AU2016200853B2 - Anti-bacterial applications of poly–n-acetylglucosamine nanofibers - Google Patents

Anti-bacterial applications of poly–n-acetylglucosamine nanofibers Download PDF

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AU2016200853B2
AU2016200853B2 AU2016200853A AU2016200853A AU2016200853B2 AU 2016200853 B2 AU2016200853 B2 AU 2016200853B2 AU 2016200853 A AU2016200853 A AU 2016200853A AU 2016200853 A AU2016200853 A AU 2016200853A AU 2016200853 B2 AU2016200853 B2 AU 2016200853B2
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bacterial
snag
snag nanofibers
fibers
acetylglucosamine
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AU2016200853A1 (en
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Sergio Finkielsztein
John N. Vournakis
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Marine Polymer Technologies Inc
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Marine Polymer Technologies Inc
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Priority claimed from AU2011239466A external-priority patent/AU2011239466B2/en
Priority claimed from AU2013202332A external-priority patent/AU2013202332B2/en
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Described herein are compositions comprising shortened fibers of poly-N-acetylglucosamine and/or a derivative thereof ("sNAG nanofibers") and anti-bacterial applications of such compositions. The sNAG nanofibers may be formulated into compositions for the prevention and/or treatment of bacterial infections and diseases associated with such infections. Regimens employing such compositions are also described.

Description

ANTI-BACTERIAL APPLICATIONS OF POLY-N-ACETYLGLUCOSAMINE
NANOFIBERS
[0001] The present application is a divisional application of Australian Patent Application No. 2013202332, itself a divisional application of Australian Patent Application No. 2011239466 and claims priority to United States provisional application no. 61/324,657, the contents of all of which are incorporated herein by reference.
1. FIELD
[0002] Described herein are compositions comprising shortened fibers of poly-N- acetylglucosamine and/or a derivative thereof (“sNAG nanofibers”) and anti-bacterial applications of such compositions. The sNAG nanofibers may be formulated into compositions for the prevention and/or treatment of bacterial infections and diseases associated with such infections. Regimens employing such compositions are also described.
2. BACKGROUND
[0003] Currently, antibiotics are a standard therapy for bacterial infections. However, some individuals have an allergic reaction to certain antibiotics, others suffer from side effects associated with antibiotics, and the continued use of antibiotics often leads to a reduction in their efficacy. In addition, antibiotic therapy often leads to the emergence of antibiotic-resistant strains of bacteria. Accordingly, there is a continuing need for new anti-bacterial agents that are effective in fighting infection without generating resistance or reducing the efficacy overtime. There is a need for non-antibiotic anti-bacterial agents that can be used in clinical settings, e.g., in the treatment of infectious diseases of the skin, digestive and respiratory tract, and in wound treatment.
[0004] Wound infection is one type of bacterial infection. Wound infection is a major complication, especially in patients with chronic disease such as diabetes or during immunosuppression. Such patients have disruptions in appropriate inflammatory responses, including the migration and recruitment of neutrophils and macrophages, which predisposes them to increased infection (Singer, A.J. and R.A. Clark, 1999, N Engl J Med 341(10): 738-46). In addition, bacterial infection can lead to impairment of wound healing and sepsis. Given the ineffectiveness of many current antibiotic treatments and the increased prevalence of antibiotic resistant bacteria such as MRSA (Methycillin-resistant S. aureus), new clinical treatments are in high demand.
3. SUMMARY
[0004a] In one aspect, the present invention particularly relates to a method for treating a disease or a condition associated with a bacterial imbalance in a subject in need thereof, comprising topically administering a composition comprising shortened fibers of poly-β-1-^4-N-acetylglucosamine (“sNAG nano fibers”) to a subject, wherein the sNAG nano fibers are less than 10 pm in length, wherein the sNAG nano fibers comprise 70% or more than 70% of N-acetylglucosamine monosaccharides, and wherein the sNAG nanofibers do not have an effect, or substantially have no effect, on bacterial growth or survival of Staphylococcus aureus bacterial cultures in vitro.
[0004b] In another aspect the present invention particularly relates to a method for treating a disease or a condition associated with a bacterial imbalance in a subject in need thereof, comprising topically administering a composition comprising shortened fibers of poly-β-Ι—»4-N-acetylglucosamine (“sNAG nano fibers”) to a subject, wherein the sNAG nano fibers are from about 1 pm to less than 10 pm in length, wherein the sNAG nano fibers comprise 70% or more than 70% of the N-acetylglucosamine monosaccharides, and wherein the sNAG nanofibers do not have an effect, or substantially have no effect, on bacterial growth or survival of Staphylococcus aureus bacterial cultures in vitro.
[0004c] In a still further aspect the present invention particularly relates to use of a composition comprising shortened fibers of poly-β-Ι—>4-N-acetylglucosamine (“sNAG nanofibers”) in the manufacture of a medicament for treating a disease or a condition associated with a bacterial imbalance in a subject in need thereof, said treating comprising topically administering the medicament to the subject, wherein the sNAG nano fibers are less than 10 pm in length, wherein the sNAG nano fibers comprise 70% or more than 70% of N-acetylglucosamine monosaccharides, and wherein the sNAG nanofibers do not have an effect, or substantially have no effect, on bacterial growth or survival of Staphylococcus aureus bacterial cultures in vitro.
[0004d] In a still further aspect, the present invention particularly relates to use of a composition comprising shortened fibers of poly-β-Ι—»4-N-acetylglucosamine (“sNAG nanofibers”) in the manufacture of a medicament for treating a disease or a condition associated with a bacterial imbalance in a subject in need thereof, said treating comprising topically administering the medicament to the subject, wherein the sNAG nanofibers are from about 1 μm to less than 10/vm in length, wherein the sNAG nanofibers comprise 70% or more than 70% of the N-acetylglucosamine monosaccharides, and wherein the sNAG nanofibers do not have an effect, or substantially have no effect, on bacterial growth or survival of Staphylococcus aureus bacterial cultures in vitro.
[0004e] These and other aspects are described in further detail below. Aspects not the subject of the present invention are described herein for completeness and may form the subject of the parent applications AU2013202332, AU2011239466 or one or more divisional applications.
[0005] In one aspect, described herein are methods for treating and/or preventing a bacterial infection(s) and/or diseases associated with or caused by a bacterial infection in a subject.
[0006] In certain embodiments, described herein are methods for treating a bacterial infection in a subject comprising topically administering a composition comprising sNAG nanofibers to a subject, hi some embodiments, the subject is diagnosed with the bacterial infection or displaying one or more symptoms of the bacterial infection. The methods of diagnosis of bacterial infection and symptoms of bacterial infection are those known in the art or described herein. The bacterial infection may be a skin infection, a gastrointestinal infection, a respiratory infection, a urinary tract infection, a reproductive tract infection, or infection of any other organ or tissue in the body of the subject as described herein, hi one embodiment, the infection is a nosocomial infection, an MRS A infection, a Pseudomonas infection, or a C. dificule infection.
[0007] hi certain embodiments, described herein are methods for treating and/or preventing a disease associated with a bacterial infection or a bacterial imbalance in a subject comprising topically administering a composition comprising sNAG nanofibers to the subject, hi one such embodiment, the method involves treating and/or preventing a disease associated with a bacterial infection, hi another embodiment, the method involves treating and/or preventing a disease associated with a bacterial imbalance, for example, an imbalance in bacterial microbiota as described herein. In certain embodiments, the methods involve treating an existing bacterial infection. In some of these embodiments, the subject to be treated is diagnosed with a disease associated with a bacterial infection or displays one or more symptoms of stich disease. In other embodiments, the subject to be treated is diagnosed with a disease associated with a bacterial imbalance or displays one or more symptoms of such imbalance, The disease may be a skin disease, a gastrointestinal disease, a respiratory disease, a urinary tract disease, a reproductive tract disease, or disease of any other organ or tissue in the body of the subject as described herein, hi some embodiments, the disease is a skin disease or a gastrointestinal disease, hi one embodiment, the disease is associated with a nosocomial infection, an MRSA infection, a Pseudomonas infection, or a C. dificule infection.
[0008] hi some embodiments, described herein are methods for preventing a bacterial infection and/or a disease associated with a bacterial infection comprising topically administering a composition comprising sNAG nanofibers to a subject, hi some embodiments, a ,n '? mistering a c"mpasrmn iermrceng s\' -0 m! io*m -«·. re >5 χο-χο t ΐπ mwo embodiments a compos?Hon comprising sNAG nunofifeer* is administered 10 s subject a; nigh risk of* bacterial Infection to prevent a disease associated with a bacterial infection. In specific embodiments, a oo'ipe-mion 'ompr-w; Ά ul ρηηοίΐΚ'χ s ,dmnsskued V . -kc -.,. wk - u wound o; „ w '-rei who has undergone a surgery, in one embedImeni, the composition is administered to an nnrmnin ompnm -s-.d νιΐχ-,. In v'mv cmkvio - re- . eomposhio' -,0:11^--1,,0 -VU1 nanofiheks is administered to o wound, whew the wound is ut hwh ri~k ofbacterial infection, in cerium embodiments., ihe wound ·.-, an op!,n wound I he open '-sound may be a cttnvPot wound, a puncture wound, a laceration wound, an abrasion, a cut, a penetration wound, a surgical wound, or any other wound. In certain embodiments, the wound may be a puncture wound, for example, i mmi u.v wound dun s e.m-ed i \ a * -.nod-a ssjs pv, eocre or a e. abets re nwu pure ee>. ;e in such embodiments, the subject to be treated may have been diagnosed with a hemodialysis-reload m rekheren '-e-cni'i, Ian, J nwk non tr <me embodim ret, d t l\k",\ \J :i\Vu-',r j o' dv .i'.' \ 'X’Wv1 ' i, ,'c4 ”k ’ wlredon o *' ' ore \-re\ < ΆΛί Co*'-" reus *'ot'. o so. , o an ones k\ o s - frem, iks \ , vou d 1 o? t mu*'', eo5 0' c bacterial infection and/or the disease associated with a bacterial infection is not at the site of a weimd i,o, fret at the ask. of„:t open n ourm t
IfilKWj In some embodiments, described herein are methods tor treating a luuuerially infected wound m a sui'ieck compmung topic diy -omnsnstesmu .¾ composition comprising d.,Ul nana fibers to ihe wound site tn a subject, in some embodiments, the subject to be treated is diagnosed with a bacterial infection or displays one or more symptom* of the bacterial infection. In cremin embodiment's ihe wound is -,n mxπ '.'.msud I h open wound t vo -¾ a guusho1 wound, a puncture wound, a laceration wound, an abrasion, a cm, a penciratiorj wound, a surgical wound, or any Cither wound. In certain embodiments, the wound may be a puncture wound, foi- example, a puncture wound that is caused by a hemodialysis procedure or a catheterization procedure, in such embodiments, the subject to he treated may have been diagnosed with a homodi-dy nsreohdod m eaiheieriwirion-i'dafed rereckon 111010} Bacterial infections to he treated or prevented using the methods described herein mi l,\v "riu'uoks o < h l m ο K'o' nose ot f'v Ί" os r . gore re's o, , A' , ,
BmcvUa, Cumpyhbucter, Chimiydia and (Ίαηα'ώψΗνΙία, Cbtsirkikmt, Cwynebactemmh F,mc^h-<\x sw, EwK¢- w\ hkk Pntth'iM'thi. ikh'i>}(tpk:kt^\ f/t hVreAk rex /.s'gm?k\%, krevoypiHk
Listeria, Mycobacterium, Mycoplasma, N&amp;meriu, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema* Vihrkt* and Yersinia. In some embodiments,a sNAG composition may be used to treat and/or prevent a disease associated with an infection by bacteria front one or more of the listed genuses of bacteria, or one or more symptoms th-greo f, [001.1 \ U^V ml .t v ,,>0,00 - 'Vo ,\ γ,ι t <itoK''v * vf v , '\\o > eKo include infections with bacteria of one or more of the following species. Barilla* anthmeis,
MmliemiM Bprreim hitrgifyriwN MmimMit a hmd0c^Ym^M.tMiisii Brtmrifd. gwMtm&amp;M·
Era el hi 001, Campehmu' imrim οκν Chiaimhim meant, m;a, i htmm ala trm m mutts, :psiiMep €1¾^ ·€ΙρρΜΜΙρρυϊΜ0^ :pprNiogcm,·:
Oostnannn tciam, Co''vm K,\ m* (¢.0¾ Jtptvrct ttn\ tmeomom 10 hue al¢. Ente*·"* cccu\ ha\ mm. Escherichia coli, Framisella ndarensls, Haemophilus in flue me, Helicobacter pylori, Legionella pm amrhihi /vovomwAhk 1 gnVvsVo 1 ,όο* m mams rhtecne,
Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae. Neisseria gt>narth>c>.\\ A, ."ovno menmghides, Pmo'>li.memn\ am ague ><α, Pom m nErabihs, R:< Icitdn rmrx asv. Salmonella trj>hi, Salmonella lyphnnuriam, Shigella m>nnci, Staphyh>t.f>t.cns oarcm.
Streptococcus pneumonia. Streptococcus pyogenes, Treponema pallidum* Fibrin cholera#, and Yersinia pestis. In some embodiments, a sNAG composition may be used to treat and-or prevent a disease associated with an infection by hisetena from one or more of the listed species of bacteria, or one or more symptoms thereof. |0(M2| In certain embodiments, the bacterial infection to be treated or prevented using the methods' described herein is an MRS A infection, a F'-euaammno infection, or a C. di/owfe infection, hi some embodiments, a sN AO composition may be used to treat and/or prevent a dev. v uvsouiutee W.SA mV* tier, ,¾ Γ > ;„f "wv, .wcuiev oi ,h ' . ,v owno 1, or on, or more symptoms thereof,symptom thereof.
[11013} hi ecrcm: embodiments, die havesuU iuiecUon to he treated or presented nsim.· the methods described herein is caused by bacteria that are known to one of ordinary skill in the an to be resistant to a standard anti-bacterial therapy, for example, resistant to one or more antibiotics, in one embodiment, the bacterial infection to be treated or prevented using the oviomts ^escribed fe mm w \tk"~ \ or ,i nivtooi ?! MRSA hv<"'t r mss' ems, Ά \s composition may be used to treat and/or prevent a disease associated with an infection by bacteria resistant to one or more antibiotics, in one embodiment, a sNAG composition may be u*cd to treat and/or prevent a disease associated with MRSA, e.g., associated with a nosocomial MRS A, {0014} The subject to be treated using the methods described herein may he a mammal. piei."\tbh .4 in man 1he -me λ i -mx 4-m 6, . I \e,s to g .¾ i hv\0t' a i ov. a px;, a :,-'at! os af.,,, a, < eoi O' t e„"\ m „n\ oJkx .urn u {0015} 1 ho A Mi «a xmho '< come'1p ax J :o tr e m.”lions no^C" > x tv. cot i v\ . - m \ai\rtg kejl.s ax!th'. nx'k, nias ^es>.A uJv^.ixve n. sk.mxt 5 k \ xr <γ. οοάιο embodiments, the majority (and m certain embodiments, at Utast or more ihan 60¾. 70¾. 80¾. 'M'1,, 'i’'' o m o omhe s\ xt n .no be-v or 106¾ of no A At x -¾ o ' mxv a>e A on m' umiut 1 to )' urn in length in some embodiments, ’be majority und m -.cruet emb'Mtm-.'nt-'i at lo ix or mme t xie "t'A, "tV , soA,'Vt 'i'ik'.nfti!, o; ,0(¾ M m*, sNAG nano fibers, are between about 2 to 10 urn. or 4 to 7 uni in length. The sNAG nano fibers o the dpsernvt length van n obtam· s., k'T e^amele, ' J '''".'«'bee be -m ,x Set tm5·' x ', " - ,. |Θ(Μ6} 5n Ci rtam embodiments the A AO nunmihcnv xeie ps Aueeu in mudetimn, e =.· , gamma tmuiktmn, of poly N-nt,erylgjucosarnire or e dens ato e thereof hi some embodiments, ihe A AG nanofmots ure raw send K trmJvo'm oAhe po \k- * x\> ,et tGe,',tkVsamo s m Ox form of dried fibers (e g„ at 500-2.000 kgy), or irradiation of the poly-p· I-+4-N aceiyiglucosamine in the form of wet fibers (e.g.., at 100-500 kgy). |Θ01 ?| In certain embodiments, the sNAG nano fibers me derived from mieroAgae. in another embodiment, the ,\XAG nnnofibers ate not domed from eatstdceaus In ν·.Ί unrulier embodiment, flu, Ah\G namyfibcm may be derived from microalgae, crustaceans te.g., shrimpy fungus or any other source. |00i8i A one embodiment, 'he ,A \G tidtkifihers , orvms. Xveo'\ -i'l.i.iKtmre monosaccharides and/or giueosaminc monosaccharides, wherein more than 60¾. 70%, 80¾. ’*»' eor ' ,00 e* Uk ",oX"~ x., "a-,vk'v'' A, A ' χοχοΆ .eo V te. η kkxemo n xe monos mtm exk-s h.ano.be? esnbo.hmeiu, ti c A Xe> xm-iumus ,u prm\ m etA '>,osoov!„ monr-saceharidc''and ot gjuensamhk monosneeh tu-k's x h-rrein mmeihan Ά"·, ol the monosueeiun sdx'i ot the A At» nanofihefs ax \-.x ei>h.-hn,o'^mnue x»'X<K,x'hhat!-k'v jfi019| Ip < ο?ΗίΡΐ cm K\j fha \SAG mTOudwTO ns,v m the ne‘TO,'.\ desc??\v TOvm do no? have an effect on bacterial growth or survival ot Stuphyivoovcu^ anrem bacterial cultures in vitro·, or substantially have to effect on bacteria! growth or survival of Stuphyhcocous aurem bacterial cultures in vwo. in some embodiments, the sNAG nimoribers reduce bacterial growth or -survival of bacterial cultures in vitro by less than 1 log, 0 75 log, 0.5 log. 0 25 log, 0.2 log or d ? log, o $:., when -to reoew I'ariTO.d > nliurov me we uoC moTOume v- id- the ,\XA< ! n~ t l'C\ 111, ?ί to! »4» ** * o \ \( ,u to,, jo \ > * v * v'v, ' \ a TO TO' o’jltidtiop of the :cm result to* Oesentwi, ?o? e\ unple. in SC'"i,vt " !, i'\ :mpk' Γ to g . Section 6.2,2.5) and Figure ! i 1:., intro, j002Mj in i en.?in ombodim'.ms, she s\ Vi- ο,λποΠΗ’· ·, u.to! in the smuhc-TO dev-nbed hc-vm Uk, i\>novaem,' n a bn,'* enp,u by test m teen Ιό* TOTOmle, d % c\ Ui' .uanp\i ·, w t ut the methods described herein may be ncm-reactive when rested in «η elution rest, an intramuscular implantation tost, an intracutanc-ous test, or a systemic test, in some embodiments, the compositions described herein are non-reacrive when rested in an elution test, an iron'i’M u ar ΰνρΊ.η1» ϋΐ,ΐί aTO, ,f nm'jTOuv'eoTO v·", v. vworro iom Ip uTO:? ο>’;ά!!1γ''γ.'', the sNAG nanofibers used in ?he methods described herein have Grade 0 or Grade ! when tested ?r er eLn on f o, m I’^ramu wu a; imrlapr mo' res; 'TOimrcuTOVOTO .ον m ' " \ to to fe vp- another enibodunem the ‘Nau oamdiKTO used m the method'» J,'m need heroin ,e-e at tnov: mildly reactive when tested in an elution test, un intramuscular implantation test, an in trace tancous test, ora systemic test. In one embodiment the sNAG nano fibers or compositions comprising such nanofibers are non~rcactivc as determined by an intramuscular n?p Gto.'sto tost In oenctr vOL"TOa>v' vt, syrup, wkoto >TO„”tK' 1 me?" dc nut or so an allergenic reaction or un irritation, c.g., at the site of application, in other ernbodirnems, the compositions described herein cause at most a mild allergenic reaction -or a mild Irritation, e.g.. at the site of application.
The contemplated modes of administration of the compositions described herein are topical, e u., topical on the skin; topical at the site of a wound, a surgery, a bacterial infection, or a symptom of an infection tc,g.„ a swelling); and topical to a body surface such as the skirt, mucous n embrancs <o vegr'A to v, fhro.r . toto - ere:, dm snrhiTO TO e?he ;!\-- to In ccrtatn embodiments, the sNAG nanofibces or compositions comprising such nanofibers arc ietmii at v, as dse-Meg a ’"VTOt >C'\ a nn a outo S'grTO a Mt-;pTOM,m a to tototo a er„ ats em'pv.nv a w amm a p.mte e Mippmnn'rp or' pel in «"m' omivmms V> ti^SAu narsoi’ibcrs or compositions comprising, such nanofibers arc formulated as a cream, a gel, an ointment, a membrane, a powder, a spray, ora suppository. |lit>22? In anothei asp-w t, described here;a arc eompoviUmts mr nsr In die methods ,lev, abed herein In a specific embodiment. the compositions comprise sMAG nanofibers, in certain n*v' v's Jiev' s v \ ίο, ' , v.e1 ,e '\vs\At ov' ,a i'j.'w* ooe additional active ingredients useful tn preventing and/or treating a bacterial infection, &amp; disease associated with a bacteria! infection, or a .symptom thereof. In some embodiments, the additional active ingredient is an ami-bacterial agem. Such additional ann-bactenai agent may be an amibiod' tn another emKld?m>jn. -.neb add-bonai ,uni-b^cteria! ;s.eern v, cure in \et number vmlv brnom, die eor"vwmvv ,VsU’\v he'cm do not commie ar an,, mme in , utbei embodiments, the compositions described herein do net conip,rise any additional ami-bacteriaI agent, in one embodiment, she compositions described herein comprise the sNAG nanofibers as h e '>il\ aen\ c mg wo. d mj do not emu msec w ijjbional ac-o.: mood c'is {0023? in O'velfk cm bods moms, a amrmmsiiicm comprises the --A nG nanoithers -mb an antibiotic. Examples of antibiotics drat can be used in the compositions of the invention Include tmerol-dcs to e erythromycin. azithromycinb ammoglycoMdes te.e annkacim gentamicin, neomycin, streptomyeirtl, cephalosporins t'e.g., oerkdroAtl, cefaclor, cefotaxime, eefepimel, tluort-puinoiones (c.y.. elprotlosacin, Looflosacmt, penicillins -mg , pemubiu, ample·!no. srnoxictHio). tetracyclines tc.g , tetracycline, doxycycUnc), and/or cacbapenems fe.g.. meropertem, mupenem). The sN AG nanofibers and agents described herein may be used sn such compositions, in some embodiments, a composition comprises the mN b l) uanoilbers and an agent effective to treat or prevent or commonly used 10 treat or prevent an 5. mmes Infection,, MRS A infection, a Pseudomonas infection, or a C didenie infection (c.g.. an antibiotic effective again»! or commonly used against such infections}. |Ο024| In other embodiments, the compositions described herein are administcrcd in coop"·!, mm web o uma rvte iimhno'vf .ml -boeV, ·, movw or an> mne" outab c Jwpy In some embodiments, the additions! anti-bacterial agent or therapy is an antibiotic (e.g„ a standard n 'i.vii (h \sp\ re! itte buemna! -mbc mm or a d mease ' λορ is d -anh ,j huetenai mfoenon m be treated, as Known i.n the art or described herein). !n some embodiments, the additional anti-Kvmrml ages" i\ an agent cftl mm-, c to u\,,u or mv-·cm e” m'-mrmnh o v-j to tn i-· prevent .a s ^ ' o w V5Rx\ , . ,' x , ^ v ' st aththtoue eueotve reum-a m , mmewiA nwJ Amme mdi mteenon*» h, -»>)Γλ oj\ vst.-eum, the addiίϊοπϋΐ therapy is administered before, simultaneously with or after administration of a s\ Us ο ,ί·'·'Κ" eotepOOttoe to set mmle * emKxxmem, <he '.wnfn'smw."' desersb, ,' V on ,ro not adminintcrcd in conjunction with any other therapy, e.g.. not administered in conjunction with :: a»: ΕΠίίΙϊ ίοR£::. : 3.1 Imuimim [0023} u uued harem, ;he terms "$\ λο muxAihor " 1 s\ *G." "Talxl''rm ' ot ' 1 a \ t xM' (ibrmeriy known as “Taiidcrm'5) are used interchangeably to refer to shortened fibers of poly-M-v. ' v. \ ό \ ' ' eo |0026| As used herein, the term "about1' mean* a range around a given value wherein the s "xhjnv, ' duv. is the same os sm'M wsalb ihe ->an; ve -.·, v 'thm 10', e- o e. A A ss fV esxes·., \ ie nod', alee In one cm t· un "about"m, wx sOms 10" > m a ye es wthteor range. in another embodiment, the term "about5'' means within 5% of a given value or range. In another embodiment, the term ‘‘about*' means within 1% of a given value or range. (00271 As used herein, the terms '‘disease,5' "disorder' or "condition"' are used interchangeably to refer to a medical condition in a subject, hi a specific embodiment, the >te pa'indogua s ve txvu-itod λΊ*1 (« w , -vei tm’ mux" or (8028( As used herein, the term "bacterial infection4' means the Invasion by, multiplication ο O '' vVi\. O' bi X' SH ' .., 05 , s, ' O'.' [0029} As used herein, the numeric term "log" refers to logic·.
