually , suggesting a rolled up configuration of the incorporated molecules , with two water molecules per ethylene glycol segment . further , aqueous dispersions of 1 , 2 - tetradecyl or 1 , 2 - hexadecyl phosphatidylcholine accept large proportions of c 16 ( peg ) 4 ( mädler et al ., 1998 ). as used herein , the terms “ to interact ” and “ interacting ” are meant as having an effect one on another either by direct contact or at a distance . surprisingly , the applicants have found that compositions comprising non - phospholipid lipid vesicles can inactivate gram - positive bacteria . this is surprising given that the effect of non - phospholipid lipid vesicles on viruses was mediated by membrane lipid exchange between non - phospholipid lipid vesicles and viral envelope , while gram - positive bacterium have a cell wall composed of peptidoglycan . examples of gram - positive bacteria species include , but are not limited to , micrococcus , peptococcus , peptostreptococcus , enterococcus , bacillus , clostridium , lactobacillus , listeria , erysipelothrix , propionibacterium , eubacterium , and corynebacterium . gram positive cocci include , for example , streptococcus ( e . g . streptococcus pneumoniae ), staphylococcus ( e . g . staphylococcus aureus ), and enterococcus . gram positive rods include , for example , corynebacteria ( i . e . corynebacterium diphtheriae ), listeria monocytogenes , bacillus anthracis ( i . e . anthrax ). gram positive branching organisms include , for example , actinomycetes . gram positive bacteria include : bacillus anthracis ; bacillus subtilis ; clostridium botulinum ; clostridium perfringens ; clostridium tetani ; corynebacterium diphtheriae ; lactobacillus spp . ; listeria monocytogenes ; mycobacterium leprae ; mycobacterium tuberculosis ; mycoplasma pneumoniae ; staphylococcus aureus ; streptococcus spp . mycobacterium stain acid - fast but phylogentically are more closely related to the gram - positives than they are to the gram - negatives . mycoplasma stain gram - negative but phylogentically are more closely related to the gram - positives than they are to the gram - negatives ( it is their lack of a cell wall which leads to this confusing state ). additional gram positive bacteria are listed , for example , in volume 2 of bergey &# 39 ; s manual , which contains six sections covering all gram - positive bacteria except the actinomycetes . bacteria are distributed among these sections on the basis of their shape , the ability to form endospores , acid fastness , oxygen relationships , the ability to temporarily form mycelia , and other properties . exemplary gram - positive bacteria relevant in human medicine also include those listed in table 1 below . a variety of gram - positive organisms are capable of causing sepsis . the most common organisms involved in sepsis are staphylococcus aureus , streptoccocus pneumoniae , coagulase - negative staphylococci , beta - hemolytic streptococci , and enterococci , but any gram - positive organism may be involved . ( see , e . g ., bone , ( 1993 ) j . critical care 8 : 51 - 59 ). thus , it is contemplated that the subject compositions and methods can be used as part of a therapeutic treatment or prevention program for sepsis involving gram - positive bacteria . it is further contemplated that the subject compositions and methods can be used as part of a therapeutic treatment or prevention program for bacteria infections caused by drug - resistant bacteria . examples of such drug - resistant bacteria include methicillin - resistant staphylococci ( mrsa ), methicillin resistant staphylococcus epidermidis ( mrse ), methicillin resistant coagulase negative staphylococci ( mrcns ), vancomycin resistant enterococcus faecalis , vancomycin resistant enterococcus faecium , and vancomycin - resistant s . aureus which have become resistant to virtually all currently used antibiotics . non - phospholipid lipid vesicle ( nplv ) disclosed herein preferably comprised of non - phospholipids that can be selected from the group comprising the compounds set forth below in table 2 . more preferably , the non phospholipids of the invention are selected from the group consisting of polyoxyethylene cetyl ether ( pce ), palmitic acid ( pa ), hexadecyl trimethylammonium bromide ( htab ), dimethyldihexadecyl ammonium bromide ( dhab ) and oleic acid ( oa ), either alone or in combination . in one embodiment , the nplv comprises a lipid comprising a quaternary ammonium head group . such a quaternary ammonium containing lipid can be represented by the structure of nr 4 + b − , wherein nr 4 + represents a quaternary ammonium cation , b − represents an anion , and r can be same or different alkyl groups . quaternary ammonium lipids are salts of quaternary ammonium cations with an anion . quaternary ammonium cations can be synthesized by methods known in the art , for example , by complete alkylation of ammonia or other amines . many quaternary ammonium lipids can be obtained commercially . exemplary quaternary ammonium lipids include hexadecyl trimethylammonium bromide ( htab ), dimethyldihexadecyl ammonium bromide ( dhab ), cetrimonium bromide , cetyl trimethylammonium bromide ( ctab ), hexadecyltrimethylammonium bromide , cetrimonium chloride , cetyl trimethylammonium chloride , hexadecyltrimethylammonium chloride , cocamidopropyl betaine ( capb ), tetra - n - butylammonium bromide ( tbab ). in one embodiment , the quaternary ammonium lipid includes hexadecyl trimethylammonium bromide ( htab ) and dimethyldihexadecyl ammonium bromide ( dhab ). the ratio ( e . g ., molar ratio ) of the non - quaternary ammonium lipid in the non - phospholipid lipid vesicle ranges from about 0 . 