[00301 's uuJ detenu 1u terms' Αχ tm. A and ' nx'rum , a; xxes to , n\ wmv oh-'s mulxxhs't om-npossstm···., ms'mohUnmx and or eeenssS dsU can be owl m -he rwxernsw nsu.l or ire;: 1 ment of a bacterial infection or a syninnnr; or condition associated therewith In certain embodiment*·. me tore} "die* ρΆ \Mefs m ,< s\ u; n,noriK'nm m ,t p.\om,xxmm a' eompes;is-m ',outp;sNt;ie , A \i* n„sof,beiys5 in othe „.ubodimems the ten', YteemA .elds u» „ Ρχ5,.ρ\ ot'tvt huts η ,-,χ Ate nutxAbou -s ot .; ο ι ΐ’Ίυ,νΐϊη,compo-on.'." v'r-poo. .. a s\A\' nano liber (si, in ssxxtti·, ombudsmenis, an 'arlrimonul therapA' and Xudmonsl therapies" refer to a id "ep\ orx" than n -A \u n«ni?fthepu o" , ohe'snac'.utse. An' ;>'ssUi'': eoo'.vtvox «. Λ u lunefbeX'.:. In a sp.,uf.e etnboemeni, t:u ·Η.,κ;ρ\ mche-w use of a A-bi nat'otsbeo w or pharmaceutical composition comprising, a aNAG nanofiberf=s) as an adjuvant therapy; for example* using a <>NAG nanofiber composition in conjunction with a drug therapy, ««eh as an mbfeieue, and m oihoi me: a pies nse,U in rwmmov and m gve c; mm et .¾ beet, nai mieemm os a symptom or e-mdition associated therewith.
HhOf? A s used hewm, die t> nit' >"Yee;.\c m mm;" m de ^miteM >" afm miei op a Ά V I nanofiber composition to a subject refers to the amount of a sNAG nanofiber that re-suKs in u \ \ Ο o ' , 0 0 1 !'WO * W'Wks Oil ' ' * ' ' ' O * A '( nanofiber refers to an amount which is sufficient to achieve at least one* two, three, four or snore ofthe following cocos ti> the sjeanm-.e of a hac-ersaf mtec-iom (n't the erndh a mm of one us mom symptom's ^sm'Gated ihete'vidi, ini) the reduction of tone :cm; red to deo a buetenG mb. mum. us ) the : eduction or arm tmr.mon own-. seventy id’a Guterm infeet ;un end os one w moiesv; pV «« C'siv sA\ iewi"', ' the'χνκΟηη' J;e dcMOo" w .shaw mi ;k'<oo' and/or one or more symptoms associated therewith; (vi) the prevention or delay of the generation of a resistant strain, or strains of bacteria or reduction of a number of resistant strains of bacteria generated i\it ? the prevention m the recurrence of a bacteria! infection and or one m more a-'M.i',\«'„b dve'Si’h, won ihetedneron m eimvnaeun m in.’ rueten « - el; population, (tx) the reduction in the severity and/or duration of a condition caused by or -ts^V'an'd w te t Kto'enai lefoeno i, i\w't, 'eejem>u m h 'Sf'.h.d'.ru'noi' of, pdf', redneoon to ho'-pnahrams leapt i m u'' dm n« κ use «, ,he «mih,il of , -u g,, k λί n'"o orwa icon·’cm ot p w; >emos"., t the maaremw etfeoi m oh οι \.. os 1, red act on n mortality; (xv) die reduction or elimination in the spread of the bacteria from one subject to irmt\s si ,'S„a, ,h mie ois m o' e owe v anodk'i ,-ρ,αΐ· m m-sn. uva the pv ', nmm m eu meteu-e m t| e rnoOs'i m iwi ! v iw ν'* t'i ''res omum of me bexoh'umv«i m « w i o a haeia-ai infection or e;v m n\n\ svmptouw asmi cued iheteouh eoui the mdikdon m the ' i" '* 5 of sv" p o 's wittw ' etw e ' * et o' ^ G Ή ’ ~e „~Wien n + , das-mm « d i'' O,"n a e ,u'i tition cause *«" oi iS"k"o,,ed w oh a beere'iel ""'eetie;, iv)>;, inhibition or reduction in production of a bacteria] toxin or toxins associated with a bacteria! mfeet-on, { wo the suduh/mion to reduction <u ushaounuiKW ;i ited wdh a h,ted'rial infection, {xsin the induction of the expression of one or more defemhn proteins and/or defensin-I'ke ps-w 'η-, i w i i the odu Opr o «ho oe' xs«en «1 -'«e or mp/e 1 o; 1 hie i e, eptor«, w''s) die induction of the expression of one or more proteins that are beuefieta; for clearance or reduction in a bacterial infection or one or more sympt.oms associated therewith; (xxvi) the reduction m mspu Indus., , wx * 0\. 'νι. , - ο. . ! ί U ο , u>\ ν·. Λ' ν , ν. ' ν ον' \\ο > ho prevention of the onset, development or recurrence of a condition caused by or associated with a bacteria: infection; sod or U^edO improvement in nuaiiiy of hie to. assessed by methods web ' 'ν' '0 V , vS ' '-v'\ l"*l Π, its, f\ NO ' \ ' , ' sNAG nanofiber refers to an amount of a sNAG nanofiber composition specified herein. ¢.:,¾.. in tseebooiAn ratm*. |0O32| As used herein,, the term ''elderly human": refers to a human 65 years or older, [00331 -\s used hetvtn, die term ' human -r-ls U ' .owns tu a human th u in y.uur· older HHB4I As used heroin, the term "human child" refers to a human that is i year to 18 yearn old,: [00^5: so-A b, r, ?n ,he totm "hunac nAnn ' - on r« u> new v-nn. \v \ . oas olo your hsmas:. 10036! As used heroin, the term "premature human imam" refers to a newborn to ! year old year human who was born, of hears than 37 weeks gestational age (e.y,, before 37 weeks. 36 weeks. 35 weeks, 34 weeks, 33 weeks, 33 weeks, 31 weeks, 30 weeks, 39 weeks, 38 weeks, or kss than 3a weeks of ptegnunevl 1003?! \s used hemin, rbe term ''hitman toddler"' refers ?o n human that ts I year to 3 years old..
[16)381 ws,v » w w kr" ' ο v a ' ν ο ν v v ' ' of %, 55%¾ m,· [00391 As used herein, the term "subject” and "padem" arc used interchangeably to refer to er imn.droj,· ,.vv,\ bow , dvcf, pc., eh-ch' r, u,w, Λ, .. ,u, non·:/-,, τmb-m m. me a pse, ,o t In a spo, 5,7c cmhoetrw, toe s,f \v· -s , me ions! 'ueh a*, a ί\ν men A, w a gt,nuk' e'.g., a human In specific embodiments, the subject is a human. See Section 5,5, A/Ar, for more information concerning patients treated in accordance with the methods provided herein. |0040[ As used herein, the term 'low expression,” in the context of expression of a gene ,, p , bn-ed on the lose! ee'puxem O' pepoo, ptoJutod p h,' ;ν\Α v,iw to an '\-nes\Or ‘,h ,* is loss than the "normal” expression of the gene. In a specific embodiment, "low expression” refers to expression of a gene that is less than 90%, less than 80%,, less than '?5%, less titan ?0%, less than 65%, less than 6054, less than 55%, less than 50%, less than 4556, less than 40%, less than 33%, less than 30%, toss than 35%, or less than ,3055 of the “norms Γ expression of the gene, fn another specific embodiment, “low expression” refers to expression of a gone that is ahous Dtoold, about lb-told, about Ηϊ-told, about 5-tokl, about 4-iokt about Mold, about 3fold, or about 1.5 foid less than the "tiorman expression of the gene. (11041 j Figure L Nanofibcss admobte Akt 1 uotivatton, an upstream icgobkn· of Fts I t Λ ? V-e-un Λίο 'ss Λ \ ' \ \ , %^ί jnu \\i( x -mi, , , e sen η mui\ed ΐ'Γ. ϊ B) RT~PCk anal>.sis of BC infected either wi-h semrnbkd eonf el t '“SCk' ϊ or Akt} \ s <Λ \ e " ’ n ises m ' e-ses m o\ ' v\', e * . f j HX* ,s a e „ - ” ml >0
Sefedrnttto: of®. »tg®®i feattodustlou: pitowity #aM|dutoig: :i sigtisb;#0to: sM A© daaillfestsitb:, AM l ,,-E-si and ©a tensing. |00421 Figure 2. Delayed wound healing in Akt! null animats is partially rescued by
Ttoiderm treatment. (A) Representative images of wounded WT and AKT I null mice with and with-ei feat me: U of Hi icon'i sBy tlXv oumeu,m lome-vuum', > cmeso ski" seenon- tom 4&amp;y:-3:-'Wbg®ik> 100431 Figure 3. sNAG nanoftbers stimulate cytokine and defertsin expression in primary endorbebai cells. iAI lmrmnio!n>toebemHty of EC treated vkh m without sV\G using :a: antibody directed against a-defensin. IB) ELISA showing that nanofiher treatment of EC results o' -he see: croe-wto defensms 1 nm source no vd -vli > s. e τ -ί *0 . g ms s\ H i (00441 Figure 4, :-N \(1 nanofibcf'·' Ximutofe dtomsm c\0:0,- saw) in rnnwrv' endotb-Hul cells to an Aki t bop:.uderu mmme·. 1 \) ;jnd ίΠ) Qmnoo'mve RT-PC. R .oudyses 0: sonm starved ECH'ss'd treated with or wuthont sNAG ("snag'to with or without PDdHOfA ; MARK inhibitor, *'PD!'|, Wortmannin (PI3K inhibitor, “‘wtm") or infec-ted with a scrambled control C'SCR”), or
\k:l 1" \k!V - -d-RN \ turns mss, -, .ss^-v-eb fm 0^----^500 o: Hie gene- uitoourcJ (00451 Figure 5. sNAG nanofibers stimulate fwdefensm 3 expression in mouse i.erMin0cy,f&amp;, fΑ| Imrpnsoito^ toiflf fsdgfgusto. 3 fGisibfe: m i;righd:sMid:p|: fto if:s uppv: oglu hand panel, see, 0 g. thick white nnvw-o and hr-otoeem antibodies ι-t |-;u,HYin embedded rnotiv’ cutaneous wound -c- lions from \V Γ and Akt J null unmvjfs on Day e ; B'i Quantification of D-dcrettsin 3 immunofluorcsccnt staining using NIHImageJ software * X oi , Ά ”ul 1 HH tom v’^eHno" s^u- s- ^ e ά - ,ni r I tv \d ind on treated keradsf" ytes null toDefc:vhi ^ {visible as hoe,hi -eunmy; --0-,. -: c,, deek while arrows) and TQPRQ-3 (nuclei staining: see. e g.. thin white arrows). Notice the increase in β-Ddensin 3 staining in WT and Αία I Talidcrrn treated wounds, |6U4fe5 figureb V\'i eepeneeni mmvtnp'mn -Vioi ''snesn; -ms Selumat ,, Ά \k;l Jcmvde w n\o* rpmv Gi to, b odiop, -..V' * \mp rc'viounx smosau', c<00 wp vi\„e m ot th transcription start site was analysed for conserved sites on the mRNA of DBF I« 4, and 5 (ETS-black ovals: FKHD-*tripcd ovals: CREB-white ovals; NFKB-checkered ovals).
[0047} Figure 7, -ΆΛΑ Ovanurrst icsuits in espies non end vXtefwm of detenstns sc Gv..; > ) RTPl. R n i \-w·. of -·,·\·Λΐ’· sta vee P w A p am ,r\ v\"dotool ,u *,elG ace r won x\AG 150;.ρρ/ίϊϊΙ > for the tunes indicated and assessed for expression of β-dcfensm 3 and. nt-defensm I. AG In i ise^duoK'seeni l ,.- hry "fen mm r rd e; k cAv' so; err :> <t\ ^ an no m etes neeee woh \u ranoidvts ; uum nn im ς ns' νΆ,νΑοχ cue Jeweled ig.m.xi o ,( A cm v \FH\\ upper of hand 'eou'l p defeo0 n i <vle\, ^ b _d, upv* iGot dev p. n„ 'f \te»e. -re to, \v woF TOPRO-3 (Blue, lower left hand panels. Lower right hand panel represents topic overlay. {€) fmmunorluorescent labeling of keraiinocytes {HaCau that arc either scrum starved (untreated) or nc, e s e^s\X't unoitbctS' A e "i! to -1 ~V G* >oJ es as droned _ ' -. e-1 ' s ί Π ft , nppu ,eP bane '» n F dekuses 11 fev.-s Red. η<ν\ i ii.Lt t mv te 1 \ wA' ax stained with TOPRO-3 (Bine, lower left hand panel}. |8l>48} Figure 8. «NAG induced defcosin expression is dependent on A.ksl. (A) O'. ' \ S v S V , U^O S 1 V. 0' 'S O'1 O'" or v\ \
Isolated from serum starved endothelial cells treated with or without sNAG for 3 hours, with or without piutvamvm w-th fTFOAGo p PD'};50:AK woitmanntn Γ Ά PvT'h iOOnm»
Gumm tuPon ;s χΟον to the Όο p^tom .stiOnou it)} t^u^-ttn.it-on cbp-dotenMo 3 x pv '\sion from total RNA isolated from serum .starved endothelial cells heated with or wltbota AT AG for 3 hours, with or without PD9K059 (50μπιΚ wortmannin i lOOnnt) and show'n as relative to S26. {{.'} W "ti re Olo, ,m,d^siv of phosplw \fc to, ^ usn -tj}\, J endotheha , ed' xS'-\ suounatsv w it t sN A.G for the times indicated. Line indicates where lanes have been removed (D) Quantitative R Γ' X X n oh ses ^ nets s',,n \oe or Jod c i.d ce K mTwo w n t,' s, run -Ra O'^nno,;St M os
AkiJ shRNA Icnhvtruscs, xcatcd with or without sNAG and assessed for a-defensin 4 expression. Quantitation ts shown relative to S3.6. ;E) Quantitation ofp-dcfensln 3 expression (torn total RNA isolated from serum starved endothelial cells infected with a scrambled control (Si k'i m ,\kt) AR\ \ letnt \uu.ves, treated with or without vN AG. Ouiinthunon ts \how-ti ?Λί IX χ· V S-?n \Π ;\p." 1-iiO'XtN 'X ΟΧ doS’C η.'' WCs' 0 i;'i\'VO urd 5000 ih v it lc<wt 4"ee independent times and p values are shown, }0049f Figure 9, sNACi induced defensin expression in vivo requires Akti, (A) Faeatfin embedded sections of cutaneous wounds harvested on day 3 post wounding from both WT ίη::ο} and A.ktl mice. Wounds were either untreated or treated with sNAG membrane, loom ·,.ί 'uetevenee w.w p, rorucJ nvue snubi-h' ί JueeioJ |ΆαΑ U'.u 3 tgwvn '•as-l'k n,\ bught mmnug ni the nppes ugbi hand panel, s„e e ι,,, s hi A 0 hi, -n'>w\ inudut. -m (Rodh rfiid Ί npio i Blue f’uei> 5 simmsm, w oi., vhno bun mo λ -,) (i\) ΡΑι.οΠο emhebdrod : I <'i", 1 v, anv ^ on Ά A' h. , *·\ d >'>' >' o e k'rmenoA„me wc,\ |\" m o\ , <,x,eg si’Kveu Amove ,s erw w v" mu' o' tv'v ' ,u t" r-,, w oi thus oboe anowsh Iv-dow iRoeh and b'pio ·,Κηο, neeA' m m\„ see e e Am wh'V mew s' i As ie'we η\χη.ν n.on s.>d\t is sn,,uded to better iAwv the -epioeo v. 1 nos optessux A doibw s A.Aeku- ^ n’t. id''dui!uei!(nrt kvv ism * e\m\wn Pom pokuTe embedded sections was performed using N1H imaged software. Experiment;* were repeated farce independent times and ρ values are shown. 100501 Figure Hi, sNAG treatment increases wound closure in wild type mice. H&amp;E staining of wound tissue sections derived front C57SIF wild type animals either untreated or no red wrh Ά \G ·ικη4’'ϋ < The Aw pos^woune ,s mA; avd to *h Ah >' i aeh vn t 1 uc solid black line follows the keratinoeyse cell layer indicating wound closure. Black arrows u dune Ιό n'.ugns of the wwune poo |005*| Figure II. sNAG treatment reduces bacterial infection in an Ala i dependent rrsurer ί x' ft-sno at an van· og of > .".>i n- unf\'ed wovtis aom A V iv, a tv f mu e woo wounded using a 4 mm biopsy punch. Immediately after wounding rntce were inoculated with 1 s Hi'* cfu/ml. 30 minutes post-lntectiom mice in the treated group were treated with Taiiderm, Skin samples were taken 5 days posMreaunent and sectioned for analysis. Tissue gram staining was performed. Dark purple staining indicates gram-poshive bacteria and neutrophils that have civaduv 'vtoi.. S'.w,.ons n"der Γ0α unM ' m.quAvetvi' ate Aw' Mi f,\-\e cram stainingol"paraffin embedded S. imrvox infected wounds from WT and Aktl null mieetn:::3}. lokwvd worn d.s we e cun.?' unt rented or fronted wrh MAG mor'erane and wennui Ivcis wete i wwAdk u ' ,J Λ ^ 1 \ M w \ Dokm s‘e sfammu u dmevs 0 ' es \o\, -pO "-0 \ v'l !li VC.\.\ \X’ * uk {ΠΟΧΧ^ ΪΚΟ UC \ ' ' Λ V' Os t ' x
\o o 4v it.^ί ιΐί li mn s » v * u-y s imu <He»t W~ f <i ·» ,λ __ ' V- ‘ ir r a \ re o' with «NAG. Scale bars ::: 50μιτη {€} CFUs derived from dav 5 post wounding wore quant! utred from S', aurcin infected wounds using, both treated and untreated WT rim 3) and Akt \ mice .n ^ Wj d t\|v mv- ΐ i e vi.ere sVul ;re nod \v>' a signtlie.nr if- U > Cecreu-v m u. visa load in the wound beds as compared to Akt I null animats. AH experiment»» w ere repeated three independent times and the p values ate shown, f D} CFG quantitated from infected wounds at day 3 post wounding in a similar fashion described in (C), sNACi treatment ofinfectcd wo unds s sigre are do reuse «- ί f'C ot tvuh vs I dee Akt' re., I .trm'.ik un 0av ) bin the WT animals show an approximate 10 fold difference compared to a 2 fold diftcrcrtce in Akt I animals. IB) Quantitation of CFUs m S. tsureuy cultures that were either untreated or treated with ' ereres ameun'» re A Ά» nem », kK'wv\' '. λ λ iv o \""».e »e ' \1,νΆ' vs and ρ values are shown. (F) Tissue grain staining of .S'. ααπΊκ infected wounds harvested on day ' re-' n^und Hmo W I mtee a G H .n were re .- J red' »»' a .«hoA Λ devos.n » ;. po le .. o rMi Area tue dev »> re -T pevuve As, rere. re revved wet "ds Hu i we e '.re nee u it- p def* r sm » rerH d (G) Quar'Amoo e't H \'Vom .s' , . winded Vv γκοα 3> re He»’, with or without pviefensin 3 peptide. Infceted wounds that were treated with peptide show a .significant decrease (p <‘.05) In CFU. Scale bars ~ 50um. Each experiment was performed three independeru times and p values are shown. jftOA?» Figure 12 k»' ^ *>di re eerere ca os d v ' ' s . < > I eku »v \ „ vs Γ'ν i * » er vt do». » .ο ν' e ' s << '» -. »»·»'. us
Ikuve.sied on dav 3, were .subjected to immunofluoreseenee nsmg amiixidies directed against IV defonsm 3 ipe r., srem c u\ ivgln eterenre m due upper π phi hand panel wj re the low s panel in the middle; see. c.g.. thick while arrows), saved uenn (reds to mark the kcratinocyte layer, and Topro (blue, nude! staining; see. e.g,. thin while arrows) front both sN AG treated WT (n~3} and ti»Ui»«u'»c W ) >'.» c,. '» \ ' A'.'U ',s o' s». t ' » ?'die Hod "» f , noare eon on which was warned ·,. p· re-order» .tntuvdy onl\ 8-: Go i re aopru CBt Gnvistlt dien »H'p-defeevn -t ^sgtession Bom ρ η'.η'οι. eswfdv. 11."0\ cvnu AH I imav ) ^ueas e O v;. u , mfeek'd »o sues the*»». sc neater, with »-N A- show a ssgnnua.". uu ev-e in» OG nt p-.Lre.wnt ! atijining. Experiments were repeated dime independent times and n values are shown.
Hgnre 13 AwHfee.cc·» a^,. cm H det-vs," » .,'.»v«es oet.lvetes.». eoe w efs\ Vv^ e met v vo "suMureoi v. * m OvOded s wrlu ,d"» v e ee with sNAG from WT mice in~3) that were harvested on Day 3. sNAG treated wounds were wif|:diti:erf~dilea#is:J :aiiPolA:OP tldiyps/ eornroi goat pk$r its iMAGr |lei5rciefiMiy«:|ms|Gi ill®w t® or eaiei: secispBliite gpix ::|sMti^e¥Slsls5feg iMmM ,n owA i i iV wooed \vs of t\n,o A, ,ned oU5 ..·; ,η".,.''-\Α dr λ ied ngem® ρνο,οηΜ i ' So . o h.t Λ*.!'!! ί B) (n.-mtiHfior oft Ft \ item Λ ," ·,μ u fee ted ^ i m.ee re nod ,ν.ϊκ" P< defe: ·. n ; anybody -, t u or eoneoi lev am Kv^ in G pn^r o' Ά A- neoin'-ent fRη in ί \ app lesi IM: JigaiSpallf; f ndtsisecl · Ify:<SM, CPtl, fOOSi| Figure i4, n\ *v ηη,οη ots{ f cd κ, - Ret, moi 'ireeiio'> e\ <e, i<i\v y -u\,-Mw„ oete vvoutided e-mg ^ 4 inn ί ιορ'-y paned, ntoc dated w.it. tr \ ν' -,\n etl t’ „ wow i 1 v.\J λο,'\ .se\ v. v", ' ^ oo N ο o o' 'Mg A 'Ά v'F .,> ' V» n »n '\isn-re, Oiu'wnnl 0-.,G wok* 'lareuee im g,a\ * v- ,n nU -,w euhno J to: 1Γ- o unnev plated, and CPUs of the untreated and treated Infected, wounds were quantitated. sNAG treated trie- -dvw a egn'be eit to G s .i,\ r,\,.,.,- 5.' i- ,-, we 0 'nod m h -, wound tOiG ,0 - ear·; ,,-ed ό «η treated m I mala, j 00551 Figure 15. Effect of irrad iation on chem ical and physical structure of pGfeNAc 'Ά 0 0 v'^0\ ? to Ov. . v ,'. 0 m'< \ X, ,-j < oOnO on '< ' Ae tot nsadiaooi iBi luxated HR} -g- Anoi os non mudur,'. evi- \ \, Go -. fop one's S'G v \ ' ni i,lni , he κοχ v \> io<r 0 of 0 4 v vAx , ' 0 dried u "OJ ¢0 ' v-.
Luo'* tG'i Foanunsg dee if on euro'-e-que APM'> nn„K-. - e''f' 'LN \o {D; 0v.us'eu'y oioot'i>'' microscopic iSEM) analyses oi\sNAO. 100561 Figure 1.6. pGIcNAc did not affect metabolic rate. For cadi time period: (hi?,., ai 24
Mddfledc>:urs|,: tFo- MeotSy :lbi' eadh: of lit©: :|duf firorf: f eft. to f iglGdy ase id IdAi;. fpridi: oarvcuon \ F^A , ud pCno\ X' ;N '0 Ant >d aid idd ,y rd
Figure i't ^doA Xf pioioeiod imoian ιηοϋη,ηί \o'n ofn^vhoFa: v,e‘i iEG; Fo.-u cell d*.ur sndunce i'\ \o ns" , ^ se < to -, jinvpM'o. , ' d ; a ore'''' R is
Os-Uins A'' O'n ' O'' he live 1 j·** s'G r-f io‘t n- ne· 'x -s j" id o^s worn "ϊηρ,.η,ο·' 'Λνχ \ i.GF,
Mi $ .pGIdN Af; |M A:0 i 4|;··5|^.:. f 0¾ .Md 5d'0:':g§| 10058| Figure iff N Xu thu Knd ;n1 vo *\e s' t < car* tooo. *y (0 0. ' 01 ts <
sO if ( r« (off 0 it \ ' ov> \ wsfitn M<n yam > , \ ' ' , \Ά\> s! GO |0O59 j Figure 19, sNAG did not protect FC from cell death induced by serum deprivation, i m Os n ' v m , m 2~ aao -Λ '. ν' me k,w> m \ o-.' o', s No urn χ i'<uv, let? ,o rights is as toUuw-s so nun star-. -^io-? {dSt x HOF, >md ,s\aG -it cm ]00 and 200 μ a, ml
5. PET Vll.EO PESCR1PTION \mm Πιο mv mom Ϊ >v\. d ν>\ο\'·Ι 'no, \U namAiK's v ,w ' ο-, >ae\ mri , ηο-Όοη id cutaneous wounds infected with Si&amp;pkyioeoccus rmreto and Pseudomonas aeitrgfnos’ut. Without being bound by any specific mechanism of action, data presented In Section 6.2 suggests that the antibacterial effect of sNAG is not due to a direct interaction of sNAG with the bacteria but I* dec to downstream affects such as, for example., the regulation, of def'imsin.N by Akt i activation. Specifically, data show that treatment of bacterial cultures with sNAG nano fibers in vitro does h-u ntKA χχηοπ d emsm in, xxnmg tn n s\ 'G ow mlG*- du not o j; th uhTw be, temri ο,ιο-Ά. In λ y volte „\ampl, dcvoabed in S-, cm-' c 2 2 0 ana rihsMiafed m Fwme l!l,A V< nanoilbeo; do not barm a duvet ci Nxl on oo'-w th or vnr, mas uf AwA, A voa an a.or w. The ms; dess need n A-, tson " 2 ' \ ' ; may f i used to tost 'he tool ofa f'\\ t ef\\ \( nanotihers on bacterial growth ;>r survival. In this example, S. ttttreu* cultures in solution were to a,cd w ids -aswn'g <m'<eomatnvs m «Ν ul ϊ',ηΐοηΚην tor P - ,v hoar- cub urn- w m dura ,\ e, v, ϊ t , ^ v mu I ~ucs a Gt ' „Uenvum,o, 'λμολρ ι I , > 1 ·ο,*-.ν' on bacterial growth or survival was observed.
Mibb it 1'he inventors of the present invention have found that sNAG oanoftberx can stimulate expression of defensios, w hich may boost the innate anti-haaerial response. It hs widely accepted that defensors are important players in innate immunity and function in. ami-hautewi „-,m.t'Cv V eeovnsomed ir th- cv nnp ,v n;, v-.nxo m Se<,iBmv ο I ene 0 2, c me ϊγ\ογ 'wot t !- p’vxcrn mcmuM"! tune fotmd th n \t\ \G mmeFtvw can η-m ,k, : he ox ores-mo m both o- und f" i>pc ecteouns n endotbeivi veil- end ρ-pw -vows n,.memo,*, - ;n \it;o and in a wound healing model in vivo. 10062f Further, a-, demon-euued io the Cv onplcv ore-.-:-me·,: In tketumv o I e-nj 0 2, ri\\but w if hoc tvtt'u hound b\ - -, ssveriv "nrehemsm ,m „et t-:-, '--el mnow to be νηρο-ί,-ηη M t\ Xv -depend, u beef, n-e? express,-m In uin> and ,ix \"-,\ m a woenJ :"e,fvn- rs-rvl <'ansi:Stently, s’MAG treatment decreased bncterinl infection t-fentaneous veounds infected with x, ' - wiki - (mpmiuo n ih e if mt n v " , - e, 1 ΛΚ r ,1 animals.
[0063! The inventors of this invention have also found that a. number of Toil-hke receptors can be tmoegu laved by sNAG treafmem of human cndot.hd.iai ceils, Toll-like receptors rTl.Rs" or 'TLR'’> arc highly conserved receptors that, recognise specific molecular patterns of bacteria; eompone \\tdov 1-¾ aetnamv m n5»,<,e smmon.rt Roc. m so;! !\s„s linked Knout' def- e-on expression to TLR activation, in particular, stimulation ofTLRs can lead to increased defensin synthesis. Thus, without being bound by any mechanism of action, sNAG munsfibers may act as a stimulator of innate immunity and bacteria! clearance via the activation of Akti, [0!M| Act m'dmyfy, described heron? the u-e of v\ li: ruoudibem a-' a 0- --.01 ov'hod for preventing arai/e-r treating bacterial infections and -diseases associated therewith. In certain ^ he,'moms c-V'",'" ’ h, 1 "de, .c"- -v Ά Xio ,« rit vo \\' , \ - v , yr, U'rfst ?n paucrtt·' In e?viwk'nems she n^e ο* -Λ Xvi ο e ebnei's u; nances Aonnd < O'-mte while simultaneously eradicating, decreasing or preventing bacteria] infection of the wound. 5-* [00651 !.)evub>,.d ηοΌ'Π a?c - \A<; n -.-5-¾ 1 .. oamo-ortons ! he s\ V1 ''enoluvi- e-m?pi.so fs'vis o? pah \ UvOi\ ti.Uteo-* rri'sv and o! a v. --5'\,no -ts't 6, too'' the o mono Hwh s' an' k",\ than 30 microns in length and at least 1 micron in length as measured by any method known to one -hilled in fk. am f-v - sample, by se-mnmg elevtroo nvs-osem's p'ShM d btsvh -,\ 31= na-saidvrs hcomam-ed loresnnple as tlesenhed neresn 100661 In certain, embodiments, the majority (and in certain embodiments. ar least 60%, 70%, 80%, 00%, 05%, 9K%, 99%. 00.5%. 99.8%, 90.0%, or 100%. or between $5% to 65%. 55% to 75%, 65% to 75%, 75% to 85%, 75% to 90%, K0% to 95%, 00% to 95%, or 95% to 09%) of the Αλα naoo;Jv'\ .a- k-siu». about K K\ A, \ 1Λ id, 6 f, ", e, Ά,5 n?n torts -n length, and at least i micron in length as measured by any method known to one skilled in the *rt, he mernpie, K SbM, h? specific embodiments, the tnajonts (and m certain embodiments, .- R ,-- 5- -, V A % '' ,-A' e KR' -'en-ieu
55% to 65%. 55% to 7553, 65% to 75%, 75% to 85%, 75% to 00%, 80% to 95%, 90% to 9534, or 95% to 99%) of the sNAG n&amp;noflhcrs are lews than about 15 microns or less than about 12 micro?'!-- in length, and at least 1 micron in length as measured by any method known to one skilled :n the (it, A esasv. dr, Is 3IA1. in spec!"' -„ CiO.-eM ovm-,, Al s. ηΆ '> -it »1 e sN V I ϊι,οο*Ίόλ aie L's- than ubu ?t I ' e?u rm s m 1-¾ pt , n d a, e ss, 1 mw’-m n h-s-A* -5s n eave-v, by any method known to one skilled in the art, for example, by SEM. In corners embodiments. Ί , ' on u <t' on ms e οΚΝΟΌ/ηκ ,u k'j"i et.*U\ V'' o, ‘'V''. Λ> A'', 90.5%: 99,8%, 99.9%, οι 100%: or between 55";· to 65%, 55"» to "5%, 65"» to %%. 75% to 85%, 75% to 90%, 80% to 95%, 90% to 95%, or 95% to 99%) of the sNAO nano fibers are equal to or less thus· 14, j 5, 52, 11, :.0, 9, a or 7 microtis irs length, and at least 1 rmeron to length as measured by any method known, io one skilled in the art, for example, by SEM. in some emknh'uems,'he n mono, mod ?n ^enan ,ί'ιΙ',υιι'λ' w \* ',\sa* mV >, "0' ,, ri'%, 20%. "S , 98%, 99%, 99,5%, 99.8%, 99.9%, or 10996, or between 55% to 6556, 55% to 7556, 65% to 7556, s a, \ ^ ό °P '* »%&amp;’"' 'ur <, too '' ' °° ηΊηΆ \l 'uv' \ are between 1 to 15, 2 io 15, 2 to 14, l to 12, 2 to 12, 1 to 10, 2 to 10, 3 to 12, 5 to 10, I io 9, 2 to ° ' e u ^ ’iiO o ' N >o ' e , - o , e ' to - \> o to ' microns in length as measured by any method known to one skilled lit the art, for example, by ^50h% lr η -,ιν,'ϊ'Κ' emhoJ'mem, t m trtMo'srt posd m vAa » et" aommensa, a. ic y eV „, %%, Mt%, 90a, os * „ ‘»o on y , ^sh, eo oy,, ;,ys ^ o; between 50 V to 55"» to 75%, 65% lo 75%, 75% to >%%, ?%;. m QOnb, 80“.: to :%%, 99% to 05%, or 955» ro 00'\n ,9 'λ, -,\ \ι,1 η,,,ίοΟ',',η» a e uK>·,* N. ", \' 'o '; ο,ννο , t 0‘Vts' ,.s "sCus.sk 2 K no method known V- on,· skilled in the on, tot example, by Si M in any her \p·. mb', emhodinsutn, the majority (and in certain embodiments, at least (=056. 70%., 80%. %}%. 95%, 98'.'?'. 0-Vy y'' o> K, ' %>% ,- - h " » ' l· ' , V<
So",, 5% to yyg, ίΛ On"„ η» ., f0 oy , ^ ,>< oy\, m o ',) o'·'the Λ 90· n.t ief.eem >re be-wsx t: about 2 to shout Hi mo-roes, about 5 to shout h om rons„ or abme. 4 to abom miomm· in lemuh :» measured b> env method know n io one skilled tn the an, for »ssmple. b> bb%. In as'otivs specs*',' ποΚχΙηηοη! '21 , 0M%' of nr e\ ''-G > uuoihxas tJ?v bywe-m timet ' to ubmU '0 tsssets''ts, uuom 6 to eoout 8 m\'ts''ts, ot about 4 ,o about ' miC'Otw ,n Uvetb d' maasnmd K ui \ nemod snowe loon ni led m m/.0-, v> evs.mcle, % sf*A1 |Θ068| In t,en ts?: esubivliments the A X(s namsilbets iifjetw ,uc n: a ttsti'..'- bciwet':: if 005 to 5 microns in thickness and/or diameter as determined by electron mieroseopy. In specific ewcoe ' 'sis s' , \\ XtsssUs -Π', s «ί d' top ,*qp u ,s, η ;'4, n t*y ,%»?*, 2 st5 o h\ P no i' 2 0 % 0 H.'h t..... e 0 o' ' ^ i*' i ' 1 „ ' ' HU, 1 ?, 1 o, 1 '' iy U% 2, 7 2, ' '· ,e λ 2s οϊ 4 m-erons in ditekness mm os du.metcron o , s w , , v , o 02 , ' ' , o ' 0O2*o 1 ",,o ' 0 92 o ' " η ν*Όη>* 55 ¢0 9 > 5"5."Οϊ’\ 0 0? s-' 0 ' I'nc’ors., 0 % ό 1 n οόρ -, 0 0: ;o 0 ηϊ^''^, 0 Oi so 0,5 microns, 0, \ to I microns, OJ to 0.75 microns, 0,1 to 0.5 microns, etc.). So specirio s" "" ' 's “h ' V V ' s ' 0 ',W V ' ΟΛ '<* ' v ^ , ' ' ' , ^ "O 0 J0t ,, O " i< -s'- ' . O'' , '' ν'-'' , 'N o-V' , % f lO '' ,99 , to O'" '0 ' \\ % uOf*<"b,'' h o.o a ibk'ks'ev. os 05'm etes to alvnU o 5k' so \ smelt's Ist mhe' -ρο- om , 'οΝν-Ο-ηοοΝ ,bc majority (and in certain embodiments, at least 60%, 70%, 8997, 90%, 955-7, 96%, 99%, 99.557, "'\ " " >n 100o! notkom ss , ,o, ' "-o' 'S Ό '' '' o\'' ^
to 90%, 8057 to 95%, 9097 to 95%, or 9550 to 9977:,} of the sNAG oanofthers have a thickness or 1 v o 0' tti J ' v o' is\ \ o'1 Ό ', ' i h , 'ο Ά 'A ns. «ο Iv's^'' ,!. 'v\ se^ οι 0?, ' so <7 about 9 o; ,o ' sme m's o month O' ο o c 'vs.", in certain embodiments the majority (and hi certain embodiments, at least 60%. 7ϋ%, K0%, " V '''' " , N " '
n v - 'n c"'" , n m s0' , v , v , ' -s o'" V 55, «0 kk’ S ' Ϊ 5 Oski 0S\ 05 n , !Vk' Ot a"!OU5 ') U > "i, ' \ Ud! >5 1 5" O''' t! O'' 0 "5 mio'otiv i) 05 to 0.5 s'Si, toms, 0 % to 0.5 itiieto" -, 0 05 to ' vk"'U"-, 0,05 to 0 77 ms- urns 0 05 to 0 5 microns, 0,1 to I mseron.o, 0.1 to 0 ,?5 niicrons, or 0 1 to 0 5 oisuoro I0069I ΪΓ: c-olcin embodiments. ‘be imsjouiy tarsd in certain ei-iK-ass-urni·-;, K,-;jo 6<j%. ‘Ό”·-. ' V' " ' '" ...... ' ' '' o Λ < ' O 0' ' o' , ' 'n ,"% m 'n 'Aha''' ",to'V H%, o'-'mo'v . 'Ήίο"' ' o'm sNAG nanofibers are between i and i 5 microns in length and have a thickness or diameter of about 0.07 to 1 microns. {00701 In certain embodiments, the molecular weiuhr of the sNAG nanofibers is less than lOOkDa. 90kDa, KOkDa, ?3kDa. 70 kDa, 65 kDa, 60kDa, 55kDa, SDkDft. 85 kDA, 40kDa, 85kDa, 30kDa, oi‘ 25kDa, In certain embodiments, the majority (mid in certain embodiments, at k ,5 st * 7%., % NO'.', '81 \, It S' N S^' » „ 0» S ' , my ^ OO .r' N (}f 100 ' „ or K'tUOO‘5 ''5' .' *o 65s' >, 5:!‘« to 5>.'f',! 5o''5'to 55%. ".'‘7'm -7V-,, sti'-,· to. '.·9'7' so 77--.. or''1.'”» m 99%) or the sNAG nanofsber* have a molecular weight of less than I OOkDa, 90k.Da, 80WDa, ',5kPa, A* ki%, <9 M)a, 601.1%, 55kDa, 50kPa, kD% 40kDa, Am-1 ia, HA Do, ,-t 25kDa tn othsT enAvdimetu-i, ¢0-- nsuioritj sand in cenasn embodimciUs, so 1 coos Gv'..,, 70¾.,, 70'6,. -?ο%, O ' sW , ' ' s'"' s ' \ Ο'λ, ' " Ό,' ' s -0 's ,,N ,, ' S , N s o ie . „ , * Λ Λ 0 Λ <s ,, 0Λ 0' s\ ,, nano fibers have a molecular weight between aboui 5kDa to IDOkDa, about iOkDa to 10OkDa, about 20kDa to 100kDa. about jOkDo to HQkDa, a bom 20kDa m kOkDa, 20kDu to '.'xkDu. about ?%l)a V , !<Ha "HlXi about *0hDa m about M\Da almd ^01- D. to „D„, ''GD , »x l -+41,-1, to about 80kDa, aboui 40kDa to about 75kDa, about 40k Da to about 4 Ok Da. about SOkDa ro about "OkDa, or about. 55kDa to about 65kDa In one embodiment, the majority tarsd in certain. cmK-hm, :ds, ,u Last fit* * „ '0%, N4%, <** . '-·>%, 4% „ \ ^ ν' , oo >» ,*« ;P0 , O N'\'v\' NS o, ^ , ‘ο ' ^ V ' s V\'' s 0 9'u' , <o ' 0fjibtO''' v 'ν' ,' eo ; f 3a Ds V ' 'u.v* be Lu'^r , \se%' ^ > „t'GkDa [tbDli it1 o i o o" !' d ” *<- e ,N o v. , ^ o' S e*o m ά m eO'b'O: the s\ -ub os. no fibers are Joaeo!>iatod. to some emboibmem.s, 1%, v\.. tAyjs·'., 20s,k %%, ot *0% of Ota s\ %> o U"o: vm ,us >tej»,,'isbfeo bt Abu e'D'e-JimeuK 1, -,s b,ni *'4',, %%, 'OD !>',,, to1' D 7'b c." tof the sN \0 nanotuvrs ats'Oet'''rD,ncb bt some embodiments, equal to or more than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, ♦ ' v%, " O'% ' '' , " S", '\' ' oo , * e"% sNAG nanotibors are deaoefyl.ued. hi other embodiments, less than 1%, 5%, !0%, 15%, 20%, 2' me te ,-,, '0 \s , ,A , N AS'' 4 , '' et 100% of the sNAG nano fibers are deaeotylatod, 00172; b ee^no CiuD'dm.e.us % VNO',,, '> , V 1,4%, V S ''G, s-°, +,, »>-',,, 4p'tj to 95%, 90%» to 99% or 95% to 100% of the sNAG nanoftbers arc acetylated. in some