01 % to about 1 %, preferably from about 0 . 1 % to about 0 . 5 %, more preferably from about 0 . 1 % to about 0 . 3 %. in one embodiment , the ratio is about 0 . 3 %. in another embodiment , the nplvs can be cholesterol - free ( or substantially free of cholesterol ), i . e . it does not comprise cholesterol ( or , respectively , only traces of cholesterol ), cholesterol derivatives such as for example peg cholesterol , ionogenic cholesterol and surface stabilizing cholesterol , beta - sitosterol , ergosterol and phytosterol . in one embodiment , the composition comprising the nplv can comprise an additional agent . in a particular embodiment , this agent is added to the composition and can be a cyclodextrin or a steroidogenic acute regulatory protein ( star ). preferably , the agent is a cyclodextrin or a derivative thereof . cyclodextrins ( cds ) are cyclic oligomers of glucose that can form water - soluble inclusion complexes with small molecules and portions of large compounds . chemically they are cyclic oligosaccharides containing at least 6 d -(+) glucopyranose units attached by α -( 1 , 4 ) glucosidic bonds . there are 3 natural cds , α -, β -, and γ - cds , which differ in their ring size and solubility . these biocompatible , cyclic oligosaccharides do not elicit immune responses and have low toxicities in animals and humans . cyclodextrins are used in pharmaceutical applications for numerous purposes , including improving the bioavailability of drugs . β - cd can selectively extract cholesterol from cellular membranes . high concentrations of β - cd show cellular toxicity and can induce either cell lysis or cellular cell death ( apoptosis ). derivatives of cd are disclosed in u . s . pat . no . 5 , 760 , 017 ( inventors : djedaini - pilard et al .) and international application wo91 / 13100 ( inventors : coates et al . ), the disclosure of which is also incorporated herein by reference . examples of cd derivatives comprise , but are not limited to dimethyl - β - cyclodextrin , trimethyl - β - cyclodextrin , randomly methylated - β - cyclodextrin , hydroxyethyl - β - cyclodextrin , 2 - hydroxypropyl - β - cyclodextrin , 3 - hydroxypropyl - β - cyclodextrin , 2 , 3 - dihydroxypropyl - β - cyclodextrin , 2 - hydroxyisobutyl - β - cyclodextrin , sulphobutylether - β - cyclodextrin , glucosyl - β - cyclodextrin and maltosyl - β - cyclodextrin . usually , the agent is added to the composition . to this end a suitable concentration of cd is prepared in water or pbs and added to the composition so as to obtain the required concentration . preferably , the concentration of cyclodextrin or cyclodextrin derivatives in the composition of the invention is between 0 . 01 mm and 50 mm . most preferably , this concentration is between 0 . 1 mm and 10 mm . at such a low concentration , β - cd has limited effect on cellular integrity . alternatively , the agent can be the steroidogenic acute regulatory protein ( star ). the steroidogenic acute regulatory ( star ) protein is an essential component in the regulation of steroid biosynthesis in adrenal and gonadal cells through camp - dependent pathways . in many cases transcriptional induction by camp is mediated through the interaction of a camp response - element binding protein ( creb ) family member with a consensus camp response element ( cre ; 5 ′- tgacgtca - 3 ′) found in the promoter of target genes ( pulak r . manna et al . ( 2002 ), molecular endocrinology 16 ( 1 ): 184 - 199 ). a truncated form of the star ( e . g ., n62 star ; tuckey et al ., 2002 and alpy et al ., 2005 ) is also envisioned for use as the additional agent . this truncated form of star has been shown enhancing the cholesterol transfer between phospholipid - liposomes by a factor of 5 to 10 times . preferably , in such as case the star protein is also in solution in the composition . the composition may be in a liquid , solid ( such as powder . . . ), or semi solid state . usually , the nplv of the composition has a diameter comprised between 0 . 2 μm to 10 μm . methods of manufacturing nplvs , and the nplvs of the invention , are also described in more detail in example 1 or in u . s . pat . no . 4 , 911 , 928 , u . s . pat . no . 5 , 147 , 723 , u . s . pat . no . 5 , 032 , 457 , u . s . pat . no . 4 , 895 , 452 , u . s . patent application ser . no . 761 , 253 , and wo 2007 / 135523 , the disclosures of which are all incorporated herein by reference in their entirety . also encompassed by the present invention is a pharmaceutical composition characterized in that it comprises a pharmaceutically acceptable amount of the nplvs disclosed herein , optionally in combination with one or more pharmaceutically acceptable carriers . in a further embodiment , the pharmaceutical composition further comprises an enhancer agent as described above . preferably , the enhancer agent is at least one cyclodextrin or derivatives thereof . the phrase “ pharmaceutically acceptable ” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction , such as gastric upset , dizziness and the like , when administered to a human . acceptable carriers , excipients , or stabilizers are non - toxic to recipients at the dosages and concentrations employed , and include buffers such as phosphate , citrate , and other organic acids ; antioxidants including ascorbic acid and methionine ; preservatives ( such as octadecyldimethylbenzyl ammonium chloride ; low molecular weight ( less than about 10 residues ) polypeptides ; proteins , such as serum albumin , gelatin , or immunoglobulins ; hydrophilic polymers such as polyvinylpyrrolidone ; amino acids such as glycine , glutamine , asparagine , histidine , arginine , or lysine ; monosaccharides , disaccharides , and other carbohydrates including glucose , mannose , or dextrins ; chelating agents such as edta ; sugars such as sucrose , mannitol , trehalose or sorbitol ; salt - forming counter - ions such as sodium ; metal complexes . the form of administration of the pharmaceutical composition may be systemic or topical . for example , administration of such a composition may be various parenteral routes such as subcutaneous , intravenous , intradermal , intramuscular , intraperitoneal , intranasal , transdermal , buccal routes , preferentially by inhalation , or via an implanted device , and may also be delivered by peristaltic means . the pharmaceutical composition , as described herein , may also be incorporated or impregnated into a bioabsorbable matrix , with the matrix being administered in the form of a suspension of matrix , a gel or a solid support . in addition the matrix may be comprised of a biopolymer . in case the formulations to be used for in vivo administration must be sterile , this is readily accomplished for example by using sterile compounds for the preparation of the composition of the invention . it is understood that the suitable dosage of the pharmaceutical composition of the present invention will be dependent upon the age , sex , health , and weight of the recipient , kind of concurrent treatment , if any and the nature of the effect desired . the appropriate dosage form will depend on the disease , the nplv , the enhancer agent and the mode of administration ; possibilities include a spray or other aerosol means of delivery to the respiratory passages . other ways of topically applying the inhibiting solution include creams , mouthwashes , dental pastes , eye drops , solutions , ointments , gels such as vaginal gels , and lubricants such as condom lubricants . an optional dose of the compound for treatment of a bacterial ( such as a gram positive bacteria ) infection is about 1 to 50 mg / kg , or 1 to 20 mg / kg , of body weight per day , more generally 0 . 1 to about 100 mg per kilogram body weight of the recipient per day . the effective dosage range of the pharmaceutically acceptable salts and prodrugs can be calculated based on the weight of the parent nucleoside to be delivered . if the salt or prodrug exhibits activity in itself , the effective dosage can be estimated as above using the weight of the salt or prodrug , or by other means known to those skilled in the art . optionally , the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0 . 2 to 70 m , e . g ., about 1 . 0 to 10 um . this may be achieved , for example , by the intravenous injection of a 0 . 1 to 5 % solution of the active ingredient , optionally in saline , or administered as a bolus of the active ingredient . the concentration of active compound in the drug composition will depend on absorption , inactivation and excretion rates of the drug as well as other factors known to those of skill in the art . it is to be further understood that for any particular subject , specific dosage regimens should be adjusted according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions , and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition . the active ingredient may be administered at once , or may be divided into a number of smaller doses to be administered at varying intervals of time . the compound is conveniently administered in unit any suitable dosage form , including but not limited to one containing 7 to 3000 mg , or 70 to 1400 mg of active ingredient per unit dosage form . a dosage of 50 - 1000 mg is optional . the present invention also provides a method of preventing , inhibiting or treating a bacterial infection , in a host , for example an animal , and typically a human , comprising administering a therapeutic amount of a nplv composition of the present invention , optionally in a pharmaceutically acceptable carrier or diluent where the bacterial infection is due to a gram - positive bacteria . the present disclosure also provides a method for inhibiting gram - positive bacteria comprising the step of contacting said gram - positive bacteria with a composition comprising one or more nplvs . inhibition of bacterial replication or treatment of an infection in a cell can be measured by showing a reduction in bacterial replication in a cell to a level lower than the level in an otherwise identical cell , which was not administered the compound or composition of the invention . the reduction can be by about 80 %, 85 %, 90 %, 95 %, about 99 . 