AsKsJene os, AY , N nA „ ? ^ ',,, %%, "s,, »e ,40--,03 104 oMbo sNAG eamenes s it>',Kv:d;‘ ,1 tu othot enlvennem.s, more but 'e' 4> bb%, A -, %i\,,e-' yei-, e oee , , \ Η n troAlvts ,e , ' „ H < ' e' ° e„, * t»e roe A3 0 ' % S , *0 O', + ' ' 0, e" '0 ' 0, 'se , N \ A >s. Ο'' ,<·ί dbt't' t oT f% 's Ao! ,n -ri',”s „e,s , e, D u ‘ 'S ,'Λ,ΙΙ.) ^ , '' V '' ' * 'D " b oN'', '0 "·%,, ue',, ,, on'',,. Os x -()01 , r·.· -he s\ \D na ?iof be-'s a caeA', atee 00173? Irt some embodiments, the sNAG na.n.ofibers con-prise at least ooo glucosamine rant·ssao-'h.%dc arD n\,:v L%·^t=cr ceom^ Ό at leas? ?Α% 2e'ki, etv-s, */,,, ssv-„ Ao°,,, bO'iL 90%, 9.5% or 90% of the N-aeefy igiueosannne rnonosacehandes. in othe- embodiments, the sNAG nmurtibei's comprise tu leas·: one N-acetylylueosunune monosacehuHdc, and may t'lUflK’: compose n Gs>a id's. ΑΆ,όϋΆ 5(,4\\ 'Vo, N0":--, vO'V !VG or 4’V. oi a \ ueosamixs c monosa ech an des, |0074i In one uspe·:· the VAi j nanofibem ineKaw die membohe mw of \,wnm-,om\ V human nrd'ihe'J otu( \γϊι enomheba' , eiU Γ fi'"! m a \G 1 as-i λ Λ Μ Π ass ο ?\ , laboratory ic-a? and a standard colorimetric assay (an assay which measures changes in color) for !' > a-, on ;; ecHn at -nobs ?a,.mt ucll mowdA 1W\, v dew Ml <" ξ Ve Vfmnoi'i\'d''a '.cm~ >h ' e dn"vu\ It ..v, .'imm bn muds, * K'uaeoV ,s icArnc, o' " n ' e Gis^ac-c *' 'h, rmVei i.m.u c os' in wo cabs 1 ins reduehon tm- s - Sac, or. \\s t rt nn\>cho~dr.a ' ,-e j:oa.-aj otvsovs au jcono, j/sj th, icfosa ., a·· vernmn .,;m be ,n;..vs > icGtco ίο the rmmi, t of stA e (living) cells. The metabolic mic of cells may be determined by other techniques commonly known ίο the skilled asusan 100751 In another aspect, the sNAG nanoiibcvs do not rescue apoptosis of scrum-starved EC w a trypan blue ev'UMmi test Λ nypiiu b:uo .,xGu\!on tee is i p\- oscinsv-n uva used m determine the number of viable cells present tn a cell suspension. It is based on the principle that live cells possess Intact cell membranes that, exclude certain dyes, such as trypan blue, Eosin, or presidium, whereas dead ceils do not. The viability of colls may be determined by other techniques commonly known to the skilled art scan. |00?6j In certain embodiments, composition··» comprising the sNAG nanedlbcrs are do\i silvd. whoK'sn the e\ T(. nuwudv-s nn team ?G. metaboh,. rale efsornm-Aarsofl human umbilical cord vein endothelial cells in a ΜΎΤ assay and/or do not rescue apoptosis of scrum- ·. τ'» tm 'V'! I'm!' 1 c.il u<i / '.or o'rmtc k 1 ,e ·. ' , >,y’fi css . o oo I'-sorv smi vdnn, ,si\, tlw Ά \G ϋΟί,ΤΚ . > seeuMse too meiAvlw Ae .η sed hemm imh H.U>\x so' csdothchd el s m ιΜ” λ.' < ideo' e\ „·, v " v .w .earv, d hr man mubd s<d t orbem ou^niuJ? J ..o is ,u a Yv\ < burn , w b;s or lev [00771 in a specific embodiment the sNAG nano fibers arc bioeotnpahble. Bioeompaiibllify n a\ sc betormm. ο Τ'· a \.nict\ O'b λ brouts 1 <c uJ'nt>, uu sot tim tod v- -mm p'o ivt.w - as the elution te&amp;t, sntramuscular implantation., or iniracutaneous or systemic injection into animal sab eeis M.cb a v .f;se s.sod in i \ ibioebb vyX'Ls.i' ο.,· i samrl; OP wh'ch :s incorporated by refereuce herein in its emircty. {11078, ir con»!!·' cmtsvimunK the Ά > nvw Ίχ-w aw' ρ fhe ί ιοΊϊ*ν\ devi.’i’xv 'U\ w are non-reaetivo in a hkicompaiibslitv test or tests. For example, the sNAG nanofibers used in the methods described herein may be ntm-reactive when tested In an elution test, an iniramuscuosr implantation test an kOraeuisneous test, and/or a systemic test. In other cmh'jn-nc u.\ me Ά -U I n t xjr.bev-. us-.d at the me-hoes cexrx'ivd fere η; have w'-ee o m wade tee v ' o a v ' eoee i an cs,k ne ,χ a' , ' a , x , a u\j < u a \v x- t, Ά w a sw\ n\e ,est h \,amolx i rmtV'hnwn -Jx' Ά \G nanotibois ns,d >o tee m, Jkxn dost mod hexm u tvos' ~m jk w,w x wt.n test, J m ,n eusoe'· x-e an "it' rmsveutm nnphxiaiion te^t. an nPra; umn-mtis f·, e, end ot a y-.stetmc twt In cmknn embodiments, the - ouxxcmxsv·. d, x;riX a hewm A- not - am·? an ait-e g?ok' non. non o:” an irntnnon m otl\n -,mU- hmenw, du ,νο.ροχηο-!"- described he: out ears, -,* most a ns d •dies gome tea, non -' t mild irritation, e.y.., at the site of application. The relevant tests and evaluation of test results are described in, e.g., U.S. Patent Ao. b,686,447, which is incorporated herein by reference in Its emmety, and In Section e b m;}.; {00791 in a speenk ernhe-nmem. the x\ AG nanoilbeix are ixm-roucn'.-' xhvn w,d m '? irdvamnsadai· implantation test, in one aspect, an inttamnseular implantation test Is an x m , v ) i ax , s SO ,,-,, τρ < , , wui d " bev+e*' N ' ' in certain crnfxxiirnenis, the sNAG nanofibers display no biological reictb dy as determined by an e utio*’ x o ihli'ant lest Gtade ' A in mux. cunv,' nxmix m. -Λ \G wee 1 -xu, ,- test score equal to OF' and/or arc at most a negligible irritant, as determined by inttucutarscous injection test, in some embodiments, the sNAG nano fibers dick no Irsfradennd reaction (t.e., G?"d· i r·.',!, ϋΐ'ϋ ; m k igni m test nnc o! 'χη e a weak ΑΚ'χχ'χη p-xw".u a\ eek rmr,c-,' b\ IGigmiurtcst, 111080! !ri certain aspects, she sNAG nunofiber.s arc tm.munoncntrai the., they do not elicit an immune response !,: {00&amp;I- 1c some , m\>droeiOs ,ho i'N wi ι'.ηηοΠΝ'ο-- ;e- iwxvgxkXib e The \ii nnovo -wee ' ,, g , ee ' ' ,\x , ,'s e w, ', ^ \ee\' s e It1 vi' ^ ", ,, \n ' t w " e," v, e\ ' ' , w w bhd -*0 du\\, I moiitb, id da'.'-, A* k.i\s, ds da>s, 5tt ,i:r,s ?' di>v t'tl da.1 month- o* dtiys, 70 days. 75 days, HO days. h$ days, 90 days, i mmuhs. 9$ days, 100 days far 4 months after adrmnistr&amp;ifon or implantation into &amp; patient. |ft082j hr ue mm oofoodtrtK ms the ν\ -χΟ no; m fives ό.ν· me erose- a Jevx-t foie foreign both reaction, Λ foreign body reaction, which may occur during wound healing, includes 'v »v> iu otc\.»c ' ' o s ν' ' .. ' vn'" >" \iftj ,000^ area, and she formation or granulation tissue. The pcfttstem presence or a foreign body can inhibit full healing Rather than the resorption and. reconstruction that occurs in wound heating, the foreign body reaction is characterised by site formation of foreign body giant cells encapsulation of the foreign object, and chronic inflammation, Encapsulation refers to the firm. eonor.d;\ ,5^ ,ονηΐ er collagen shed Jepe^md nxujnd -a foreign body offoehx oK wjfomng n -mm the host tissues, in one embodiment, treatment of a site (e.g.. a wound or a site of a bacteria i ! Let on n , o' -. I x*' 'tnorbor‘ doe no m v ee , ' o , ' v reaction in I day, b day,'., 5 days, 7 days, 10 days or H days after i-eatmem. !rs one such emhiKhnseiO, u'o.nnvm of a sm to e. a wounds vuh dv xV\G vnu-fovv d-.-C' not eh, o a foreign body encapsulations in 1 day, i days, 5 days, 7 days, 10 days or 14 days after treatment. ips'n i< h>,\\e ·> <->,'* the". * , " ' -m v ' . o ' foe bbe;s ,iU booxeen about : and i 7 roue η -> nr fongri:, end fn'» sat -rvrvi'-v the rnciebohe one ofsemm-starved EC in a Mil' assay and/or do not rescue apoptosis of serum-starved EC in a trypan Nuc exclusion test, and (h) arc non-reactive when tested in an intramuscular implantation w In ,etti'u on uofonn ms, On xG xfo η.ιηοίϋ-π*. t-' e^m-mse ''bets, vh, ;,\n ma'-nm, ofo v obem arc |"-,w en dwd 1 nr.d *nncoms m length and .,t t pO uvsetse On1 metabolic tap of serum-starved EC in a MTT assay and/or do not rescue apoptosis of scrum-starved EC’ in a Uvpa s bine cv/foskiK ,est, end s d „.o vm-,e .Awe when VetoP n; an -unarms, Mat -mofoifoshor test m .co.rn on ννώ nents, -he x\‘ \G nanoblw ν', so.vmve -'foe s, xxhov-n mas-v-ee, ofoh tVoets ,ne bv.w. or \sU> h 4 sod " -05,.,0.-5,-. n le^pn -.nd tubn nvtease the sn„; fool·, \-ne .n semm-sfarved EC' in a MTT assay and/or do not rescue apoptosis of scrum-starved EC in a $r\p ,-5 bine ..si lews ,es, ,nd sfo av * on-.v ,. 55-.0 when wee m an un;aruw, nlai onphm.nsm' tiMt {11084j In certain embodiments. the sNAG nanofiboo, do not have a direct effect on the prow ,h o- xn i- x i of h ,. tew', ,N,t,'h i.> . w.. ,s-ieV-cn-ned h\ on, wfi -,1 -.s n,' ju In οιντ embodiments. sNAG nanotlbcrs do not have a direct effect on the growth or survival of bacteria, v·. s . ,, , rr i ^ - , v v, ,v- . iish ^ V\ , o ' --. ‘ii vit' end'oeiou ns, m. s\ W n„ vn 0,,.,-. do r.ot ntxc a Joe,t „-.foe-m - uo on wevd gww s o. s ", a I lu ere ."n'Kvh'nent tK''\U newlUX'rs do neg Ρ.ν, e > ;f' .; ;; ; , ·; \ Urol on growth or survival of grarn-tiegahve bacteria, in another embodiment, file sNAG rtanoribets do not have a direct effect (e.g„ in vitro) on growth or .survival of gram-positive bacteria. In yet another embodiment the sNAG rmnofibera do not have a diveer effect (o.g., in vitro) on growth or vs.-rvival of either gtanvposltivo or gram-negative bacteria In some embodiments* the sNAG naoo 'tv- or a -N \t * nanottlvi , onp,'S.'von does no, >»nu, back na u g , et at, 'p-'sKo e beer rut, gram-negative bacteria, or both types oi bacteria?, in some embodiments, incubation of a b.skrt eulntrewnhli Ά \G no,’ ' . , v<u '20 , e wb ' ,, * χ s po ·, not reduce bacteria) load in 10 minutes, 30 minutes, 1 hour, 2 hours. 3 hours, 6 hours, 12 hours, '1 Htuis, 4\ hours, " hot* sot A hot‘s cf meuba’ mt ί\"ϊχο,'ν~, he bar' o tal eu-ae,' mas ''s' jun',iue Ό nsoree 'Koe \ \ xnk.no’ 'i m t sen u < with sNAG nanofibers (e.g., about HOgg-GOOgg, or about 100-200 p.g of s'NAG naoitbef-sj in s’, s' s ,s ) l iOs'cee oae.eo.d oao t w , ' , s, , a "4 ho , s d u*, ο mt t ' other embodiments, the sNAG nanofibers reduce bacterial growth or survival in vitro by less than I log, 0.9 log. 0.K log, 0.0 log, 0.7 log, 0.6 log. 0.5 log, 0.4 log. 0.3 log, 0.25 logs 0.7 log, 0 I leg, 0.05 hw,oi 0 4\'s kx, O' example, when v,r,''.se.' t. ,,a. t,' "· i,,t,'t;.!. eultui,'-» at, ireate<i',n"uha:ed tv rh the sWi ntmoilbex m x ore In some embodiments, the s\ -\v nanofilvt's room e barter tut vumth m our. χ a; m x ero hx i^s that! I ,. ΙΟ'" 2 \ b>\5 \ lu', 4 s 'dr " x gi\ o \ |p\ ' s ίο', ν' ·, to1 or lu ·, 16’ eff ml, t,v , \„mp.e, χ\ ; ,Vi„-Of:r.i.'.','i.xe.'fs iVJi'-viis h.jGorvjl cultures are treated -‘incubated ο ith d;e sNAG nauofibers m vitro. The tests of the effect of sNAG nanofibers on bacterial growth or survival and the esalu.m.’i x of'he t," t tesu ts urn do.serdvd ’or ex to pie m b\. mp-e ' ;e ; Sermon ; " 2 A n d I'UgUtC: Id 1:2:: rs/t'U:. (888^ b sum. e ternd·*( ukn > srx Hi i ur.otd „ss j , " ' o\,, , xeete tl e f b ne bevsse,"; about ' ana ) s rumen', i oci I' t" "mts o' ' „'t I ' w "mts - long'd,
do \* t v fee oe u ,t ,,O" os'' ^ , ' , I e.'h'.’i a, "» ϊ Ό,:, atulere'ion- e.i'"i'0 os'cn ’even m ,t bVi.empa.ib’ tp, tests- g, e tsutattaiseniisr tsuphutiainni tost'} f80861 In certain embrjdtmenta, the s'N.AG nanofib-ers induce u certain pattern of gene '.'spK'ss'Ou sP,>s \ ο- ’!,’tc'ts espresso*,’ as deturtunee e s; . RTOt K. t'Xt,‘s’ s''«ο or I I !.\ \t in a cell, tissue or organ treated with or exposed to a sNAG nanofiber composition. Specifically, in some embodiments, the sNAG nsnoFbers or a composition comprising the sNAG rusoofibera mdnco expression of on:: m more defersssn moiemv one or more deienvU'-bkc protons and or ops :0¾ more/ TdlGifcdf eedpiorG ln/f et: 0tif r#0: #1AG: ARnrifiherif: 0¾ e composition comprising the sNAG nanofibers induce expression of one or mo re proteins that are
InppG toTbAUAri aSt:Gb8«toriil;0®€t. 10067) In uersum omNvhmonK the s\ XO ηννήΐνοοί a eompoGmsu :.o:npnssm; the A H-nanoftbevs induce expression of one m move α-defcnsirsn m,g., Off El:;.·,:.. n-defensin 1), DFFAGB, DEf A3, DEFA4, DFFA5, DIEFA6), one or more p-defmsms <e.g., DEFBi Do., β~ defcnsin 1), DEFB2, DEF84, DEPB1G3A, DEEBI04A, DEPB105B, DEFBi 07B, DEFBKGB. DEED) 10. DEFBi I 7, DEFBI :4, DFFBi: P, DEFBI :9,, DFFBI23, 0EFBI24. DEFBI 25. DEFB 126,, DFFBi27, DEFBI 28, OEFBi29, DEFBI3 L DFFBi36), and/or one or more H-neiensmx!,'; , All f? in so no, .noocrnc"'s λΑΆ,ι,Λ' ,v \ on oo.'"w in i co lipusry t 0' \\ Η* η,ο 4n 1 vrs ns,ns >· cxpvs·· vs oGve ,.: mo;,' v DEE \ DFI \ χ IM F \ 1 OEFAS, DEFBi, DEFB3, DEFBI 93 A, DEFBI 04 A, DEFBI 08B. ΟΕΡΒΠ2, OEFBi 14, DEFB: IN DEFBi 19, OEFBI23., DFFBi2*1, DEFBI23., DFFBI26. DEFBI78, DFFBi2^ and DEPB13! In certain embodiments, use xNE-H’s rsanoiibers os a composition comprising the sNAG nanofTbem induce expression of one or more Toil receptors (e..m, TLR 1, TLR2, TI..R3, TLR4, TERN TI..R6, TLR7, TER8, TLR9, TLR HE TLR 11. and/or TLRi 2 :. in other embodiments, the $NAG nanofibers or a composition comprising the sNAG nanofibers induce :λ p:-,ss;on mV-ne or more of H E OF Ή a 61E SE ·\υ I E EiGtRR Gl i 4 Ac reeep'Or), tk AkL 1RA.K2, IRaK.4, TBKl. TRAF6 and IKKj, In some embodiments, the sNAG rumoftbers or a composition comprising the sNAG nanotihetx Induce expression of one or more of IRAK2, SiGIKK, Ϊ L R i. 11 R2,1LR4,1 i.KG, Π R8. PiRK'Und f'RAi 6. Its otv esoFodiscem, the Ή Λν !':oofK"\ ο 8 comne-huon cos; p;w v dv Ή A' n.mef v\ os' sec expsessu·^ oGn JMst one of ;|hA: alsvpsIMed:: genpiprodMG,: }0088| In some embodiments, the sNAG nauofibers or a composition comprising the·sNAG mnoA cm n'dn, e m premvc msc or mme oHhe ,fxv., rive i ϊ ,m,s m '64' mwst oeea to 0/ n'ou ' on 0 \h r'"'vc ' ' tole, v: , Mold 0 ' ' Ola, ' ' s 0 e c ' 0 ; o J si.. Ιοίο, ο ,, ^ V d u {o d I i to d 1 {e d 1s U'd IT 0 , " \o,d ^ «’v te# I AT:k$0 mmmM ifee; onemr mm e-of tM'aboM-IBted:. gtnef in.. &amp; cell^-tkpgE.'Of o|gdn,.0f :¾¾. s’4h 4,} l>4 to’, '4 mow %0h f i: \\ A '40utm !' u , ,itno’o" A,i,^'le,GGv;x,,t>im the one or more of the above-listed genes). In some embodiments,, the sN AG nanofibers or a c^'ep'\sshv\ ,ompnxu'£ no ν\,\η innmboors induce e^'tos-vi oGvoo nonm th; dv^e-in d ,ov\ .Tea vh e1' , ,όι note**(»»' ttv5<i ,- , , too ', ^ till,: .1¾¾ lllllelllf Gllll,; |?ll| .3111^1:5:11,41(11,: 4511,1:(111:53(¾ A ϊ. ' , x.V , h>0% ,n 10(10” ( L" ' o expos'', ' o''he-Tv. e '' <,o"'e ΤοχοΊνορ genes η. a , oil, nvM<e os on an οί i v, u, ,w K-vw η at n -> * wT" iv ΛΆΙ nanoiibcrs (e.g„ « known average level of expression of the one or mom of the above-listed gene*!, |8089) In some embodiments, the sNAG nanofibets but not long poiy-N-acefylgiucosaminc,
Ainu one or efukwjfji uvi,'.e -o ^ „'.νοη of tG ολοί roo g -,,:·. sslee, dw,, i- eeiermeoJ A a method known to one skilled, in the art, or described herein, in some of these embodiments, hngpoh. \ ^enluitee* acme on inn and m eh.nwen do no' ( niece exp;,, worn a -e the ,-ηοοΊ mov . - hoe' e\" o ho \ - o< , e, 'snoJ'^o eA , ’ ' ,' old, 1 N foul 3 fold, 3.3 roll 4 ibid, 4.5 fold, I fold, 6 fold, 7 fold, '6 fold. 4 fold, or 10 fold lower; of expression of toe one or more genes listed above as compared to the level of expression of the tone or moie guww I ivied ako „ induced hx- the sN Αίο nunoiiivis, us deiesnoiicd K a methyl known to one skilled in the cm or described herein 18090} in ceAsw emk'duucms, 'he s\ V,| n,no.''he's e? a compo-unoc e*' np"sw ,e fm s\ Ά nanofibers induce a gene expression profile that is consistent with, similar ic. about the same as, ot eqiiwel- ot :: cue o? more gene ,'χρΐ'."Μοη p= oilί-x’ - demon urged in 1 aH, , ί, 11, 111, x , Vill tod i\, f'Cviams o f-o 5, icroe In ,->ome embodiment g the ‘X.XG rnmonlytsor s cmnposikm comprising the sNAG nano fibers induce expression of one or more of the genes shown to be upreguluied by sNAG treatment in Tables 1, II, Ilk V. V11 i and IX, Sections 6.2-6.5, infra. in Moreem.wt.ment' me A Xm c muidvisc'; ec-mpm-m ,m u\nri snw me -.VxG ηηΆίχι.ν induce expression of the majority or all of the genes shown. to be upregulaied by sNAG treatment in Tables L II, ill, V, V1H end IX, Sections 6.2.-6.5, in/ra. hi some of these- embodiments, gene expression levels are measured at 1 hour, 2 hours, A hours, 5 hours, 6 hours, 8 hours. 1.0 hours, 12 hours, 14 hours, 16 hours, IK hours, 20 hours. 24 hours, 48 bouts: 3 days or S days after * , «‘f ,o ,, ' (i] ^ ?ί , Λ \u \ o v .o t',vj i o Ov , meouv-% or osie 'Tnicd 'η die aa, ot des0t< w ivieei |009Ι| In >, enam omtHxttηκ"ιΚ the A Ά π mAivv -m a cvncAismon oornpnsine ‘he A At . tunokhem imovu :·. gem- -,χρ·cs-noe pmide'.hm Afters been dk p:oh'.e induced m ίο:»ν poA A- 'V V v.'\'' VV^C'N ' ' S *P - O Ov'\\\ '"0 X _ W 0\Sv ' v o induced w rv s\ Ά o,nvA>es\ is omsisvc, Ah, annual v, .(if-H the <αη,' a- ο m ΌΧηνη to that shown In Tables I, ii, Hi, A Vί 11 and IX, Sections 6.2-0.5, infhi, whereas gene expression e Ah ndmvd i \ hvg nets A .v.vkh n> οά me Aseki m-' Ac - λ on isss's v ** , sooHss to, about the same with, or equivalent to that shewn so Table VHi and-or IX, Section 6,5, mfra. in n;tte emhochmew -> *he AHm ni-mnlibers o . v\' ηρΆχν sviwmmje^ hv A AO v un-fibers induce a gene expression profile that diiicrs from the gene expression profile induced by chi on m chtmxan::. |00^2ί is' ,t sp, v s is on A o ; no no A u! 'raconeeis -tie obt t neh Ia mud .meg pA A acciylgiueosamme and/or a dcrivanTe thereof. Sec Section 5.1.1, irt/ra, regarding polyA acctylgiucosarmnc end derivatives thereof and Section 5.2, infra, regarding methods for produomg ibe sNAG nanoftbers using irradiation, irradiation nmv be used to reduce ihe length οΓpciiy-N··acctyIgiucosarnute fibers and/or pdy-N-aceryigiucoaurninc derivative fibers to form shortened poiy-β-1 ~-*4-N-accty igu icosaminc fibers and/or shortened noiy-N-acety!gl ucosaroinc do m ?me a, .'f",', A A n mAhvrs Ssve. ' al', n, mm mas m i. v, 4 ιο redo _ nt: length and oinks ui,k weight A poh A-,u eixlgiv osaocm or ,i deo\ Ui-.e ihereof v\ uhmu disturbing its microstructure. The infrared spectrum (IR) of sNAG nanoiiherx is similar to. about d c sa ne as, s.n eqo'A Ή to ihu of the non emmuk'd pen, A ' --' \ aeeuk ok'-wum or or >s deri v stive: thereof, pUW3* 1 CO me, n ns'. ,. , A x, Ad , s i < > ved rer cl '' e A , -