9 % or more . the level of bacterial replication in a cell can be assessed by any known methods . for example , the level of bacterial replication in a cell can be assessed by evaluating the number of bacterial particles or amount of a bacterial component , such as a bacterial protein , a bacterial enzyme , or bacterial nucleic acid , in the cell or in fluid or debris associated with the cell . the number of infectious bacteria in a cell can be evaluated , for example , in a plaque assay . the level of a bacterial component such as a bacterial protein or enzyme in a cell can be evaluated using standard analytical techniques of protein biochemistry , such as , for example , using an activity assay for a bacterial enzyme , or using western blotting or quantitative gel electrophoresis for a bacterial protein . bacterial nucleic acid levels in a cell can be evaluated using standard analytical techniques such as northern blotting and southern blotting or quantitation by polymerase chain reaction ( pcr ). nplvs that can be used in the methods disclosed herein include those that are disclosed above . in one embodiment , the nplvs used for inhibiting gram - positive bacteria comprise a quaternary ammonium head group . such quaternary ammonium containing lipids are disclosed above . in another embodiment , the quaternary ammonium containing lipids contained in the nplvs include hexadecyl trimethylammonium bromide ( htab ) and dimethyldihexadecyl ammonium bromide ( dhab ). in a further embodiment , the nplv can comprise an additional agent which is added to the composition . in a particular embodiment , this agent can be a cyclodextrin or a steroidogenic acute regulatory protein ( star ). preferably , the agent is a cyclodextrin or a derivative thereof . cyclodextrins or derivatives thereof are disclosed above . in still a further embodiment , the nplvs can be cholesterol - free . the gram - positive bacteria that can be inhibited by the nplvs include those disclosed above , such as staphylococcus , streptococcus , micrococcus , peptococcus , peptostreptococcus , enterococcus , bacillus , clostridium , lactobacillus , listeria , erysipelothrix , propionibacterium , eubacterium , and corynebacterium , streptococcus ( e . g . streptococcus pneumoniae ), staphylococcus ( e . g . staphylococcus aureus ), and enterococcus . gram positive rods include , for example , corynebacteria ( i . e . corynebacterium diphtheriae ), listeria monocytogenes , bacillus anthracis ( i . e . anthrax ) and erysipelothrix rhusiopathiae . gram positive branching organisms include , for example , actinomycetes . gram positive bacteria include : bacillus anthracis ; bacillus subtilis ; clostridium botulinum ; clostridium perfringens ; clostridium tetani ; corynebacterium diphtheriae ; lactobacillus spp . ; listeria monocytogenes ; mycobacterium leprae ; mycobacterium tuberculosis ; mycoplasma pneumoniae ; staphylococcus aureus ; streptococcus spp . mycobacterium stain acid - fast but phylogentically are more closely related to the gram - positives than they are to the gram - negatives . mycoplasma stain gram - negative but phylogentically are more closely related to the gram - positives than they are to the gram - negatives ( it is their lack of a cell wall which leads to this confusing state ). additional gram positive bacteria are listed , for example , in volume 2 of bergey &# 39 ; s manual , which contains six sections covering all gram - positive bacteria except the actinomycetes . bacteria are distributed among these sections on the basis of their shape , the ability to form endospores , acid fastness , oxygen relationships , the ability to temporarily form mycelia , and other properties . in one embodiment , the gram - positive bacteria that can be inactivated includes those relevant in human medicine ( e . g ., those listed in table 1 ). in particular embodiment , the nplvs can be used to inactivate drug - resistant bacteria including drug - resistant bacteria include methicillin - resistant staphylococci ( mrsa ), methicillin resistant staphylococcus epidermidis ( mrse ), methicillin resistant coagulase negative staphylococci ( mrcns ), vancomycin resistant enterococcus faecalis , vancomycin resistant enterococcus faecium , and vancomycin - resistant s . aureus . minimum inhibitory concentration ( mic ) is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation . generally , mics can be determined by agar or broth ( e . g ., lb broth ) dilution methods . a standard method of determining mic for a composition comprising nplvs includes : growing inoculums of bacteria ( e . g ., 5 × 10 5 bacteria / ml ) overnight in lb broth medium in presence of various concentration of a composition comprising nplvs , each culture sample is then serially diluted and plated on an agar plate , the agar plate is then incubated overnight at 37 ° c . for titer determination . disclosure of such a method can be found in “ determination of minimum inhibitory concentrations ” by jennifer m . andrews in journal of antimicrobial chemotherapy ( 2001 ) 48 , suppl . s1 , 5 - 16 . in addition , guidelines determination of mics can be found from organizations such as the clinical and laboratory standards institute ( clsi ), the british society for antimicrobial chemistry ( bsac ) or the european society of clinical microbiology and infectious disease ( eucast ). there are also several commercial methods available , including the etest strips and the oxoid micevaluator method . mics are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent . clinically , the minimum inhibitory concentrations can be used to determine the amount of antibiotic that a patient will receive , as well as the type of antibiotic used . such determinations can lower the opportunity for microbial resistance to specific antimicrobial agents . mics for nplvs against various gram - positive bacteria can be determined using standard methods described above or other methods known in the art . these mics can be used to determine the minimum amount of the respective nplv that can be used for inhibiting gram - positive bacteria . the present disclosure further provides a method of treating or preventing a disease associated with gram - positive bacteria in a subject comprising the step of administering to said subject the pharmaceutical composition as described herein , to a