Whereas in another embodimem, the eomposhtons described herein may be derived front diiurt or chitosan. or the sNAG nmtoiibers may be derived from ehitin or chitosan. 5,1.,1 Poly-M-Atelyiglneossmlne and Derivatives Tin.1 reef l&amp;m* w v.ulv's \- A ,'b'> , ' A.' \aa v, s^x ,so 0 Oi"o >X ' ' vss am ' ^ < v’lthi AO HA ' , ν'. ,. s v. " , ι V Ο o by returcnce) describe the fro I y - N-a Cc ry i glua >s3 tr:;« e and dcvivA-vcs thereof, and tnethods of pm'-dnOOs tv'.νηο 6' ν«ν ο nbod!ovaU:-, A,; pi-a \ ae> w !gbV''s.n",n.' has a ρ t configuration. In other embodiments, the pory-N-aectydgiueosamine has a «-'»--*4 configuration. Γ ,. pol· ~Ν ..... ο 'ο hr 0".η'ηιν νο1 ί Vow μλ - :·. *hotoof m.i\ bo ‘η me ί'οιτι .π'.» ;vi\;v 'γ ,·' ϊγ Ίν term, ©fa frf>m I00^5j IV- \ \ uoor, igkeos.ton to cut kvcvtntp'a οο ρ ooucoO b> ιιο^,;·, 'eor-e'beO from, microalgrie. preferably diatoms. The diatoms which may be used an «tailing sources lot the production of the poiv-N-acetylglucosafrune include, but arc nut limited to members of the r 'v, ' ' '' s ,no ' ' ' ,., λ !, . , \ o ms l\ , \ acetyl glucosamine may be obtained from diatom cultures via a number of different methods, nvhiduig sho meeb .nn-.ni fore.' me!hovl and ehomk .0 brbvgsc.v method know r in dv art ,se .\
, , v \ 'jtoriK.'s s- % 'ί \'V, ^ ο 'V •\o^<\bs", unn \ 1 I >, 'sb, - non .,,' which irkVfpsuawl bote r* !'\ tofo'ceo; ,n Us ouhrop » in voitam o'ubi'vitoio'Uv die pc \. Vue n igloooso'ii.'ie is mv, JrrwV. uo.n wteo; o i^v''die following: a shell fish» a crustacean, an insect, a fungi or yeasts. 100961 in one embodiment, poly- f -I --4-N-acetylgluaisarninc is derived from a process comprising a) treating a microalgae comprising a cell body and a potv- f · 1 -"4-N-wo'.sf j. , ,wan on ροΚοιο fthoi wu.i u "Μ,Ι,.,,,ί, ul agem :«ne i „«* io.Nub.suw'* cal'- d. .if sepal at ng the \ ooo’Vehk’s'ts.u mv polvno' oo r not 11: %. e·. I " \y, gu a mi 'tkr;;© tone so Hi o the posy. I1 -1 ···♦ -i'N-acetylgluco.viminc polymer fiber is released from the cell body; b) segregating the poly·· f -1 --» 4 -N-acery Iglueosa mi us polymer fiber from the cell body: and c} removing contaminants from the segregated poly- h -1 — 4-N-acctylglucosarnisc polymer fiber, so that the poly- r- Ί •••^N-acetylglucosamine polymer is isolated and purified. 1000% Jr ottvr erfvd'rv'fUs rv --: \ ,v'Mdgoovsnr.'U'o s "ms 1 dented for, one or more of the following: a shell fish, a crustacean, an insect, a fungi or yeasts in certain {.nbodiirteniM ibo rotnpoMhons a-wcrUK.u hetem do not co-nprn5·; ehbin or onUosan. |Θ0981 One or more of the monosaccharide units of the poly-N-scciylglucosamine may be oca,. I. Lik d In »-<\tin onfvd'nvo's , :o 5' \ -' ό ί,Ί* k' \r\ k' o' Γ"" , s(J ' "'s \ vO" ν' 'v . w\” v v i u t,'U v it* , on 's 10%, 15%. 20%, 25%. or 50% of the poly-N-acetylglucosamine is deacctyluted. In other S' o S 0 v v s ' t% d1 ' ^ , " o , to ' s \ \igb..o»\san;,.»i,'. us do ©ok 1. ted ft soeto οη.οοο.ΐ'.οη.ν eou-d to ,-r moto I'n-n *'*,», 5il!-,-, s 0 s . ΐ ' , s' k ♦ s,, v , o w.s x \s 's Νϊ w 95% or 99%, or all {100%), of the poly~N? * aeety igl ucosamine is dcactctylatcd. In other O' ' U ns 0-0,50..0 N \ 0 's ,\° ' , '' , , N ' "N '9 65%, 70%, 75%, 80%, K$%\ 90%, 93%, 99%, or 109% of the poly-N-acety Igluco-s&amp;mtne is -dea-cetylMed, [09991 In certain embodiments, a poly-N-acelyigkcosaminc; composition comprises ?0% to 'N^iONf N , 0N 'S i00 vs/ , 0'>' o" <,o jo% o
-Λ\, :0 Co,nso os, x e , N-aeexdyk eo-jimne' i^oiii-sae-JuixIcN hi Sv'5s„ eml,-,no, ,'tv; p,n S ' χ s, ' v '-} pX O '0' ' " s , ΝΛ X \ s N , OO
o5 10%,' 0%5·, ',· -. L',,0. 0, SO-Xsarm',-, t, , .. V-;;,. Ch'U:· >0.0 vs'isSO.et n;0,'VXViO,N\..xJe,, O. ofcOS embodiments, a potv-N-acetyiglucoaaromc composition comprises more than 70%. 75%, 80%,
Ά% 4'"\ , ON ' , nO' ,, 00 S' , ,,r sot u'\ 05%.V ο , O', ,X\' ,0'5, i-MsONW h V.55NC emhodsmavtv a poly- \ vighjeo.vmm;-: composition comp· ί-χο -.-quo! to or more than fx, 3%, 10%, 1556.. 2036, 25%, 30%, 35%, 40%, 4556, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, X)% %% or or el! (100"%, of hx kotyhtoct ghixosamuu· In od<x>r emlxxiinxnxs.»viy-S',x""v'eiviCo\„nitK> χΝ>η"ν"η<ο,Ί svn ;''!^c,n te\s > ,,, -91 15 ' 7,",, i ' ,4,' . % % ns ,.-/ ,,s , ν' , 's v/, Ny m,’o, %' -° %, %0°< <- 'it acetylated gin cosam ine. |00!09| In some embodiments, a poly -N -acety Igl ucosamiue composition comprises at least· one glocosamine monosaccharide, and may farther comprise at least 10%, 29%, 30%, 40%, % " , \ v' , % x' , \ o -. O' ' , „i x ,s%x{ n holm embodiments, a poly-N-acctylglucosamine composition comprises at least one N~ a<.'"\!g!t'cov4n'}ne ronowab: xx\ and t:\ fi-rttvi ix’-mpris-. A kv-j %%, ,0V , Vv ,, k's\, 50*'a, ?%%. %% n%%%, nyx os -%% of of 5050,,5 jo ux mno.-uwchandes [99101J Derivatives of poly-N-ueetylgiueosaminc may also be used in a composition -.kwt x\t h, ό n IX'i \ t'NO 05 go y -V,s,\n\U- 5i, o-.itm'X' <«V, toot ,,xS ot n A mo νϊ i l derivatives are described in D.S. Patent No. 5,623,064 (see, c.g , Section 5.4k which Is tiv-'i'xir.xedl·) roloioux'' ivicn 6' ns emm 5\ Ο,χχ,ηο, s /%·5·q Ovm-vsusno i sxq, include, but arc not limited to, partially or completely deacetyiated poty-N-aectylgiueosamme, or its dcaceiylated derivatives. Further, poly-N-acety Igl ucosamine may bcdcrivatized by being Mjlfas,,·,!, :'lx>'pisofNΙλΧχΙ vud t-i 5mmxJ. if s i\ - Vcox·^yIio.O",;iiuoo dertxato ,,-o nvhxk\ : y, ejithU' I ,x9\ N ,x\'t\ ty ixx>s.ennv f,''r\a;o. -, rvfXs, m -., "v ' \ v.eti tg,u. os tmix derivatives, or nitrated poly-N -aeetylglucos&amp;mitio derivatives. Additionally, one or more of she monosaccharide units of the poly-N -acetyl glucosarrrine may contain one or more suubnyl groups one or more O-acyl groups. In addition, one or more of the monosaccharides of the deacetylated po '.-Vue· r. Ιο,ΚϊΟ'Κ,γο.οο m.tv eouta u ,m 1 yonp One >u nano of 1«< f'uuK'.,,!, oh asm . of the poiy-N-acetylglueosaroine or of its deacetylated derivative, rnay contain an O-alkyl group. One or more of the monosaccharide units of the poly -Nwcetylglucosarnme may he an alkali derivative. One or more of the monosaccharide units of the deacetylated derivative ofpoly-N-ac"'\i;fii\s,!s.’!5.'ioc re ο mi \ ,t t,\ gso ;p O' e w nano ο1'» i > '^οο<> ιοπ,πΛ’ m ns of Ι'ν.'νΐ,νΟ4·'. dtchcLtuam' ot pok-No.,onh, η^οΑηπι.ν max vvmmo n k,-mve ,'.v,'\vhel,'0C5. derivative. One or more· of the monosaccharide units of the deacetylated derivative of poly- NS ' 'S' ' v ki” ' ' v O \ ' O' * ' tv Ou ! ‘-i < 1 f'kS ίϊ t " deacetylated derivative of poly-N-acetylglueosamine may form a metal chelate, la a specific embodiment, the metal is zinc. One or more of the monosaccharide units of the deacetylated derivative of poly-N -acetyIglueosamine may contain an N-aikyfidcne or an N-arylidene group.
In uv of' S'·. :, no dvϊn,n v is no ,vekne den' a*t\i lo n-otucs Of-iL'Ovki"idie demame r- not an aeuam doxo -m'-e, In mm embodiment the pok-Vi. ·. LUeJavVsamshv m vV ee v*' N ee^ 5 o o' eo * w 4 v j mho4·', ti" o. ' ' ne, u u embodiment, the donvaiiso is not dcrivanaed niih belie acid MHI102| Thepok \ aoonJe v.OO\ im; ό ΐ’ί'ί',Όν'-ϊν 0? fhv's .no m.· il/'nmtoe·, r pek V acetylglticosavninc polymers or fibers described above, can be irradiated as dry polymers or fibers or polymer or fiber membranes. Alternatively. poly-N-aceiylgiucosamme polymers or fibers, and arty derivatives of poly-N-acety I g Iucos&amp;roitie polymers or fibers described above, can be irradiated when wet. The methods of making sNAO tumoflbers by irradiation and the sN AG iieio^lv m> p i, . ",'vobuidov ''v ; Ί \ bat mi i\sb \e ""00° 0 ' ' ίο i ' incorporated by reference herein in its entirety jihlH*3{ lr corun'etnmvk ηοΊΚ, the pe.v N cctsbru ο,','ίι'ί; 'vSt'Ots w fvms are ib miniated into a suspsaxsion/sluny or wet cake for irradiation, irradiation can be performed prior to, concurrently with or following the formulation of the polymers or fibers Into its final ibrmuknmo. smh a dresOO.; Uenemlh, the iVKmet or ium emnev' οϊ'ν^ροοΜοη··, «Ιϋΐι,ν nod λ „ι. akos can xau, ka example lion· uhmt ok to jjKmi id mp 0t ookmes w fslvi ;v4 t v; ; οl JwniKd 'λ λ'ο" sor Hemes vr! fion about >2 roc m ahem 1062 n^of ?v vroe1 m fiber per 1 ml of distilled water arc use for we? cake formulations. The polymer or fiber may firs- he lyopihlized, frozen in liquid nitrogen, and pulverized, to make it more susceptible to forming a auspen&amp;iost/slsmy or we? cake. Also, the suspentdona'slurries car; be filtered to remove v it, -,,eh sp o i ,-. e* cm 5> -oni^d 1.¾ ceiW.n , -oem-, οχ- \\Λ! ,-- dee i- .omb sVe >-w .- su-pe imoo v ebom 0 $ mg, \ w.;,;, 2 cm, ' -m , - use, ς mg, a 'ey, " n; n my,2't -.-, 12 trig, 12 tug, 15 mg, IH mg, 20 mg, 25 mg or 50 mg of polymer or fiber per mi of dusulled w aier, or'on 'ary-' n between f κ I or eye!"> u ..-w dad; v, ers t , i ί g 1-10 mg ml,: ^ a m. τ 2 h roy nt, 20-50 mg/rni, trie.) in other aspects, the polymer or fiber is irradiated as a wmi cake, comprising ikatf Mt I,-.-CM my pXym ; or '- - " pe~ ' ml re J-vdTJ w.uei in -pc me eovvem-vm,-, the w -,,sle era Sip1'abrnsl 56, 100, 2,00, '00, 506, 500, 600, 700, K00, °00 {,; i 006 ^g of polymer or fiber per I ml distilled water, or any range in between (e.g., 100-500 mg/nil, 200--600 mg/ml, 50 -1000 mg/'mi, ere.). ΗΗ1Ί04Ι The irradiation is preferably in the form of gamma radiation, c-bcam rntdltfibn, or x-t'jys, Tw-o sources of Irradiation are prehttred rsvTotmsrve nucf d-m and e 0:--,. Mens In specific .mhodiosem the uuiioaefive nncKie-s esc ooball-iO ,md ersmm:'" Both as these neelm,--, com gemma rays, which are photons containing no mass. The gamma rays have energies irmn 0.66 ti' I 5 \?e\ s. vng -,'f v'f s-, c .-Urorw ? c g-. rwm'ed end .s.^c','-stcr v enctg -.- ip to 0 MeY o be W'm med *v, os'C's -. c vm, r ·" s,c, u'°v d, moo s to-e , n,v account is that the depth of penetration of materials w ith densities similar to water hv 10 Me V e ccrwins is homed r>s aeon1 ' " ers wch o-V"'ded c\pov,-> or vvn? 8 o - s s -vsd; 'vm-stdee exposure. Depth of penetration decreases at lower electron energies. Electron energy can be converted to x-rays by placing a rnclal (usually tungcbm or tantalum) targe? in the electron bears-path. Conversion to x-rays is limited to electrons with energies up to 5 McY. X-rays am photons with no mass, and can penetrate polymers or fibers similar to gamma rays. There is only about CX efficiency in the cots version of electron energy to x-ray energy. High powered electron bean's ? '>t or, -¾ am ,\-cd m ' tr\ 'tod -,t.a." --. n.evs to ae-,o'un ?b' the loo, c-1'-ctvs.'' ettb'ccc-. |iS0l05) In a -.pc i'T: emhod’mcni, so - nTadiatiiV1 ?- gassm-u nrafOc'nm |00H)h| The absorbed dose of radiation is the energy absorbed per unit weight of product, mc-AMucd it; ;j,n icylos keoinvo tko ? fm'dauJ poly'uc-'.s >»{ iliwts the ptufeocc s-Psorbed f sp "ΧΚ,-’Ο \ ^ · " ν ν. χ ^ 1 '' \^ν Λ\ 'J0(i- ! JOO key of radiation, fm wet polymers ot fib-erx she preferred absorbed dose is about i00-500 kgy of radiation, most preferably about 150-250 kgy or about 200-250 kgs of radiation.
[00107] The dose o< sadunon cut? be desenix’ti in terms of :K t fee: the length of tbs' polymers or fibers in specific cmlXKiirnenu», the dose of radiation used preferably reduces the length oft he polymer or fiber by anywhere from about 10% to 00% of the starting length of the po'ser ror ->e\ fes'vetw eK in spceov esKwcsoeutv the a a eraao emph 1- t,\h >,,v K about 10%, by about 20%, by about 30%, by about 40%, by about 5045, by about 00%, by about 70%, w „ o, ^e w i a , ',,e vs,\ ' *o ' ul so o , ' so forth). Ahernu lively, the dose of radiation used preferably reduces the length of the polymer o ' e . ' w ,s w worn) o fth't'i, om >ts n\ ’\\ \'s , d cef o ! 't;> r>5t f'c siatling fiber length, the average length of the polymer or fiber is reduced to less than about 15 ' , s os Mm fame ' " os \ s\ %m a be ' ' \ e ' ssbrnoavu ' 's Λ'', ,\s than about 11 microns, less than about )0 microns, less than about 5' microns, less than about 7 microns, less than about 5 microns, less than about 4 microns, less than about 3 microns, less than 2 microns, or leas than i microns. In certain embodiments, the length of the majority tancl In certain embodiments, at least 60%», 70%, 80%, 00%, 95%„ 985% 99%, 99.5%, 99,87% 99.9%, or ol% o s m , s s " , " to'" ,'OAto s 0'< to s ,χ o yv<' , „, ^ ' v , v so % s ' , ,o v *' ·, ' «bout 20 microns, no greater than about I 5 microns, no greater than about 12 microns, no greater than o >ont On s, uo , c , , o,\,u's i.MsVMry e 'o' , mil, ' s euro o,
I i 5iX>uS ' v ' s I i vv. c 1’ (V X ιί «V art Ο a- V v "0 \ ' ' ' , ^s i <C , V '' ' V r ,0 , ' ,. » * 0' x S > '' !«X*v„v>s betweew%% son'%,.%% to %%, % ^
VS' ' to \ s ,90* K N , ,v' ' U) II \ s , , W,\M between about I so 20 microns, between about I to 15 microns, between about 2 to 15 microns, between about s to I 7 msesoos, between about 2 to I 3 nnesmss. between about I to 1st mu.svos between about 2 to 1.0 microns, between about 1 to 8 microns, between about 2 to 8 microns, between about 1 to 7 microns, between about 2 to 7 microns. between about j to 8 microns, between about 4 to 7 microns,, between about 1 to 5 microns, berween about 2 to 5 microns. ibmn 3 to 3 S'-UCiX'-t-s, ivtWWOi: about &amp; :-3 *0 ρΐΟνΠ,\, Οΐ ,m\ : mpCS bol'^eCU tl-0 tote eon to, I'/eUis, wind' urn ai>* 1001081 Ϊ ho A"0o* taJia*»<v s < a>v''\ «3-utlvd », o."'\v v.' ,' \νι>ολ ι n v\x 'ginw-t 'ho t'i r -at ''i' ;* K '-m it fie omhixiirnm'-, the do-e of' νηΙχΠί?" used pc’ ;r mv reduces the molecular weight of the polymer or fiber by anywhere from about 10¾ to 90% of the s rt'oe w i i^hU'Uho ·ν!\ηνΓ ,w-1- : fr, sp-'e/l err. -m w-v*' 15 «-.er ee m-\ in ttw-edts :- seduced iy ubod !>,"' \ . v d-osn 2d''- . m l,vm fi'l-, Iw about 10' -, b\ abort 56" - ον anon oO'-s 'v-iboa: 0s s\ ebe, o' ,-w thui'' > o- \ ό be wee', 0 s'-' -,1, hi 'V"\- s'd ‘O t!'< aid W> tOtf·/ X 'sO-Vs-it's s s', the de‘W' o''\td 'iPO-V \',C s'K J-VisOsSv' molecular weight of the polymer or fiber to anywhere from 1.000 to 1.000,000 daltons. in bo -.'ntwlsmeits and dopenesng on the st-,ft'ne xin-r v-.e em, p e a\,.=--- -yse'vwhs weight of the polymer or fiber h reduced to less than 1,000,000 daltons. less than 750.000 daltons I os- than. 3 00 /00 j.;tovw-,, tew tr an ^00,000 a liter-·, I- w dee: 700 00/ debmis -es- tbs' 100.000 daUoriSs less titan 90, 000 datums, less titan 80,000 daltons. less titan 70,000 daltons, less than 60.000 daltons, lea;, than 50,000 daltons, ie-.,-, dr-m 75,000 daltons, les-' than 10,000 daltons, or less than 5,000 da lions. In certain embodiments. the average molecular weight is reduced to no less than 500 da!tons, sto leas than 1,000 dadoes, no less thrift 2,000 dadoes, no loss 1500 ds.lt.ons, no less than 5,000 daltons, no loss than 7,500 daltons. no loss than 10,000 da!tons, no las- titan 25,0:)0 daltons, no les- than 50 600 dahnns. η-* ies- than m), 00*0 daltons m Ui- tes- titan 100.000 da!tons. Any ranges between the foregoing average molecular weights are also encompassed: for example, in certain embodimems. irradiation of the polymer or fiber reduces ti"-. ,Ην"Ίχ: etolecnto: wextot to . tu w 1 etc hcPweto 0,600 t.< lOO.OOO d.-bents ee tween L00O and Γ ' <W Λ i nvw, be*wex't /6000 and 500.000 .tokens, ","w ecu 2, \-'O0 mtd 00,600 daltons, ., ,v ee V -'00 id iJd 0(m dJkns- /v-ee' e,v * -0 0,'O uu< fOOtoidniVts be.wee * ,-tv' * 25.000 and 75,000 daltons. between about 50,000 and 70.00(1 daltons, or between about 55,000 and 65,000 daltons and so on and so forth. in certain embodiment, irradiation of the polymers os fiiv>s:edu -"v ihe'Uxdeeu’.-*''w-."gb: tbe w\,j:ono; and ntcum em. - r-'\, 0 . am fib, 76%. 50°1 'tifo 9^,,9^1- 9!11, 9155m- Od srn,., dd.dh·, imv., m ben-me;; -''Ί to il"-,