location proximate to said gram - positive bacteria . possible modes of delivery include a spray or other aerosol means of delivery to the respiratory passages , creams , mouthwashes , dental pastes , solutions , ointments , gels such as vaginal gels , eyes drops and lubricants such as condom lubricants , creams , lotions , or solutions for external application to skin and mucosal surfaces , including the cornea , dermal cuts and abrasions , burns , and sites of bacterial infection . interaction of the composition of the invention with gram - positive bacteria in the airways blocks bacteria infectivity , thereby , propagation in the lungs ( individual prophylaxis - treatment ). physiological processes flush out the composition and inactivated bacteria . moreover , being partially or completely inactive , bacteria are limited in their spread in the surrounding population when expelled by coughing or sneezing ( population prophylaxis ). the term “ treating ” refers to therapeutic treatment , while the term “ preventing ” refers to prophylactic or preventative measures . those ( subjects ) in need of treatment may include those already with a disorder as well as those in which a disorder is to be prevented . the subject to be treated herein may have been diagnosed as having a disorder or may be susceptible to a disorder . most preferably the term animal refers to domestic and farm animals ( e . g . poultries ), and zoo , sports , or pet animals , such as dogs , horses , pigs , cats , cows , monkeys etc . . . . embraced by the scope of the present invention is also the use of the pharmaceutical composition , as described herein , in the preparation of a medicament for the treatment or prevention of a gram - positive bacteria - associated disease . also encompassed in the present invention is the use of the composition of the invention in the preparation of a large - scale biocompatible disinfectant . accordingly , the composition of the invention is diluted as needed to prepare simple aqueous suspensions , or formulated as dispersions in hydrogels , pastes or sprays . in addition to the described active components , the disinfectant preparations of the invention may contain other typical components depending on the desired use of the formulation . in particular , an acidifier may be used to keep the ph range of the disinfection solution below 6 . suitable solvents for the protonated compounds and / or the other active ingredients may be employed , and preferably are water or water miscible organic solvents . solutions such as these may be readily sprayed using compressed air or any other propellants known by those in the art . these disinfectant preparations of the invention are suitable for surface disinfection in medically - related environments , such as hospitals , veterinary clinics , dental and medical offices and the like . use of solutions of the invention in the sterilization of surgical instruments is especially preferred . these disinfectant preparations are also useful in public areas such as schools , public transport , restaurants , hotels and laundries . the disinfectants also find use in home as sanitizers for toilets , basins , and kitchen areas . the disinfectant preparations comprising the compositions disclosed herein can be used to disinfect medical devices , surgical instruments , surgical glows or personal masks . the disinfectants may also be useful in surgical settings , both for the medical staff and to sterilize the area of surgery on the patient . for example , surgical instruments can be coated with the compositions of the invention to provide for a sterile coating prior to surgery . the present disclosure also provides the use of the composition of the invention , in the preparation of an antibacterial coating agent . this coating agent may then be used to cover , for example , surgical glows , surgical devices , surgical apparatus , male condoms and personal masks , or for coating indwelling catheters and similar implants which are susceptible to harboring bacterial infection . another aspect of the present invention is to provide a kit for inhibiting gram - positive bacteria , said kit comprising an nplv composition of the invention , optionally with reagents and / or instructions for use . it is possible that drug - resistant variants of bacteria can emerge after prolonged treatment with an anti - bacterial agent . the efficacy of a drug against the bacterial infection can be prolonged , augmented , or restored by administering the compound in combination or alternation with a second , and perhaps third , anti - bacterial agent , for example with a different site of activity than the principle drug . alternatively , the pharmacokinetics , biodistribution or other parameter of the drug can be altered by such combination or alternation therapy . in general , combination therapy is typical because it induces multiple simultaneous stresses on the bacteria . accordingly , the kit of the present invention may further comprise a separate pharmaceutical dosage form comprising an additional anti - bacteria agent . suitable antibiotic agents are disclosed , e . g . in physician &# 39 ; s desk 30 reference ( pdr ), medical economics company ( montvale , n . j . ), ( 53rd ed . ), 1999 ; mayo medical center formulary , unabridged version , mayo clinic ( rochester , minn . ), january 1998 ; merck index an encyclopedia of chemicals , drugs and biologicals , ( 11th ed . ), merck & amp ; co ., inc . ( rahway , n . j . ), 1989 ; university of wisconsin antimicrobial use guide , http :// www . medsch . wisc . edu / clinsci / 5amcg / amcg . html ; introduction on the use of the antibiotics guideline , of specific antibiotic classes , thomas jefferson university , http :// jeffiine . tju . edu / cwis / oac / antibiotics_guide / intro . html ; and references cited therein . non - limiting examples of antibiotics classes include sulfonamides , penicillins , cephalosporins , aminoglycosides , chloramphenicol , tetracyclines , macrolides , lincosamides , streptogramins , glycopeptides , rifamycins , nitroimidiazoles , quinolones , trimethoprim , oxazolidinones , lipopeptides . specific compounds include , for example , amikacin ( amikacin sulfate ); craramyein ( gentamicin sulfate ); nebcin ( tobramycin sulfate ); netromycin ( netilmicin sulfate ); streptomycin sulfate ; and tobi ( tobramycin ), azactam ( aztreonam ); cefotan ( cefotetan ); lorabid ( loracarbef ); mefoxin ( cefoxitin ); merrem ( meropenem ); and primaxin ( imipenem and cilastatin for injectable suspension ); ancef ( cefazolin ); ceclor ( cefaclor ); cedax ( ceffibuten ); cefizox ( ceffizoxime sodium ); cefobid ( cefoperazone sodium ); ceftin ( cefuroxime axetil ); cefzil ( cefprozil ); ceptaz ( ceftazidime ); claforan ( cefotaxime ); duricef ( cefadroxil monohydrate ); fortaz ( ceftazidime ); keflex ( cephalexin ); keftab ( cephalexin hcl ); kefurox ( cefuroxime ); kefzol ( cefazolin ); mandol ( cefamandole nafate ); maxipime ( cefepime hcl ); monocid ( cefonicidsodium ); omnicef ( cefdinir ); rocephin ( ceftriaxone ); suprax ( cefixime ); tazicef ( ceftazidime ); tazidime ( ceftazidime ); vantin ( cefpodoxime proxetil ); and zinacef5 ( cefuroxime ); biaxin ( clarithromycin ); dynabac ( dirithromycin ); e . e . s . 200 ( erythromycin ethylsuccinate ); e . e . s . 400 ( erythromycin ethylsuccinate ); ery - ped 200 ( erythromycin ethylsuccinate ); eryped 400 ( erythromycin ethylsuccinate ); ery - tab ( erythromycin delayed - release tablets ); erythrocin stearate ( erythromycin stearate ); ilosone ( erythromycinestolate ); pce dispertab ( erythromycin particles in tablets ); pediazole ( erythromycin ethylsuccinate and sulfisoxazole acetyl for oral suspension ); tao ( troleandomycin ); zithromax ( azithromycin ); and erythromycin ; cleocin hcl ( clindamycin hydrochloride ); cleotin phosphate ( elindamycin phosphate ); coly - mycin m ( colistimethate sodium ); and vancocin hcl ( vancomycin hydrochloride ); amoxil ( amoxicillin ); augmentin ( amoxicillin / clavulanate potassium ); bicillin c - r 900 / 300 ( penicillin g benzathine and penicillin g procaine suspension ); bicillin c - r ( penicillin g benzathine and penicillin g procaine suspension ); bicillin l - a ( penicillin g benzathine suspension ); geoeillin ( carbencillin indanyl sodium ); mezlin ( sterile mezlocillinsodium ); omnipen ( ampicillin ); pen - vee k ( penicillin v potassium ); pfizerpen ( penicillin g potassium ); pipracil ( piperacillin sodium ); speetrobid ( bacampicillin - hcl ); ticar ( tiearcillin disodium ); timentin ( ticarcillin disodium and clavulanate potassium ); unasyn ( ampicillin sodium / sulbactam sodium ); zosyn ( piperacillin sodium and tazobactam sodium ); and dicloxacillin sodium ; achromycin v ( tetracycline hcl ); declomycin ( demeclo - cycline hcl ); dynacin ( minocylcine hcl ); minocin ( minocycline hydrochloride ); monodox ( doxycycline monohydrate capsules ); terramycin ( oxytetracyline ); vectrin ( minocycline hydrochloride ); vibramycin calcium ( doxycycline sodium ); vibramycin hyclate ( doxycycline hyclate ); vibramycin monohydrate ( doxycycline monohydrate ); vibra - tabs ( doxycycline - hydrate ); declomycin ( demeclocycline hcl ); vibramycin ( doxycycline ); dynacin ( minocyline hcl ); terramycin ( oxytetracycline hcl ); achromycin v capsules5 ( tetracycline hcl ); linco - mycins ; and cleotin hcl ( clindamycin hcl ); abelcet ( amphotericin b lipid complex ); ambisome ( amphotericin b ); amphotec ( amphotericin b cholesterol sulfatecomplex ); ancobon ( flucytosine ); diflucan ( fluconazole ); fulvicin p / gamma ( ultramicrosize griseofulvin ); fulvicin p / g 165 and 330 ( ultramicrosize griseofulvin ); grifulvin v ( griseofulvin ); gals - peg ( gxiseofulvin ultramicrosize ); lamisil ( terbinafine hydrochloride ); nizoral ( ketoconazole ); amphotericin b ; lotrimin ( clotrimazole ); dapsone tablets ( dapsone ); diflucan ( fluconazole ); monistat - derm cream ( miconazole ); mycostalin crc am ( nystatin ); and sporanox ( itraconazole ); aralen hydrochloride ( chloroquine hcl ); aralen phosphate ( chloroquine phosphate ); dataprim ( pyrimethamine ); ladam ( mefloquine hcl ); and plaquenil ( hydroxychloroqnine sulfate ); capastat sulfate ( capreomycinsulfate ); myambutol ( ethambutol hydrochloride ); mycobutin ( rifabutin capsules ); nydrazid ( isoniazid injection ); paser ( aminosalicylic acid ); prifiin ( rifapentine ); pyrazinamide tablets ( pyrazinamide ); rifadin ( rifampin capsules ); rifadin iv ( rifampin for injection ); rifamate ( rifampin and isoniazid ); rifater ( rifampin , isoniazid and pyrazinamide ); seromycin ( cycloserine capsules ); streptomycin - sulfate ; tice bcg ( 3cg vaccine ); cycloserine ( seromycin capsules ); urised ( methenamine ); and trecator - sc ( ethionamide tablets ); alferon n ( interferon alfa - n3 ); crixivan ( indinavir sulfate ); cytovene ( ganciclovir ); cytovene - iv ( ganciclovir sodium ); epivir ( lamivudine ); famvir ( famciclovir ); flumadine ( rimantadine hcl ); foscavir ( foscamet sodium ); hivid ( zalcitabine ); intron a ( interferon alfa - 2b ); invirase ( saquinavir mesylate ); norvir ( ritonavir ); rebetron combination therapy , which contains rebetrol ( ribavirin ) and intron a ( inteferon alfa - 2b ); rescriptor ( delavirdine mesylate ); retrovir ( ziduvudine ); retrovir iv ( ziduvudine ); symmetrel ( amantadine hcl ); synagis ( palivizumab ); valtrex ( valacyclovir hcl ); videx ( didanosine ); viracept ( nelfinavir mesylate ); viramune ( nevirapine ); virazole ( ribavirin ); vistide ( cidofovir ); zerit ( stavudine ( d4t )); symmetrel syrup ( amantadine hcl ); combivir tablets ( lamiduvine ); and zovirax ( acyclovir ); dapsone tablets ( dapsone ); daraprim ( pyrimethamine ); flagyl 375 ( metronidazole ); flagyl er tablets ( metronidazole ); flagyl i . v . ( metronidazole ); furoxone ( furazolidone ); mepron ( atovaquone ); and neutrexin ( tfimetrexate glucuronate ); cipro ( ciprofloxacin hcl ); floxin ( ofloxacin ); levaquin ( levofloxacin ); mazaquin ( lomefioxacin hcl ); noroxin ( norfloxacin ); penetrex ( enoxacin ); raxar ( grepafloxacin hcl ); trovan ( trovafioxacin mesylate ); and zagam ( sparfloxacin ); bactrim . ( trimethoprim and sulfamethoxazole ); bactrim ds ( irimethoprim and sulfamethoxazole double strength ); pediazole ( erythromycin ethylsuccinate and sulfisoxazole acetyl ); septra ( trimethoprim and sulfamethoxazole ); septra ds ( trimethoprim and sulfamethoxazole ); co - trimoxazole , sulfadiazine , battrim i . v . infusion ( sulfamethoxazole ); sulfapyridine and pediazole ( erythromycin ethylsuccinate and sulfisoxazole acetyl ); furadantin ( nitrofurantoin ); macrobid ( nitrofurantoin monohydrate macrocrystals ); macrodantin ( nitrofurantoin macrocrystals ); monurol sachet ( fosfomycin tromethamine ); neggram caplets ( nalidixic acid ); septra ( trimethoprim and sulfamethoxazole ); septra ds ( trimethoprim and sulfamethoxazole ); urised ( a combination of the antisepticsmethenamine , methylene blue , phenyl salicylate , benzoic acid and parasympatholytics ( atropine sulfate ) hyoscyamine ); ( oxytetracycline hcl , sulfamethizole and phenazopyridine hcl ); ( methenamine mandelate ); bactroban ( mupirocin ); chloromycetin opthalmic ( chloramphenical ); cortisporin ( neomycin and polymyxin [ 3 sulfates and hydrocortisone acetate cream ); ilotycin ( erythromycin opthabnic ointment ); neodecadron ( neomycin sulfate - dexamethasone sodium phosphate ); polytrim ( tfimethoprim and polythyxin [ 3 sulfate opthalmic solution ); terra - cortril ( oxytetracycline hcl and hydrocortisone acetate ); terramycin ( oxytetracycline ); and tobradex ( tobramycin and dexamethasone opthalmic suspension and ointment ); vita - a opthalmic ointment , ( vidatabine ); ( norfloxacinopthalmic solution ; ciloxan opthalmic solution and ointment ( ciprofloxacin hcl ); and ocuflox opthalmic solution ( ofioxacin ), blephamide opthalmicointment ( sulfacetamide sodium and prednisolone acetate ); and blephamideopthalmic suspension ( sulfacetamide sodium and predrdsolone acetate ); a / t / s ( erythromycin ); bactroban ( mupirocin ); benzamycin ( erythromycin - benzoyl peroxide topical gel ); betadine ( povidone - odine ); cleotin t ( clindamy cinphosphate topical solution ); clindets ( clindamycin phosphate pledgets ); cortispofin ( neomycin , polymyxin b sulfates and hydrocortisone acetate cream ); emgel ( erythromycin ); erycette ( erythromycin topical solution ); garamycin ( gentamicin sulfate ); klaron ( sodium sulfacetamide lotion ); mycostatin ( nystatin cream ); theramycin z ( erythromycin topical solution ); t - stat ( erythromycin ); chloromycetin ( chloramphenicol opthalmic ointment ); cortisporin ( neomycin and polymyxin b sulfates , bacitracin zinc and hydrocortisone opthalmic ointment ); ilotycin ( erythromycin ); neodeeadron ( neomycin sulfate - dexamethasone sodium phosphate ); polytrim ( trimethoprim and polymyxin b sulfate ); terra - cortril ( oxytetracycline hcl and hydrocortisone acetate ); terramycin ( oxytetracycline ); exelderm ( sulconazole nitrate ); fungizone ( amphotericin b oral suspension ); lamisil ( terbinafine hydrochloride cream ); loprox ( ciclopiroxolamine ); lotrimin ( clotrimazole ); lotrisone ( clotrimazole and betamethasone diproprionate ); mentax ( butenafine hcl ); monistat - denn ( miconazole nitrate ); mycelex ( clotrimazole ); mycostatin ( nystatin ); naffin ( nattifine hcl ); nizoral ocetoconazole ); nystop ( nystatin ); oxistat ( oxiconazole nitrate ); selsun r . sup . x ( 2 . 5 % selenium sulfide lotion ); and spectazole ( econazole nitrate ); denavir ( penciclovir cream ); and zovirax ( acyclovir ); benzashave coenzoyl peroxide ); betadine ( povidone - iodine ); betasept ( chlorhexidine gluconate ); cetaphil ( soap substitute ); clorpactin wcs - 90 ( sodium oxychlorosene ); dapsone tablets ( dapsone ); desquam - e coenzoyl peroxide ); desquam - x ( benzoyl peroxide ); hibiclens ( chlorhexidine gluconate ); hibistat ( ehlorhexidine gluconate ); impregon ( tetrachlorosalicylanilide 2 %); metrocream ( metronidazole ); metrogel ( metronidazole ); noritate ( metronidazole ); phisohex ( hexachlorophene detergent cleanser ); sulfacet - r ( sodium sulfacetamide 10 % and sulfur 5 %); sulfamylon ( materfide acetate ); tfiaz coenzoyl peroxide ); and vanoxide - hc coenzoyl peroxide hydrocortisone ); acticin ( permethrin ); elimite ( permethrin ); eurax ( crotamiton ); efudex ( fluoro - uracil ); fluoroplex , rifapentine , quinupristin / dalfopristin , moxifloxacin , gatifloxacin , linezolid , cefditoren pivoxil , ertapenem , gemifloxacin , daptomycin , telithromycin , and combinations thereof . alternatively , the nplv composition of the invention may further comprise