w U' '' -V "' , 'N to S'' , '' V ν' Λ6 - U' ,^0 V'"°o, ' "V O of .he *'’k\' ,-' -er- 'vbeie u.tw e'i -, stnt 20,(0161 and 10O 000 (bt'.o ',··, maust 2/000 75.000 daltons. about 50,000 and 90,000 dal tons, about 40,000 and HO,000 dahons, about 50,000 •md "0,000 dahons. or about f·?,000 da! tons. in eerOnn w.rsbodhoems. irradiation of the polymers or fibers reduces the molecular weight of the majority and in certain embodiments, at least 60%, 70%, *0%* 90%, 95%, %t%, 9960, 00.5%, 99.0%, 99.9%, or 10050, or between '*% 50 OV'„, ^y, ,Λ l0 , "Ό , u> S-\ , "Ό . U> -%', , S*V , K·, Λ , Of) ,, <0 U"-\ , ¢,, 95*% to 9v%) of 5be fibers to about 90,000 d&amp;lton-s, |O01J9| , \ , , -< , ut > i e e-' arm o? o-< u < no , ses , . > v e »ed o 6η·ηι 1050:00^1005^ ;0 e,, dressing-; and other ·, oinpo*mio5i« de^.iibod hemm* -hat are 50-065! m die practice of the invention. 5.3 fgfimoih^ 1001 lit) I he -Λ \e> η cm Ϊ ''‘'"a oar, ho hr tr eluted ό , \,O50t\ ,vV w 'S'suums o* Vy a' .idmun-tmuon a·- iesosibod hemm [IHI1 Ilf -\ lOtnpoVion eompti^ng dm sKAG sumoFber-- nui\ o ibrrnul. ited as -i oiown, a membrane, a film, a liquid sohitiosi, a suspension, it powder, a paste, an ointment, a suppository, a geiatmious eompositiem, an aerosol, a gek or a spray. In one embodiment, a composition. <.oospo-sne,560 v\A<j na not-Ivr-. 5s lorrmddod :;.a <15; ednj-Hnu memi-nme in sotne embodiments, a composition comprising the sN AG nanofibers Is formulated as a dressing, a mat, 05 >i beavueo Soon %rp5uhii5vm\ sun able t05 soliitsor; m, or suspes'·- x-a ·η 5 5. 5-w p* mr to administration aic also contemplated. li is also posuhlc thut snob compositions am incorporak-d in or coated on implantable devices, such as orthopedic implants (for hip, knee, shoulder; pins, screws, etc |, cardiovascular implants f stems, catheters, etc.} and the like where she antibacterial activity would he of benefit. |IHM-x ,0; nv-wam -,c -ho s\ \G u,mofbe -< o'.ts 'rwlcdeom orrinreof plu55'aooi.ncGlx ,e,epu'd, osO'pu'ms Meumb, exe'pvnts r ,ty .1506-0-, * dot s,dme, sac sokumn '-os'fvso, c.;·00;-'7, ethanol and the hho or eombm,st5ew\ thvsem 'scumble - ή ipieot-. also include starch, glucose, lactase, sucrose, gelatin, mult, rice, dour, chalk, silica gel, sodium stearate. glycerol monostearate, oil (including those of petroleum, animal, vegetable or .synthetic origin, -men as pc-utct oil soybean οϊ!, amend oil m.m.mv od and dm tikef tab , uxikun chloride, dried skim milk, propylene, glycol and the like, in addition, a composition comprising the sNAG nanofibers may include one or more of wetting agents, eratdsifymg agems, pH buffering agents, and other agents. The sNAG nanofiber compositions may also be incorporated u a poyxsob'gK\! 1γ uceoptub ο varnc' fot evmovvr a pb\ ammo', u% .see p'able cur !;' suitable for ropjeai application. The term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the ITS. Pharmacopeia or of vs p*"ietoib. o.. oum.vd p t ,rs.a, ο·\\; tot ,. v m aetm , -y end more f' i t\ „1 e 1\ to nmun ·. Examples of suitable pharmaceutical carriers are describe a in "Remingtons Pharmaceutical V et u,v-' by Γ \\ \Grtm 100113 j The final amount of the sNAG nanobhers in a composition rna.y vary. For example, the amount o' the s\ Λν tv V''bo \ m a e-Otpa-vum U' g , men mu lot ndrunmnfu..cm v a pv.u.m' eu\ 1, 0-o no. mm os o. t s m about ' , about ,< p About VG. atvtu ' , „>os * "'T'T,, about "G"-, ebons G'G, .Gent ΰ'" , atv:·.· ΰν\ o' a emu > xo ah- b\ \o jro l's era embodiment the amount os the Λ','ηΐ rvnofibeo m a emnp<minoo G about °> v, about ^s·1.,, ebon! 99, or about 100%. Also, the amount of the sNAG nanoftbers in a. composition ie.g , ptepareo tor adnniUxtraoos: to u pattern\ iter, bo about '*0",.- hid";., about nd".,-!(GT·, Tons "0%-100%, about ?3%-J0Q%, about 80%~ 100%. about 90%-100%, about 95%» 100%, about ?0%~ 0\'\, abous ""'b.-'-'T'o. ,'>uu S»'»'0.ο*>' , ubet ' SiG' '% shorn 'd .ibotn ΡΑΆ'' . ot ,G"Ut ,χΟ'νΆΡ', ^ejojn vd\ η Λ coetpovemn mm eve p' sse teo'. Gm %' ., -sd ' ,, >d%, emG, 70%, ?5%, 80%. 90%t 05% or 00%« solution of the sNAG nanofiber*. E00U41 A sNAG nanofther composttion may be mrasuiafed into a wound dressing, in certain embodiments, a sNAG nanofiber composition is formulated as a. wound dressing in the form of a barrier, a membrane, or a Hint, Alternatively, a sNAG nano fiber composition may he added to dressing hackings, such m barriers, membranes, or turns, A 'earner, membrane, or film car; be supplied m a ' Gog··. G -iarGurh v.vs wlmfu cun lv tart her cur wd -5 - of V the av.t K toe he„Nd t he bsek.ne, vers be a cm t cottons! mess toe mates.G, xeeh a- a wUec ot e,„ t,\ to which a polymer or filter is added or coated on, prior io application to the patient. Alternatively, the sNAG nanofsbers can be formulated as a barrier, membrane, or film made out of smugs, mierofn-ads. otierospheses, or mtcmflltrils, or the composition can be formulated as a. barrier-formim; ora In cett,nu embodiments at lo-j^t P G,oU sut Vast up·', or [oust n'v , of a du.\sV';!y ' -i .O' ','Wf. of Π0 A AG 'ΛΟΟο Vts IP i . tGU. ,i.xpG t.\. a dtOsx tip a,a„S ,'Oi '\,"UaU' ,c recount ' ev; t tu <t, t .Gt,··, eu oe\ hue oooO' v"o bexNv n^mdUv' use if o. Ibtsoulated ax ; v\muvi dresonj.. }001 ^ compost'.on eemnos'ng 'lie ,\f\ Ή) oanefiLvrs may ΰην γτ^ν ' tr> sm"f.sle natural or synthetic polymers or fibers. Examples of suitable polymers or libers include cellulose polymers, xamhan. polyammid&amp;s, polyamides, polyimidcs, polyamidebrnides, poiyarnidehydraxidea. polvhydrazides, polyimidazoles, ροIybertzox&amp;zoI es, polyester/amIde, poiyester/imide, poiycarbonate/arnides, ρ«Iycarbonate/itmdes, polysuUbnemm;de,s, polysulfone imides, and the like, copolymers and blonds thereof. Other suitable classes of polymers or fibers include polyemyiedeoe fluorides and polyacrylonitriles. .Examples of these polymers or fibers teJ u s* *hw· eesenlvj n t \ P.nuV K04 kf "V1·''', a+A ' ' ' '4ΐ c , '-m 4,f.m ηρο 4 om -EVg mhHuOrn 4 vG 4G- b ohu ev *nj Ϊ taopuun
Patent Application 0 2\9 H?8, all of which are incorporated by reference. The polymers or fibers „,>η include at least one m mtoer of es Setose po\ mem eu:ss ooSs,n η:ηΛ -, ρο4 n d„ t md,w οι pels .m-JIn ce'tarn end .'dmur is, f s„ "oU mens u IKi-' mck dc pe ram oak"·· pot-cmos oroihan ,md poplemoKooutlw lone h, one 000--1000:0 :he compositions described herein comprise more than cate type of polymer (e g., the sNAG naocuiher and .vSlnipses }0iH i6| h" s eoan aspects, bu \N \G mteomur s- the oot\ cet'\e mem.hi m 0 a , ot "Vsiooo 100117} In other embodiments, a composition comprises one or more additional active K'predKmt-, e p. to promot.; an aim -hucton.il m lee- and m nee boy, t e g , w ounf be ro -) tr >or c ee md r ηκ " add t onal 0 m ;,'e s - 0¾ no *. - been uteeeiK, e »n ecimmoe e ,kfe,tsm cpiwte, t u"'o wm Use we.ide, m r |oH ocemv 1 G. pom kh ο- e e,ro\vi laemr In sp., 5 b emeu 0 > ms, ϊ to adeumnal ,e , \e ing.voot,* ,s i g;owtn 'benn v.eh as one or more of PDGF-AA, PDGF-AB. PDGP-BB. FDGF-CC. PDGF-DLX FGF-1, S“GF-2, h<sh,s RsF-\ ΠΐΓ-by Fed', fld'-u iHB-Efsht, ao.p nnumn\ umegam , Sukeebu.m ncijrcgnhns, cuigen, VEGE-.4, VEGb-B, VEGF-C, YEGF'-D, VEGF'-ll placenta etcmb factor ,Pf Gf) 0 m!s>re'C!n' k „uji οροί."*'·»-’', tGF-ί, iu* 11 hope mem- g-owt', Gem* Plot \ are macrophage-st irnu lating protein 1 MSP)· In other embodiments, the additional active ingredient is an seem shat boost 'be immune sy' terx a 's"'n .chef cum or a fe\er fs'l'-el'.uo m jltiM 1H1 In e-,-0.510 rmUninnems, the -ddifionai aefi\e i-uoeliem i- an «mtibtoii», oibine of-he following classes of antibiotics: I'merolldes le.g , erythrumydtt, axithromycia), a-mnoglycosidcw (e.g., amikacin, gcntamictn, neomycin, sircpromycinj, cephalosporins (c.g., ecfadro.Gi, cefaclor, e-k-ta'nvm, eGbpimc?, iluoixsauinolone-' te p , up!ofio\.;cm leu-bosecsm, foineiUnts to p . orrmei in, ou'x. v:i i \« hm's ' -mescmv dew v, t \'\ in»' carbapcnems (c g., meropencm, irnipenem). In some specific embodiments, the additional active n'gfodu'srf ,-s, ivo oxvo of vamvmsx'ni, sUfa ,hx,g $..: u, c- vmex.wxe it v.ethopt n sulfamethoxazole?, teinicychne ie g., Jcsycychne, rnmneychoeh dmd&amp;rnycm, oxnoohdmnncs u a , ImcxroKh, »jnmm-m,'m, k'seupiurdn, gmm.p^om'daiihpriAm u:m i\ aG ugc, ,-, mic, thicim h ,π,γ,κό, i ηΐοΑηνηηοη. hwh-kyn )¾. jvxnu.. ' wehioci' , ικ.ΆΙγόι $' nvdtxh'hi'X J, -ve deierixinri, tobramycin, ehiorhexidinc dighiconafc.. ehlorhexidme gluconate, ievoilos.aCiTi, cine, ar\t m \ ; !r χο*"ο err bod t.owv, ' e,'.nrp»>\e.itn t,os i 'py* l "sc sN-tu at'»; nr additional active ingredient effective ro ireat or prevent or comrnovhy used to treat or prevent an 0. , 'nan nnbcn\tn, MRSa mlecmrn, a Av.-v mm-rimm or a <' Jr'> < v mfeciion g; an m dmom ethxuso agantst >n- t mmeono «v\i tcnnKyl. η [001 19] A. sNAG nanoiiber composition may contain collagen, although in certain aspects a sNaG nano liber composition does not contain eoHngcn. (00120{ In certain embodiments, a sNAG nanofiber composition does not comprise any additional therapy, hi certain embodiments, 3 sNAG nano fiber composition does not comprise u. y eJchdond enn-Un to* ed ag.,;d, a 'sewde, a ee coor-Lhe p, 'Mote, s bod ο.,,Νο - like peptide, or a growth factor. In some embodiments, a sNAG nanofiber composition does not comprise an antibiotic. In yet other embodiments,, a sNAG nano fiber composition, may comprise ,n sc d wap' $0 1 , ,to nnbbim ,\n ,vv wed r1" vf wem, b $ lemof a' lows $0 1, an antibiotic) is not encapsulated, immobilized or formulated in the sNAG nanofibers. 190121 j in other aspects, a sNAG nanofiber composition does not comprise a significant amount of protein material. In specific embodiments, the protein content of a sNAG nanofiber composition is no greater than 0.1%, 0.5% or 1% by weight. In other embodiments, the protein content of the composition is undetectable by Coomaasie staining, [00122| In one embodiment, rune is also included in a sNAG nanofiber composition. In » Gttvs v ' ,s , " , v ' \, ^ „ss , c *»' wtuu'C ». u\ sex \nb“e«,,, al, IW. Adv Wound Care I.2ri3'?-b). The zinc is preferably added in rite form of a salt, such as < n ’ owC' r na sibpn no, r w' t > on 0 mw gU-vn "0 5,4 And-Bacicml Uses of sN AG Compositions jtMH21{ \ w 4,' v,!tk'*\ of baa,\i d .'iio live's aoe 4vj^ kvi tamJ ihewm' va> K treated and/or prevented by the administration of the sNAG nano fiber compositions described hew! \,v e ' . S..-U nns ' i \ a no c> 1 A oyw os one <rdb,A,noce·, the - mnptvtvns dae-CM vd herein ere bacteriostatic. in another embodiment, the compositions described herein arc IveSenc:d'd. hi an embodiment, ’be eompoMfioos deserdvd berem rr;i\ f··; u-vd to trees end oi prevent infections by Gram-positive bacteria and/or any diseases associated therewith, in another embodiment, the compositions described herein, may be used to treat and/or prevent infections by Gram-negative bacteria and/or arty diseases associated therewith. In yet another embodiment, rite compositions described heroin may be used to beat and/or prevent infections by K'th iii\wv;ve,m\> haebvta and Ivu icon eii m ,00 Civav\ associated flvmwirh. |80124j Bae’e-ud nvonvw that may K o.'.ned .am oi owmeri m-e y , ompo-riO"s-desenbed herein include mthcoons b> hauevs of tin A/nnyno ihan family, -t.;o^?>όί/^ί?ί, family. Amtobactcmcceu: family, Bmieroidoccm family, BurtoneUa species, Bdelhvibrio family, t ,r*n.f i ή./',·, v.· avevs, ΓηΙνο-rib \poev,-G y triv,miy/sw:!V';i·'G,ch doM-idium, Entvrvbactef'ieeeef; family (eg,, Citmbacter species. Edwardsioila, Entemhucter aeroguns, l'v ch,',, H.t'Via .ovens, ν', m y'G sve a, dVv I, -ρο-, vs ' ' , v idene, i, ,\ "* ,, ce C, ,, X,, , V _ , , ' (msMmidM ifarblly, MdPmPpMidi f d0M&amp;Pm, :tdpfyBudMtdMidiC iSfbiiyd bt&amp;PemBpAtde Jhrif iy y / ·>·ί; -wdsavoi. farntiy, / Go. ov ep·., tes rid th\k\ v -a famsiv, mwobv tm v (c.y . Myeohoctcnmn tubvff'idvxh.), Neisserkiceu^ family, Oceanoi pint lam family, Paste oreBaceae family, Pncmnococcm species. Pseudomonas species, Rhimtnaceoe family, Spinthm. family, Spdxnomaceac family, Staphylococcus fc.yy, metliiciilin rest slant Staphyk *ccn xus aureus and v , ' ' ' , , ' , V ' V , , , , ,N V ' , , \ , X V , ' , , , s X, , and Stnp^\h\'ii'· /Avmmmho b l atnptr>>\ dm Pcho>haeu r f'antly, and or t mn/amiwnm i "Γ'ρ In a spec t\ c^rv bmev, d,va.\0\ cn w 1α m awvwe xvim « ιΑνοοη-· -a,, h bAtcs a eu'. ;d\,' be p'm emcc wd v hei'cd usi,v me -, ompo'-dans c:c\, abed g,k in jiUM25j fia, vn<ri or ecne'is 0 ,n mm, tv i 'cmcd ,οϊ,Ι ο» ρ- ; rtec na '· y am ViM-nens described herein also meludc Inn.·, mms by bactetta oi'dm.. ibHow inn ye eves &amp; -oA rWG, Borreiia, Brueei/a, Campylobacter, Cidamydia and Ciamidctphyiia^ Ciostridhmi, ('orynehacterhim, £t?ieivcoccus\ Escherichia, Franctielhj, Naetnophiim* i-leb'ccbccier, Icghmcih, Leptospira, Listeria, Myct>hdCteriunt, 'Mycoplasma A\ms.vc?vm Pseudomonas, Rickettsia, Salmonella* Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, und er w em, in a speeds embodiment. m-osm-; caused b> ot e-wooi-ned sinn mieePous by such bacteria may «do be prevented and-'or treated using the emTiposmmis described herein.
[001.26} Bacterial infections that may be nested and/or pm vented usir ;g compositions iesCo v l how n s m .me mt,\ ,,e s ' ,e^oi+ie ,ok^'\'A\ - o , , dordetelm p*.rtussj.s, #« t\ha hmgdn\fcrt, F* <»< iifa ahonu^, drm ctla tpmo, Pan t>7u «η brrvx. bon < 7.-,: w<v, mom-r/ohm .MgaS'em, Ch'ano'Jta rmeucr»ud, C >όοίΟ d.'..< <το· Aon?JA.s, C'lamidaphUa pslttaci, Clostridium hotulimtm, Clostridium dijficuk, Ciosoidium perfh'ngetts, Ciostr* id,.'?: tmant, (d-TUc K,s ft’· oett Jtp*vhctme, I .«OtO'Oi'iVi ubo dofimw fUVtn toe, ia'm,
Escherichia coii, Francisella tulorensis* fiaemopkilus inftuenae, Helicobacter pyiort, Legionella
Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae* Neissetta gonorrhoeas* SeP-seria meniagiddes* Pseudomonas aeruginosa, Pnthws mimhihs, Rickettsia tiiliil Mdtmmeiiii Iy0k ^SalmtMelip 7 Ϊ ' ' ' , ' . .. " V , " " V V ^ ' ' . . ·. V iV v .
Streptococcus pneumonia, Sfreptoatccus pyogenes, Treponema pallidum, Vihria chaleme, and or iemmb? peais. In a specific embodiment, diseases caused by or associated with Infections by such bacteria may «(so be prevented and-Or treated using the compositions dosed, bed teem.
[00127} tr M.-mc embodiments, dm com ssitios Jescr.bcd hem' n mu'- tv wed to tmw and'ot prevent irdccuons by aerobic bacteria anchor diseases associated (herewith. In other embodiments, the compositions described herein may be used to treat and'or prevent Udeetions m ^ ee.o e .vC.^ . , a'-me ee . os, ' }001281 In certain embodiments, the compositions described herein are used to most aod-or prevent Pseudomonas aeruginosa infections Pseudomonas is n got no negative aerobic bacteria fotnsd in soil: water, other moist environments; plants and anuria Is. clinical Isolates of which produce the bhmmmen pigment pyocyumu and a charaet.cri.Ntic sweet order. itecudamonu\ a, ';vdm\u's\i awn m „anso sunn, imo' nslcetson*. pneumonia, Ospo Par, wskm. I'Mect'otts \V , i λ »t i ! , e'miifi bo ΐ 5Π(' sow* rdf , t (Vs W 0s ' '0. ' .VO1 ' *! J U \ " SCp sA -S Sk'mn is £ i i OWS !i 'S ktVW t iu be ss if'}}'''. V it 0 'UX' lA i"KVt'.Or«s particularly in patients wtih hurn,s. patients with cystic fibroma, patients who arc immunosuppressed (e.g., AIDS and cancer patients}, and in patients who have been ho&amp;pdalized tVr b's'ccs 'ban I week R 5¾ a be }.:oe,. no, et '}.'v\ oif'i J o oci.v\ -eib ,w Sat 'Ό ί mued to pneumonia, urinary tract infections and hucterimia. Any one or all of these infections may he r e\e it. „ .in.' -a sic η,,ΐ N ,'ie compo-", mw eesoOvd ' urm 100129} In jjorne ernbodirnenb. the compositions; described herein arc used to treat uivd or ' ΟΌ' s "Ov v ' mb '0 v \ , , ' fri com s, , " o VAu nanofiber composition in this embodiment and other embodiments described herein may pf'\ me t.i- goivf Aon .’it'reMsi.nn otxemvr.;. ,w i ,!·, .¾ bo οτ 'h.s s Ό'ΐ'Χ i 'ndope"kom -. on ok', ot ο htovnai miiVuvs 100130} In certain embodiments, the compositions described herein may be used to combat 1 oot-.ra 'hat ci\ k's wit to ume 01 mme ,ηη.Φ ,-ιο, ι .λίμ Γη; xve ιρ ο Jv ,ινηρο-ίΠο-'··, described herein may be used to treat bacteria -hat are resistant to one or more antibiotics, for v \'b ν' v omnomii f n m nbierns so, o „ MRSA ;n ’ h c ! u .Ο"'.,"’
Suiphxioo.^-t. m omt, VR$A {Yaneomyein-tesLMam S ova asg VRL {Vaneotnyem-feststam Eotemeocifsp Penicillin-resistant Enterococcus, PRSP (Penicillin-resistant Streptococcus pr-t i*}.'.·>τνι, isoina ί>1 nbirminuyVebon nVt.dt?t Aomu γ,Ύ >\ and o-hcrautd'mde- resistani strains of bacteria (e.g., resistant strains of £, eo/n Saimoneihs €&amp;mpyhhucter, and Strt'pfocot'd). in one embodiment, the compositions disclosed herein may be used to treat multiple drug resistant bacteria, [00131} In. .some specific embodiment'., the compositions described herein may be used to ti.ee' „nd >n pwvtin ''b\h,-,<ii,''es,vi'is sc,; *' v ,\ c,n m.;, i' i"\tl"\ \ it nr also oeeai ec1 multidrug-resistant Sfaphyhcoccus enu'em or oxacillin-resistant Stapkyhcoccus a/n'eu.s ("ORS t ')) \ΙΚ,\Λ $.s cη, str sin of e fv.\< mu em th.it It as >1r\ Doped lesisnnter :.e n,nn lactam antibiotic,",, which include but are not limited m the penicillins ipemciww ntethicditn didoxacilHn, rtafcillirg oxacillin, etc.) and the cephalosporins Some of the known strains of MlxSA luetude PAI RS Λ!5 and RMRS MotiKo kn<win as Vm;> ΜΌ, whkh ate m,-:- eon ns erythromycin and eiproilosacm; CCH (also brown as STA;US A3fK)K Vi I A.S.V-00, STfcUSASQO; ST59:USA100Q; ST03 strains; ST80 strains; ami ST59 strains, MRS A is esx so o i in οι Οι i tiO' \ u «η n SIRS ^ s » senous hel j( sc ' v \ approximately M/A of heulih-s are .wsouaied staph Infeenous, in do. I'.v, oio:o mm 04,000 oem'i oC'.ojo i x' ^,ιλ MESA * uoeuor u\d aooin a 004 > >„ , o.n ' r„i. rn,<\ ' \ x
Especially prevalent MRS A is in hospitals; where risk factors for MRSA infection include peer antibiotic exposure (e.g., quinoione antibiotics?, admission to an intensive care unit, surgery arid exposure to an MRS A-colonized patient. Patients with open wounds, irnrminocomprorniscd p .'terns { tee 10,, g , HIX \tl>S e etc, t, nu'Kpbnn puwvhne eo'.o e-d-mak xomig chdAroc to e, human infant ami human fuddiery and fee eULtK e g , cMu K banian I are at high n-k of dovekipmg an MESA mfeehou. Higher n,4. sates for MRS A infection are also observed m injection drug users, persons with diabetes, patients with dermatologic conditions, patients with invasive, devices ie.g , intravascular catheters), and health care workers and other people who W'\i moe w wndnunt spaCu ' ipn^m mme.cs p e\n\s n is-'oU'Xi h s'jgear,
i act hues, such ns nuvsing homes) λ .^uja most > omnywk colonircs do s on. nor cams phe novum st. nhhonwi nor ο, o of Mo resp;r,mv> hoc- opened wounds, numxenons e-nheiem and urinary tract are also potential sites for infection. Most of community-associated MRSA n tVn ons som kV’dic,',! m the sk η n t wf, ns^n Γν mb i \\ of X'RSA ,n> iu,? mr btrnps the* tcscmbL pn'U'k' xp'd,' bn.'-oi coils 1„A n.s\ be eecofmunxd N tem' ,nv mslvs, die X'Uinr"- inns inter develop into pus-idled h-"-ds Common manifestation' of commnmn -associated MRS \ aw 4 m ·ηιθίθη.νν-, omi us cee-'-ovnig t omu > >n peon y'suis, veixei ',*; pneumonia, infective endocarditis, bone or joint infections. Some MRSA leads to sepsis and toxic shock syndrome, which may be due to toxics carried by such strains (c..g., EVE, PS Mi. MRSA may cause cellulitis. Any of the above-listed or known in the art strains of MRSA, patient* diagnosed with MR.SA, symptoms of MRSA, patient, populations a- risk of MRSA, uej o diseases ..ssvciav.,; oho MRSA m.-x |\ nested well die ..0:000 noon eLreifve Ίοόό In sonic embodiment1!, the compositions described herein prevent onset or development of one or more of the symptom.^ of MRSA, or reduce duration and/or severity of one or more of these ,\Y!'-rCo ns leg, .>·. omm ns Λ -νοΗ. I '* Crete's jliOOlj The compositions described herein may be used as bactericidal agents to kill or damage unwanted bacteria. For example, the compositions described herein may be used to treat established bacterial infections, prophylactically for the prevention of bacterial infectious, or aOmmisteteJ dun·, alb. to arcus of ? sidy·..*.! that ate MisiepitHe M infection ot v aieusM' ihc body that are likely sues for bacterial growth (e.g., the gums, open wounds, bed sores, and virginal or groin roe a:··.). 1001331 Inc " mbcxto.vnv '.be ον*\'„Ν tv* - 2.,-: toco "vos· toeece ',>nck"x2 y- \w." and/or bacteria) survival by more than about 0.1 log, 0.2 log, 0.25 log, 0.3 log, 0.4 log, 0.5 log, 0.6 log, 0.7 log, 0.75 log, 0.8 log, 0.9 log, 1 log. 1.25 log, 1.5 log, 1.75 log, 2 log, 2.25 log, 2.5 log, 2 "5 log. i U>g, /.25 log, 3 5 log, 3.75 log, I log, 4.5 log. 5 log, 5.5 log, <' log, 6.5 log, 7 log, 7,5 tag, 8 log, 8,5 log, 9 log, 9.5 log, 10 tag, 10.5 log, 11 tag, 11.5 log, 12 tog, 12,5 log, 13 log, /."log, M tag, = -4 5 log ot I 5 tog sta ,otao\ nv-nung usm-6 Ή 3 ml In , oomo cmtavlmu m·-, 0v vota'Vi-tason' 3, Ki'l, d hesen; u\U ee itacug-g. lb .suito' bdetenta * n ^,v J b- mom 9 2 log to 15 log, 0.7 log to 10 log, 0.2 log to 5 log. 0.5 log to 15 log. 0.5 log to 10 log, 0.5 log to 5 log, O 5 log ta / big 1 tag re 15 log, 1 log m I 5 log, 1 by, 50 10 tog, I log ;< " log, I log m ' log, I log to 3 log, 1.5 log ro 5 log, 2 log to 15 log, 2 log to 10 log. 2 log to 5 log, 3 log to ! 5 log. 3 log ro ' 0 tap s ity Ui x og, 4 log lu ' 0 tag, 2 by, '0 8 og, > tag m 5 by, : by ro x y , ' log M 7 log. 3 log to 7 log, 2 log to 6 log of colony forming units (CFUVmL, and any value In between ds.-e''·.’!*! v hr ego ϋϊ'enb'.'.nnr go npovgbiTi.s t.igsg: nxta i o-gin rediu\ wmtaual g;-e'„ m artator bacterial survival by equal to or more than 1 x 10’'’, 0.5 x ID”. 1 x Kb1.5 x 10!:,2x ID”, 2.5 x !9!i,3 x 10", 1 \ iii'\ 5 x I0:'. · x I0i:. 1 x |ht;. 1 5 x UV\ 7. x KV\ 3 χ Ub\ 5 x 10K\ 7 x ltb\ 8 x l(f'\ .1 x 10! \ .1.5 x 10 or 2 x 10** (CFUj/mL, or any range of values so between these values. In some embodiments, such reduction in bacterial growth and/or survival ,\e \ o ta\i 8,50 „to ό !" ' i , Kv w ^ , ,, g\ s v.'' o wr*>, ' hours, 8 hours, 9 hours. 10 hours, 11 houts, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours, 24 hours. 36 hours, 48 hours, 60 hours, 72 hours, one day. two days, three days, four days, five days, seven days, ten days, one week, two weeks, three weeks, four weeks. I month or 2 months after treatment of a bacterial infection with a single application/dose or multiple rypliotaiotodosesora sNAG nanofsber composition. )00134} {In one embodiment, the infection to be treated with a sNAG nanofiber composition is not a viral infection, a fungal Infection, a parasite Infection,, or an yeast infection [891351 A variety of diseases or disease conditions associated with bacteria) infections may be treated and/or prevented with the sNAG nanofiber compositions described herein {.see, o.g.. Section 5.4.2, m/nt). in one embodiment, methods for treating an cxistutg bacterial infection os a dixeas·;' associated with a bacterial infection are contemplated fCIOO&amp;j In some embodiments. the compositions described herein nuty be used to treat . w s o\ ν' ΐ , ' s ,. iruiM r tiiU t -comr'- -ms oon \e \ *!v ΐ'ΉΟ' , as v ,ί„; na' \ ,'Λνχν w cures m οηκ** ec \\ e cv*-. me .m Te- tm is dev '«bed herein are used te prevent bacteria! infection of w ounds. in some embodiments, the compositions described herein are used to treat bacteria known to be associated with wound n-f-Λ $ ''ns, ,md sp- , ? '.ohb used te feat λ\"'\ - «,-> m-'. - \mb 0 mw5, > ,, ; , pyn>genesc Ememcc.fcci and/or Psitdmn*>ms miiruginma infections, and diseases associated with such: bd'ection·’-:. (00137( In other embodiments, the compositions described herein arc not used to treat wounds. In one endxxi intern, the compositions described herein are not used to treat chronic wounds. In another embodiment, the compositions described her ein are not used to treat hum wounds. In yet another embodiment, the compositions described herein are not used to treat surgical wounds In one embodiment. the compositions described herein are not used io treat chronic wounds, burn wounds and surgical wounds. In another embodiment, the compositions described herem are not used to -reus a wound and,n? n burn, tu >of another en-bodinienn die cv nO'WUi.ws dewi toee betesn e;e * m u - „o ί$„χ in,- uhx tCu wound. (00138( In sonic embodiments, the compositions described herein are not used to treat a 1 eewradv infected wound hi another die compos tow's dev nVu Ocwnn vre not used to treat a bacteria! infection associated with or caused by a wound, in sonic embodiments, the compositions described herein are not used to treat bacteria known to be associated with ten ' u'teetoushi.n ' s , , , >· ' v^w s . . , , (08139( It: some embodiment, the compositions described herein may be used io treat or prevent a variety of bacterial inieeitons and diseases caused by or associated with bacteria! infections, which are nor associated with a wound (see Section 5 4.2, hijha) (00140( In * "a"' cod ;\v> ;va,o\ ''V ", , s"V\4 cr w \,v', r 4νηκ-ι comprises administration of one of the compositions described herein to a subject os1 a population of subjects to treat the disease or to obtain a beneftemi or therapeutic effect. In spec! fie - unvJn'ivov wee 'icon cut sK'O wv two three, foe5, fur m -nor.",ρ n ,,- toi -"sme efto- is in a subject or a population of subjects; Ci 5 reduction or amelioration of the severity of a disease 0’ " c< λ ,"o os d' t n5>,5 tu<" >5 he n '0' o o-ve.-se i e -oripto^ χνχ\ uvP m," ν d χ px’xmuo" t ί mom·"*;'ae-¾ an soisoe' a Xjmm: x avac eoe d ,κχ oh, < t\ 5 s, m, ssmu ot a disease os a syruunm us Oeiaeu tm ,ewxK ;\ ' mx emmu A me develop meal or onset of a symptom associated therewith; t'vi) prevention of she recurrence of a s\ emt-m n\soex;ed the row sds, < \n' px'\> s mvi or - vtemOon of dm seiwm o· s dwe ,se fum· the subject or population of subjects ίο another subject or population of subjects'; tvm) reduction in '' ge x'xhne uvsoc mee xu ; o ;v ,χ tpO άί,mm <v dm e > ve've o'""0'pi\dmx m; ;\' reduction of the hospitalization length; S.xl) an increase she survival; (>;h) elimination of a dKcavX s, \n-i -a uir-i emem or mo'-^ e^x sa xf'he pmoh\ eX'c ^>ΐ ί'ίο'ηροηοο - ffect|\) O' another therapy; (x«v'} improvement in quality of life as assessed by methods well known in she an, t g , a nuesuonodu-x ; ά reduction of Urn number ol «smp;<mv> el a disease, and m ί.\Μ· rednedon m monelin in remc em^nf nxuK tm-annem campuses ms dmiusx irenx; compositions described herein jMOI $1 j in ,· emlvdenan', oeaurem oxt tswxkd όχχχχs ,'(Οϊ.λ;ι\χ semen exmon of one of the compositions described herein to a subject or a population of sub]cent to treat the b'A tern ru'-iUr.rs.iT^^uvxtwcii'a s juvr a u f <,\'v-r· in qve'de ombodmvnss, s;m i treatment achieves one. two, three, tour, li ve or more of the following effects in a subject or a population of όΡ· acts x) tiro < lea recce <n a b cter·.'; infect on, {u < the eradication of „ uc o; more symptoms associated with a bacterial infection, (iii) the reduction of time required to clear a vkuco d inils 0mx x t tt;c redteu m or emeherut on uf the χο ,xx ,e a b.u’te't d mfc; ,mo ,u e, o' one >'» 'non s\ np*ons esreicimco dx^exun s\ i f"o t dm tu'·*' m 'he dm Urn5" o' a bacterial infection and/or one or more symptoms associated Therewith; (vii die prevention or delay of the generation of a resistant strain, or strains of'bacteria, or reduction of a number of resistant strains of bacteria generated; ;'vii) the prevention in the recurrence of one or more symptoms associated therewith; (viiit the reduction or elimination in the bacterial edl population su, ' *s „onUomr baa ? a ,n!i!r uohqn,v ", e , \ χ , „x i H!, ml, or a lee i eduenon lx <me of the mothmb' kuox n m the art m devxibcci hcreuo, p\ ι iho redueii.on in hosnitaliaariort of a subsect; ts'i ih.e reduction i.u hoaoitali.xnion length; tsi) the iivtoat·' td \!nse,;d>u e soheex ·, die eeueeeemx^oi x'pu'su ι,.ί,ί ,be exas'cam fbaot i'Vt vt 'Ixxaxv, tvht ,i jeds'Cfior m norvf;\v^ s 5 the redm >!on f>' e.nmmatuv" x me spread of the bacteria from one subject to another subject, orotic organ or tissue to another ofgun or tv-MV i\s ' iho rTO', r'ii'io ,χ' n uvx' xc 1'0 c m'.r.'boi s.v bac· 'u ; v\ O me rs'c-\cmτ o' me j."Λ ’νρην-η w m -'ν'' os'''-sv w score o-r-p'orns ' smc 'tvr .’with, t\\ui 'ho :n s;v number of symptoms associated with a bacteria! infection; txviii} the inhibition or reduction in production nf a bacterial toxin ei Ηνοη.χ as μλ. feted with a bacterial iafeehoe, (<10 -be ' ' <o rii" o is'innon ' wx V * s e 1 n e , " ' in organ failure associated with a bacterial infection or a disease associated therewith; and/or vx\d V ' v \ i,' ΐίί^ (} i e o- as emeo w " w .m wo ' b< a , ciuesbonnasr®, |0014J-f in e-.Tiom - mlvdimeoiv aOroimsu .umo of in·- ee-nsposmoiw nevrsbob m >ν!ϊΐ lu -i ' sh^i i KMji'- n one ,-s ino'c m tm m! o\m'\c fe5 .he mduetmi' u, tru >mp '.wen e· eiso os n<ne defers mu pO'ieem .sskI or d-'iko ,οι-ηΐ"' piosomoils'» she iodneoon o; do. expfessien of''-no m ionic Toll-dike ic» epsor-·, osd sn sisis she isfenUson os the esp-ovdem of one or neve piU, sno -h;si are beneficial for clearance or reduction of a bacterial infection or one or more symptoms asiioeia ted· there w i the |00143{ In certain embodiments. prevention of a bacterial infection comprises administration o' one os' die eompov.s ous deven! -ed n esc in ;o .¾ -005.0 s ot a population o· sve;eeO' 'o s '*ηο'Ό one os uvie ot the Sohoo mg cl feeis. s*l b c .nb.buson ol dk newlm n. so >>t .*\sei o' a km. ico.d si'tect'on, o> a sv nvoo vt ,5V\.v aied femeo. oh; aee or * s'k infebnon of the reo-ητο ice of a ksen ss,s; inleeoo?:, <u a svioptosn amoeian d iiserowid! | O0144| In other embodiments, prevention of a bacterial infection comprises administration of one of Use eomposissofi\ described hendn so a •'uluee5 : a popul.ufen of sokeeis to puvens a disease associated with a bacterial infection, in specific embodiments, such prevention achieves osie or ηκην os'd o so'slowsrg nY ms is ο sub vet or . population of- nk λ 5\ j s t:’c nhOi'ion of the dcvdonnieni or onset of a disease mkou-ikd with a buckiem! infection or a symptom tliesvof. aod-'or (ti> the inhibition of the recurrence of a disease associated with a bacterial infection or a sympsom aksoe sated therewitk 5.4.1 T naimmi or Prevention oi Bacteria! ί nfecrion in Wounds [00145| In s ersusn enskvisns' :,κ. she s\AO n^n'fe·.; coin pods son., described hc!c<n m.i\ oc useful for treating a wide variety ofbactenaliy infected wounds affeeting any tissue of the body os pV'-ounsm sf'vei’on o1'wot'ids r sk ''-s' v'"0!''g rPcidOv v>'tn been s".s 1001461 There arc two types of 'wounds, open ami closed Open wounds arc classified according to the object that caused the wound. For example* incisions or incised wounds m"rm s.i mwa? νfe-i ate !..ηι- ο b\ ivfe.>;\ fee. o ,',χοο-'t' λκύ*1 v','.\ „ ~>ο·ιυ o; a glass splinter. Lacerations arc irregular wounds caused by a burnt impact to soft tissue which lies over hard tissue ia.g, laceration of the skin covering the skull s or tearing of skin and other w > ,. ' , s v*fe < h it m ! λ ' ' ·, x ,s s rounds ' v v. " e topmost layer of the skin {the epidermis) is scraped off. Puncture wounds are caused by an object puncturing the skm, such as n nail or needle. Penetration wounds ate caused by an object such as a knife entering the body. Gunshot wounds are caused by a bullet or similar projectile 0 , W V v , O' ' Ok ", ONllli'1 OOk' ,, ' ·,' V 1' 0 * c' 0 0' all stab wounds and gunshot wounds arc considered open wounds. Open wounds also include hut"* 'aounj-> ii-dtsC' b e> m.”nod, ,Ί'οηη,,ι,, ,v · ‘oob'.oal in nr\ t lose·' woniv ·, snJede O'-Uksu'·!'·. rnov * mnieouh known a*, a t' a.so cm-sod m bluut mice u turn i dud e,touv-tissue under the skin), hematoma (also celled a blood tumor, caused by damage to a blood vessel iKu in :usn causes blood to , οΓλ.1 mbe* the sktnh and -'mshe'e uw\ * cans; J K a g;o,u o; extreme amount of force applied over a long period of time).
[Θ0Ί.47| in certain embodiment», the compositions described herein are used to treat a bacterial infected open wound or prevent a bacterial infection in art open wound. In certain emh<feim'rvb\ fh. oomp,)\nmriN describe*’ von max be uw to treat m otexeet a b.-eter.s infection of a gunshot wound, a puncture wound and/or a penetration wound, in certain, embodiments. the com pom ion ^ dasemvd heron m.o be used m treat or me' cm ; posi-yvmhw bvetemn iui' mum, .i minionl sin buetet nil infection. u eathei .-avkued burner sal mieehou oi a hemodlaiysis-rehbed bacterial infection. In yet another embodiment, the compositions described herein are not used to treat or prevent a bacrenui infection in an open wound, a gunshot wound., a puncture wound and'or a penetration wound. In certain embodiments, the compositions described herein are not used to treat or prevent a post-operative bacteria! infection, a surgical sire bacterial infection, a catheter-·elated bacterial infection or hemodialysis-related bacterial infection:.
[hbl4H| h son e e-fesfe.meut··,, 'he wmatd w ,m hivon wmati fehro ue wmmd can he ntl\ wound shat lad- to deal pwipeds, including a -ury-efe wound tea;, a skin yeah donor so·.), a i/aoeou,'· n eet ic g , a bio tie usee*, ,e νο- sc-ss ,ikef a eg n\o' an coomb u\\ino, κ-η,ρ ulcer, or a pressure ulcer), or a burn wound, in one embodiment, the corn positions described herein are used to treat or prevent chronic wound infections (e.g. a.ri infection assoc feted with a. dsabem. :sk'> r, η serou- v,wi.:, idee*, .x log ukor an anenJ uisuff-erow \ ulec",;; proN-roc ukek a sumkat wound, or a barn), hi yd another embodiment, the compositions described hot cm are not u^cd to treat or prevent chronic wound bacterial infections (e.g. not used to prevent a hd i i v JMK, \ , i » ,. Mi % \ , s » v. insui Fteieney ulcer, a pressure ulcer, a surgical wound, ora burn).
[44144} h o , oe w ',. -, ,ne eo 'V \ oev nx\! u , , ,j <>'t e ίο proroul no'-oeoraial baeieoul womuons {it the nosocomial budennl nueumus, sergsC-b o onrnj bjetcra' nfoeuor-a Moo route, ana sinks'vs showing up to v > -o at' -ns-gK'n· p rvra t he direct cost of these types of infections is approximately 4.5 billion dollars pc? year. Many of
Uospoal romr.V),, Kvk'!v i.,. \-doped ssiM.nrv no nr-.,. ,r"'cn.O\'s rfuj ,’hes ren entih>'ue· based tmamsenis are desired. Use of the sN AG compositions described herein in a hospital setting could defray much of the cost and markedly reduce the production of antibiotic resistant species. In yet another embodiment, the compositions described heroin are not used to treat or prevent nosocomial bacterial Infections, such as surgical bacterial Infections.