an additional anti - bacteria agent listed above and combinations thereof . in combination therapy , effective dosages of two or more agents are administered together , whereas in alternation or sequential - step therapy , an effective dosage of each agent is administered serially or sequentially . the dosages given will depend on absorption , inactivation and excretion rates of the drug as well as other factors known to those of skill in the art . it is to be noted that dosage values will also vary with the severity of the condition to be alleviated . it is to be further understood that for any particular subject , specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions . in some embodiments , an anti - bacterial agent that exhibits an ec 50 of 10 - 15 μm or less , or typically less than 1 - 5 μm , is desirable . generally , the kit comprises a container and a label or package insert on or associated with the container . suitable containers include , for example , bottles , vials , syringes , etc . the containers may be formed from a variety of materials such as glass or plastic . the container holds a composition which is effective for treating the condition and may have a sterile access port ( for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle or an aerosol spray device ). the label or package insert indicates that the composition is used for treating the condition of choice , such as bacteria infections . alternatively , or additionally , the kit may further comprise a second ( or third ) container comprising a pharmaceutically acceptable buffer , such as bacteriostatic water for injection ( bwfi ), phosphate - buffered saline , ringer &# 39 ; s solution and dextrose solution . it may further include other materials desirable from a commercial and user standpoint , including other buffers , diluents , filters , needles , and syringes . those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described . it is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof . the invention also includes all of the steps , features , compositions and compounds referred to or indicated in this specification , individually or collectively , and any and all combinations or any two or more of said steps or features . the present disclosure is therefore to be considered as in all aspects illustrated and not restrictive , the scope of the invention being indicated by the appended claims , and all changes which come within the meaning and range of equivalency are intended to be embraced therein . various references are cited throughout this specification , each of which is incorporated herein by reference in its entirety . the foregoing description will be more fully understood with reference to the following examples . such examples , are , however , exemplary of methods of practicing the present invention and are not intended to limit the scope of the invention . different strains of gram - positive bacteria , e . g ., staphylococcus aureus , bacillus subtilis , micrococcus luteus , and different strains of gram - negative bacteria , e . g ., escherichia coli , pseudomonas aeruginosa , were used . bacterium were grown and maintained according to standard protocols known in the art . the primary lipid used was polyoxyethylene cetyl ether ( pce ), either alone or in combination with palmitic acid ( pa ), hexadecyl trimethylammonium bromide ( htab ) or dimethyldihexadecyl ammonium bromide ( dhab ) at the molar ratios set forth below . the lipid mixture was heated to 50 ° c . and mixed with phosphate buffer saline ( pbs ), also pre - heated to 50 ° c ., using the 2 - syringes method . briefly , a 10 - ml syringe , containing 0 . 5 g of the lipid mixture , was connected to a second 10 - ml syringe containing 10 ml of phosphate buffer saline ( pbs ) ( 5 % final lipid concentration ). the lipid blend was then injected into the pbs syringe , and the resulting mixture was rapidly passed forth and back about 20 times , until a homogeneous suspension was obtained . the preparation was subsequently checked for nplv quality by phase - contrast microscopy . four types of nplvs were prepared : nplv01 : 100 % polyoxyethylene cetyl ether ( pce ); nplv02 : 99 . 9 % pce and 0 . 1 % palmitic acid ( pa ); nplv03 99 . 9 % pce and 0 . 1 % hexadecyl trimethylammonium bromide ( htab ); nplvo4 99 . 9 % pce and 0 . 1 % dimethyldihexadecyl ammonium bromide ( dhab ). two sets of each type of nplvs were prepared : one with 5 mm cyclodextrin and the other containing no cyclodextrin . following plating on agar of the indicated bacteria , a drop of nplv compositions , with or without cyclodextrin , was deposited on the plates . for each product a volume of 100 μl was deposited at a concentration of 0 . 5 mg / ml ( 50 mg total lipid ). agar plates were then incubated at 37 ° c . overnight . white plaques indicated an inhibition of growth . inoculums of 5 × 10 5 bacteria / ml ( b . subtilis ) were grown overnight in lb broth medium in presence of various concentration of nplv03 ( 99 . 9 % pce and 0 . 1 % hexadecyl trimethylammonium bromide ( htab )). each culture sample was then serially diluted and plated on agar for titer determination . in order to explore the possibility of inhibiting bacteria using the composition disclosed herein , applicants tested the inhibitory effect of the composition on both gram - positive and gram - negative bacteria . applicants synthesized four different nplvs using different lipid compounds : nplv01 : 100 % polyoxyethylene cetyl ether ( pce ); 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