[001.54) in one embodiment, the compositions described herein may he used to treat or pro^cTt beet so aI inkvhem - in He, dire vro-unds ro g., hleceng. ; n >w.v ", cuts5. In one embodiment» the compositions described herein may be used to treat a gunshot wound, a puncture wound, a penetration wound or a. surgical wound in order to treat or prevent bacterial infection in such wound. In yet another embodiment, the compositions described heroin are not used to neat or ;\ ' rave') d η Ανηοο.m .'hveing wou: cK ;; g , H o cm; mof ao woo* s. s') [401511 The compositions described herein may he useful for treadng or preventing a Kvteuai rofoekero in uuiunecHks rounds, roof w wounds ,n venng the ,-ρκΙοητ* J and dr real layers of the sain, as wdi as injuries to the corner and cp it hei ia-lined organs, in order to beat or proven! a bacteria! Infection in such -wounds. The wounds may be caused by a wide variety of phy-v,d ,t,mo a, un k.dn"g sin ,mo"x Vu w, d'-.nuv expest-ro, seigic.J p*o,v U; es \o i , surgical incisions, skin grafting?. In one embodiment. the compositions described heroin may be used tor treating corneal and «clora wounds, including wounds which, afreet the epithelial layer, stromal layer and endothelial layers of the eye in ardor to ro.'&amp;t o*· prevent « bacterial infection in such wounds. In yet another embodiment, the compositions described heroin arc not used to treat or prevent bacterial infections in cutaneous wouuiis. |0Oi52| In some embodiment the compositions described herein nt&amp;y be used to treat a wound in a patient diagnosed with a bacterid i.n lection, In certain embodiments, where the compositions described herein are used to treat a bacterial infected wound, a wound is dew nessed to k k0 we .dh n kefed k, e vv o. uu ,wso tin die \ r> - re e a tre* to , e wU'wen In one embixiimeni, a wound culture is performed to detect a bacterial ίmeet ion in the wound of n patient. In yet other embodiments, a 'wound ta determined to be infected due to the presence of one or more symptoms of bacterial infection, (0015.1} In od’cr "i"boJ'nvnis me wovv-'. tves ire-,, -dvd knew· rere o>- u.\ed ’ treat i w-ouud tn a patient when a patient displays one or more of the -symptom» of bacterial infection such as: a wound Is slow to heal; bent, redness and/ot swelling a? the site cn the wound; s nb, m, »s u U t \ne o''die wound, fe mu go m *\uo m pus u rk v.,e s* he warn'd. ,mdui tore's v'i v w ot i ti , tinn i e'jdeht/< v. o ' dmkv<keo " ' loads,' 'J pom, feed teed k a, ec U few, orekrww ,tK, cm otv.mu.go wine i may v \ tv ini'-discolored and purulent, delayed of wound healing, discoloration of tissues both within and/or at woni v o re, ' c ,v, e k S' ,, *fforru sne K ^ ro η w " in site, unexpected pain and/or tenderness si the site of dressing change, lymphangitis (i.e,, a red line originating from the wound and leading to swollen tender lymph glands draining the affected ,ηο λ and wound breakdown ,re*wrened w u i wumwl poekebm brdfe'v at base o' reomn, < t, ., wound develops strips of granulation tissue in the base as opposed to a uniform spread of granulation tissue across the whole of the wound bed). In some embodiments, the compositions described herein prevent the onset or development of one or mote of the above-listed symptoms, or sww'.ee delation and ,s smelly *0'one nr tm''. m' tW-<e ,\\ m-nom·* |80I54| In one embodiment, the compositions described herein may be used (dr wound healing and treatment of a -wound bacterial Infection, or for wound healing and prevention of a saw , uo-um mb e>\m lnoirumoirv', orreoeviiOee is n , , , v,j enhance wound healing while concurrently treating or preventing a wound bacterial infection. 1 iris κ oft w s\ \fe n u *ni »er eempovt rev on oo^u d I cuowe ,re,i some <»' the ’«tv> el ti e Λα(ι awnofibew'. m wound he,bum application^ lure, been JorenKd ns t ,S. Ikuen·' Pub No. ?009 01 1 -s' seh o. weo;pu: ecu re rev tore'e rexem n mm.. re re e e e . 5 vatrk 5 i" 5,4.2 Treatment. nr Pmenikin si Other Bacterial tnkznmvi [80155( in certain embodiments. the sNAG nanoilher compositions described heroin may be used to treat and/or prevent bacterial infections of the skin, gastrointestinal tract, respiratory tract, urinary tract., reproductive tract, blood, throat, oars, eye, sinus or any other organ or tissue of the body. In another embodiment, the s'NAG nanofiber compos-lions described herein may be itv, 2 so tseat os ρο" etn <vr a-v\imwo- ea-eunw estomi e ' 'dt.to'ts swornutor^ -summers
and/or conditions of any other organ or tissue assoc iated with a bacterial infection. in some embodiments, the .$NaG nano ft her compoaitionN dc-Ncribed herein arc applied topical(y on the skin, mouth, ear, eye, anus or groin areas of a patient to treat or prevent a bacterial infection. In some embodiments, the compositions described herein are used to treat and/or prevent a bacterial si'seetvsi os' o osyas* o' t-ssuo -n s''v !-ν> i.,n -s um d t sc sue of a mo.md, and or m not assoedaf ed with or ca used by a; wono cL |00J5h| h' t,anam oosbods wont-, the s\ n em’-he- aowpowno''-, desorsbeC ',e<\”r oc a.-mb to treat art existing bacterial infection. For example, such composition» may be used to treat a s'.shy v s bwgs'w-'Cd voth s baewial meeoon b\ s tost rt an „,x\u\, .:,-vh u, o! -· os's.·,,' w-ts described herein or known sit the art. Alternatively, such compositions may be used to treat a sob ee* c»sp. t\ or;, on: ,n nunc wmpnv"!-. of a i tera. i f:, s on ο» a e m. -: .-s-'-vwtob -mdt 5 F a tem fp. s u-;-w oj> j one or mote -, oaetesrp micc-ors knov, r: so a shdied artisan so g., detennined by a beating doctor/physician to be a symptom of a bacteria; infection? and/or described herein [00157( Irt certain embodiments, the compositions desertb i>'ii ilwi cm are used to treat a condition associated with an imbalance is? bacterial microbtota, or a condition associated with ass abisesrnsid or altered bacteria! rniemhtota. For example, such compositions may be used to treat' a skin condition in a patient whose sHn bacteria! mierobiota differs from that in control subjects ίο e., subjects with no svmntoms of the skin condition*. In odser examples, such compositions t \w A s. 'i.,! m t eat ass nth cttnul condition ,,-¾ a . ,-η-.Α-ν m civ. e.hct tis-ae -, s o;e„ro as a patent wit-we sotes's A Ivevtial ('ae'-dr-'-.w sot we le-mo:,; of we ed et si--sea w ww a. 5¾ re 'or -«rulin' .etnuoi-t a -. λ ids n ---- '\rntK nest i'h"'rU"i [8015N| Ir oriv et voJmvn.s ska u'er-s/vr^ oa-"d:-aj Awa' ten; t- used to front mw disease known to be associated with or exacerbated by a bacterial infection (c,g., acnct. In one embodiment the compositions described herein may be used to treat a cystic fibres in patient }00159} In some embodiments, ibe compositions described herein are effective against toxins .secreted or excreted by bacteria, in one embodiment, the compositions described herein: may be used to mhi bit/red ace a bacterial toxin (and/or a condition or symptom caused by a bacterial toxinκ for example toxins produced by Baciihts' mthmt'ir, OostmHum Βΐβιαία, v , , ·, ' , ν'- ' ^ ooom'n an ot die χ o χχ V ,. detes’xsrsx are an e o nb 1 i m ο i J ' \o χ m std np mo-v priA x v Ov ' χ . , v
OmfHdium difficile, Cmynehacterium diphtheria, PaeiHiommas aentginma* and the cytotysins -, "'id-'iO\ ns ροχίροχΑ '> Omm γά'λ , \txdorut tre. οm e·,· blood rob··' Cnscn those 'ί"νηο\χ ofdetensim, activation of pathways rextotinp in defenam expre.salon and secretion may allow for A e .. e . \x ov ' v e e '\ met I ,χ " >e . bacterial: retostooco, 1001601 In certain embodiments, the compositions described herein may be used to treat ί d o' ptc'- cm mm or more haei t>a! inrivnoi χ ot the .Am., ot o,senses ot t· o skip .isxai emm v, nb a bacterial infection, in some embodiments, the compositions described herein are used to treat I- valmcd skm infection-> and 'or diffuse xksn directions hi xome embodiment, the compositions devrdvd hemm .no used, to treat or present -Asn mfeenonxor div-.a-o.·* of the Am ,;-, -oetoeo w ith riaetoruri mA-too-fx affectose the omoereus t. ornux and o. xe. reneges ri\ pAct uscoox o! du A la some to these cmbeuhmemx, the affAtcd Acm m tK xl.m m·. UiJu one m n.ose L" ,"s m t ό ! p'd, 'tc.s A e , x.stoum oaxtoe, xtsatvt' pinos-, ;n, .dtatu.n |.mt ,Ι,ο, m xtatsm ti.se tsr. ' n-itursAOx to\>jfvot smse ' >p·, χ x? -voO K de ,χ , c o , _ ' elastic tisstse, and retieubsr fibers), one or snore layers oi'thc deartis ti.e., ibe stoper, papillary Ae a id r.c lodes tot, :,h' Aeril, and o. one or mom t\ pcs o'' nxsti. ofthr h^Ad.ur so: is.?, ehxtsn and consultive tissue), in an embodiment, the compositions described herein are used to treat or prevent bacterial infections on the skin surface, in another embodiment, the oomposirioi's described herein are used to treat, or pres'em a SiitphyitsCiKTiis {"Staph''} infection , , ·, An - \ o , χ , x srfec'· on η ' χχ < In yet another embodiment, the eomooxruuix viese* b.-.j heretic are used to ncot λ\. A . ,χ .’riunj os AvAAvoto 1 χ A\io o* A x hi x« ns*, o sf * JsmUetoAx' ' χ χ ..χ,χχ 'u i„„e '0 ow.0 m'ree or ''rexes't lo’iuhn-x tnvcmv! ΆγΑΊ -. Oixtb'axma, cmOeoch y f'rurtk'x ,'S, ι i\x \ v. . ν' - "1' nivK' m\ 'v^ iK v Λ v " do\ \o he'cm jio ,weu m o. at m movent cethi m·, keJums at reef * u o '.e-rsa de"5.N and Mil'esifanoous Kssjiw and issue to affect' dv t η ο. arms, and levs and .dmov .boη·.\ occurs duo $ο a break in the .skin that leads to a bacteria;, infection. The aymptom.v of cellulitis include one or more of' swelling of toe skin around the break in the skio, pain, tenderness, outward signs of nh-4, me, iud ')V<s ku'soo"! n v ,\o 'to r (iJos, -oro! a '.J , n ilK I"! n on >,r οοίΚνοοο1'·*, toe composition. described heroin are used to treat or prevent a bacteria! infection of the skin at the bob fid ick>< ;e p totos'uto ;,'s The sYmrtoe'R" of fed.- o m-' miton'e melto'*,, peonies surrounding ihc hair,, bard nodules, and pair;, in another embodiment, the compositions d -set s'd bom n :na> h u- .d m 'teat os Oomn" o.o>"c mpe n-.mo of ,s.no t' m ,u t rdy a sv?np$om os'a nuclei :to mfenmo in owe entombnisnt, the sOinpostoosss ds-ercvd heroin a<e used ίο treat or prevent dermatitis associated with or caused by a bacterial infection, in some embodiments, the compositions described herein prevent die onset or development of one or more of the above-listed symptoms, or reduce duration and/or severity of one or more of these symptoms:. |0OI611 In certain embodiments, the compositions described herein may be used to -rear and/or prevent one ov more intestinal ''digestive bacterial infections or gastrointestinal diseases as-eeeaedw ο η,κη.Γ,ί! mteotvs'- ΓΚ: eon moo tom·'· <o'i'iev,t\,d - s term* e ketov n, k.ou Jf/Pblff W&amp;MMdtMMt Jispfele ''Tidsi;:feadterid:. cause diunheu and irdlarnmatiors of the stomach and intestines, si so known ns gnstTooneterims. Symptoms of an intestinal bacterial infection include but are not bruited to abdominal cramps and pain, bloody feces, loss of appetite, nausea sometimes accompanied by vomiting, fever, and diarrhea. In some embodiments, the compositions described herein are used to treat a patient displaying one or more symptoms of food poisoning, which is often associated with a bacterial mfociion,: 100162} to v 'a \\\ v"s ,ν ο 'vs ο ,* ve " ' , s , ο o ' disease associated with a Staph infection (e.g,5 a Staphylncticcas tutrea^ infection}. fn some of toes. cm! f-dmvew, th, tosetse S'- a ,\x w utooor oj Pn. ston, n<we mouth and et ee'eta' atea. In μ»nc co I ' 'it, / v , m" c o v\ ' \, os. \v.,s. ' and/or septicemia. In an embodiment, the compositions described herein may be used to treat huetona restof,jro to one m mom uuto;onex in? ewnpie n'cmk dlto testatum y.m'' t ,. 'VR\A, ' i KVrtcJ1^ ν,τ,,’ί' V' S iC ον V"- * Κί iV 0 >ι < lO *, ' '' \V’e* h' a Ox ..,- k'soa tvu 5 „ -.' „' 'ufeasoo, οι m a sub vv, o, em., e. mom -v'.pv:,..* o'' a S rapii infection f e.g , presence' of one or more of: small rod bumps, crusty red bumps* pus idled or ansocss, bods a\o m ds. ex's bh.o.c's ,n d o' roe v mix Aon s.tc- as red scabby skin around -he nose and mouth, or symptorn/s of a Toxic Shock Syndrome). in some ensKsJinv'Os, the M''np.wm,u s go-cnKb '".von poo·.. m da ο"μ ϊ os Λ fen 'em ofew or more of the above-bated symptoms, or reduce duration aod/or severity of one or more of these [001631 In certain embodiments, the compositions described herem may be used to treat or p c\ ."d a cole ..-.xx , rfee n 'th « :st ό ,.d ή ef ho" to’ e\ nv'fe 'he i o''i;xv>u<,>'.- Λ < u f hemm nt η, iv nsed m η cut m pxx.,nt ; cold den pervsts despite me wx eu\tac,iUio p on "chef medications, in one embodiment, toe compositions described herein are used to treat or prevent a bacteria! infection of sinus, eat'or throat. The symptoms of sued bacteria! infections include localized pair; and swelling. Bacterial infection in the sinus may lead to nasal discharge and ui ute ρ,ηη hi part;., of the b.xv or forehead, m one embodiment, the ce-mpixh-,on\ efe.x π bod herein are used ιο treat strep throat {Str^ivcwcuspyogvntts} in some embodiments, the compositions described herein prevent the onset or development of one or men'·;, of the above-listed symptoms, or reduce duration and/or severity of one or more of these symptoms |f>01 d4[ In some embodiments, the compositions described herein may be used to ires.· or o ' ,t κ ' i,i > i < ! i m { ,uerx ml „ o - x' x' ' * ' ' " tract associated with a bacterial infection, in some of these embodiments, the compositions box race he-- m m\ be usee 'urn:,η ; sexo.nK mm :1:,1 bxoux. ...wow n, d ..\an , baemre infection. Symptoms of such infections include but are not limited to painful unnation, cloudy discharge, and/or pain during intercourse, in some of these embodiments, the compositions described heroin am used to treat or prevent one or more of syphilis, gonorrhea, clamydia, and trichamoniii.sis. in one embodiment, the compositions described herein are used to treat ChUmidva. In anolher embed uncut, the described heroin arc used to treat gonorrhea.
Symptoms of gonorrhea include localized pelvic pain, itching and irritation, painful urination, a thick yellow or green discharge, bleeding between menstrual periods. In one embodiment, the - v s' n kvi - XX 0 x0 . -, ' ' .,,'Xfe 1 „ v ' . . r mem1- i'f fvcm'ud \,xg ms. h'dc vacmed s. l- $ eon , odm, wgmfe m I nig, <ux ulvonural p un. fn some '"mboih meets, me f.emnustnon.> d- scoled herem ρη",era dm onset e-development of one or more of the above-listed symptoms, or reduce duration and/or .severity of one or more of these symptoms. |601o5| lx otlvf ' n'.vJmvnts, me compos Kms S'u'!·, may uvs) to treat o; prevent a respiratory tract infection (e.g , a bacterml infection of the lungs), or a respiratory disease associated with a bacterial infection In some embodiments, such compositions are used to treat or prevent upper respiratory tract m lections, In one embodiment such compositions are uv'd m treat or present mbemulosm Vui-erenloMs ts iausc-i b\ nmeobaetunum ml-'raimri \ tee; n ss a Ivgldv infection- disease bus o '-rmsd from person to pernon b1, sneering or sals a. fhu.>, *K emoposnmns deserted herein -tvs he used to r- at not onK vebme···' Jmgrtcwed vuh dihercuioass or displaying symptoms of tebercalosis, but also individuals in contact with such subjects {e.g, family members, caretakers or medical personnel). Symptoms of tuberculosis include coughing Mood. e\eevn\ o esghi lo-s, fanpoe. K>ss of mperiie end persistent fever 1st some embodiments, the compositions described herein prevent the onset or development of one m more :! 'be O'^odoueh sv'ngnerK, m 0.-00:e de uno5 aul/o' se- ev. \ o''o"e o" s vne ^ mes, symptoms, in one embodiment. Lbs: eoospositmns described .herein arc used to treat pneumonia ' d b , ' , uMt " r anod'C? t rod b rent vj„ ' οοτ"3\ηκ' \ used to treat bronchitis. in one embodiment, the compostrions described herein, are used to treat Aiomwfht carawW/.v, Sovpmcoectft psammoma and/or Hutifuophiim injiuenz>j, |0θί661 in some embodiments, the compositions described herein are used to treat or prevent bacteria! infections of a mucosal surface to.g,. oral mucosa), or a disca.sc/condition of mucosa) siom· c ..: 'veut.huJ v.stir a bacteria! mfeetmn. in on.; embi-eMn-.rd, tire v compositions de-vnped herein are used to treat or prevent bacteria! infections of the oral cavity, for example, such o'7'\ v o'v ' v ,v ' 0 0 ' eve' 0 , "v v ' w e v e em 0 the mouth such as gingivitis, caries, and/or' tooth decay. In one embodiment, the compositions described herein may be used in oral hygiene products 1601671 In one embodiment, the compositions described herein are used to treat a bacterial infection of the car, such as middle ear infection, or a disease associated with such infection, la one emhodmtem, > compos'd . u a^nbeo heteo is used tu tv.n 0' ov.kc e,o -ed ,-\ a bacterial infection. j 0«168j In some embodiments, the compositions described herein are used to treat or prevent bactersal infections of implanted prosthesis, such as hearts valves and catheters. In some O' " ' ' 's fu C ' \ ΌΧχ. x\.~ ,,. v ' vO O 1C x \, Os x^ ' ' x or dog hue. In order to prevent a bacterial infection [0O169| The compositions described herein may be used to treat or prevent a variety of diseases associated with a bacterial infection including but not I uni ted to leprosy (Hansen's diseases choleta, arnhrto se g. cut-memix urnrhav pulmonary andnuv , ;.,as m u rim··, to xd arubmO, pc 'n w- s grans,! one met u .do, hncvmU vegm.Wk go. vrr· o opsi'b.isnn.t ve-" ifunn;, so hk arthritis, syphilis, congenita! syphilis,, whooping cough, mycobacterium avmm complex, tnehodoaia» leptospirosis, tetanus. scarlet fever, strep infections, invasive group A Sirepfoc*/ccai disease, Strrptiwccat Toxic shock syndrome, meningococcal disease, bmuerirnia, strep dtroat. 's'\ Ο O s Vs Vi , s s, SSO'CS O' S Si m* ulnssi^sJ S V' ,s sVlo-'to'I'i S Kk' I xi.s gss, 'ViOgs sW to dssi ?itx'rs , x, vxltow, t p to ' rto CiiOeWoux if]dnhx'ia respiratory diphtheria, Legionnaires' disease, tuberculosis, latent tuberculosis, hemophilus to fit;cm rue B, typhoid fever, vibrio piirahacmolyricits, vibiio vulnificus, vibrio, yersiniosus. Whipple's disease, acute appendicitis, meningitis, eneepioditis, impetigo, relhitios. carbuncle, bod, acre, vpsis, septicemia pneumonia mycoplasma pneumonia, meningococcal dmcasc men up, ns, v, Λΐοόνϊ!-θ'Γικ\ν ο: son syklremo pktoktoto foou rv'-xtoto u toup'" t.sv pxUsOto.ng, Toxic shock .syndrome, necrotizing pneumonia, septicemia, acute Infective endocarditis, an Infection of sweat glands (e.g... Hidradeuibs suppurativa), a bacterial diacanc tomsruh.ied by a tick (e.g,, Rocky Mountain Spotted Fever, Lyme disease), botulism, plague tc.g,, bubonic plague, pneurk^nv "'Lsguo' κ narr- a, orueeHo*?s aeon, stoAnm nougxukvoee x. to to.} s'is lymphogranuloma venerium. trachoma, inclusion conjunctivitis or the newborn, psittacosis, pseudomembranous colitis, gas gangrene, acute food poisoning, diarrhea, traveller's diarrhea, diarrhea In infants, hemorrhagic colitis, hemolytic-uremic syndrome, bronchitis, listeriosis, anaerobic cellulitis, peptic ulcer, Pontiac fever, cystitis, endometritis, otitis media, sinusitis, streptococcal pharyngitis, rheumatic fever, erysipelas* puerperal fever, necrotizing fasciitis, nosocomial infections, pseudomonas infection, and/or eat scratch disease. 5.5 Patient Ptnmhnkue [00170| In. certain embodiments, a sNAO nanofiber composition described herein may be administered to a naive subject, i.e, a subject' that does nor have a bacterial infection, in one embodiment, a composition described herein is administered to a naive subject that is at risk of acquiring a bacteria! infection. |001?l | 'In one embodiment, a sNAG nanofiber composition described herein may he administered to a patient who has been diagnosed with a bacteria! ejection. hi another embodiment, a composition described herein may be administered to a patient who displays one m moo, w" gk\"- of , e,-cn re vice on [00172} In certain embodiments, the compositions described herein arc administered to pat toms dugnos' d with a bacteria: bifeenon to ccrrsn vm-hedm-onis a p .mem o, <1? ^me-vM wnh a bacterial infection prior to administration of a composition described herein. For example, die -,.onekssnions d',wet med here»it awo, n,. aUnunNered to a p meet o hen a hnctoK d emcee .w deveke m a h eto,p« A '-an ole to!, η Tom the p .Pent fr- one op l odenem, a -no Cue, a! sample is obtained tforn the sue or area to be freakd by the composition-; described herein or an area to v,ha h the composm-vs dost need vivs't «re v iv a hMiitotoivd 1" -me embodiment, a v-ab w used to collect ceils or pus from the site of tec suspected infection to detect a bacterial infection. In another embodiment a fluid is aspirated iron" the suspected site of an infection <e.g.« a wound? to detect a bacterial infection. In yet another embodiment, a tissue biopsy Is performed to detect a leu tci sal infection In an embodiment \\ here the -vUspet. tec site of ,m Infer firm A a wound, n - tv -. v ' v " , m u\i mu,,. no ‘v \«v in ό vf ' w t biological sample is obtained front blood, urine, sputum or (ecus of the pattern, in some 1 o w <j h O'V > ^ a , e 'on v or vo >o ee-. eve i uletoo' o, -to" a bacterial infection is suspected to have spread into the blood or other ti vsues/org&amp;os}. In some
Oil VO ' >N I.udiu . j-i, '«*, , s s w,'hio' > ON od O ,, h\A UO «. tt> methods such as PCR for presence of one or more types of bacteria. In other embodiments, Siiintiroitiio'ov nee as absw, so?ο,οc'"«v.Uto Ό m c., n -me ace* eirm-e5 0' r'·· oik", tost known and/or practiced in die ast may be used for laboratory diagnosis of bacteria! infection, [00173} In other specific embodiments, the compositions described herein may be -Cm, ' C\, ,,\ N ,N Ο N N N , O' ' ' o' O - 's ,- asaoeiated with a bacterial infection. In certain embodiments, a patient is diagnosed with a disease associated with a bacterial infection or displays one or more symptoms of a disease jowako -.-,-tb ,s haekmd urieetov pu-n to adem e-t ,mec o! c m- im-witn-', ,L «.reed bet cm A disease assoeaUed with a b,ieien-.d imeeoon m e, be diagnosed by am, m-touto \ rmwrs m a skdied a risen mehiving w-sluanmi of fee pam R11 s\ mpmrm·» arm m Jeff* non m a s-sotor-m 'mt«gcp m a biological sample of the paticun (e.g., «a described above), in one example» the compositions described herein may be administered to a patient diagnosed with a disease associated with a bacterial infection, by a treating physician or another medics! professional, in another example, a patient may use the compositions described herein upon detection of one or more -symptoms of a disease associated with a bacterial infection |0O174j 3 c Cvttan.,mboJtmeois a , otmv-moa uevifeca hmmo - acn\n»siC'cm to „ pat cot who ία- Κόί o ag'i- scs' wifti »m ,00- or too / Ateinn jnftv'iun by ,'·\» \ ,s i, d< '}\L'h Uu perm s w\, ih η vs ha har^vhwhn y ifem elia aHasu i Bra*. . IL· < and. drm vf/a mclitcos th. Brucella stay Compykibocier jejutd, Chlamydia psitioci, Chlamydia pnemmoam. Chlamydia rarfe>mn/ri, Chmddophiia pditot /, t 'kcandatm hmdimtm. i 'hrutmlm-n difficaie, i 'ioandnim perftingeifx, Clostridium tmom. Coryaebottenam diphtheria^ Emerocaccm, fae<abm iEsokericMppPlE. Cetzmiseiip; ϊμΜμμ1¾ HmmmpMiMs igjMeomC Helicobacter pylori, Legionella poeumphiio. Leptospira pneumophila. Leptospira imercogam, ' >· v a , » " , ' , \ v ' , ,» ', s », ,. ,' » pscudimtona·* oenigmosa. Proteus m'c..;od-<. PneamoceoEius η\\α. Phd'eoCa r\ kepB:, α.ίΟΟΟ’Ί bo oy ' , y , ,.. b V»’ , .. ..,,,„ 0,,--,
Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agaloot kte, Streptococcus pneumonia. Streptococcus pyogenes, Treponema pallidum·. Pihna choieme, >, r a ' ϊ o\ and -n ,wv otb.u bo, iottai nriV<. don described bevem o; known m the art It·. one embodiment, a composition described herein, is administered to a patient. who has been diagnosed with the bacterial infection by MRS A or Pseudomonas aeruginosa. |00!75| in certain embodiments, a composition described herein is administered to a patient who has beers diagnosed with n disease associated with bacterial infection, o g., leprosy {Hansen's disease), cholera, anthrax (e g.. cutaneous antrhax, pulmonary anthrax , gastrointestinal anthrax), pertussis, granuloma inguinale, bacterial vaginosis, gonorrhea, ophthalmm neonatorum, septic atthntis, syphilis, congenital syphilis, whooping cough, mycobactcri.um avium complex, meliodosls. leptospirosis, tetanus, scarlet fever, strep infections,
' S ' ,» n 1 \ ^ » - s , , \ S .0,- Ν' ' N disease, bactcrimia, strep throat. Typhoid fever type salmonellosis, dysentery, colitis. S ϊϊ'ϋ < 1 'Μ Α) Π \ Ov'\ V. ' ν Λ. ' s ' ( V' Ον ' ν. \ ο >. s'W.C , s s ,Η lilt ΚΗ ,0/. K'OJiJ flit I, Π .tesUluiv sufi'iU -. ' t , , " ,s tuberculosis, latent tuberculosis, hemophilus influenzae B, typhoid fever, vibrio pa-aba,m vbtv vb' m e\ wlv-so \,\'"5nos»\ \\ h,pp,>-7>h v.t*", „mne app, iim, my meningitis, encephalitis, impetigo, cellulitis, carbuncle, boil, acne, sepsis, septicemia, pneumonia, mycoplasma pneumonic, menmgocoecai disease, meningitis, Waterhouse-Friderichsen syndrome, ptomaine food poisotung, Staph food poisoning, Toxic shock syndrome, iVCsiVi.·! vy ρ-ί^ί-Ίί,ϊΓs , χ'λϊ ϊ ο"! ί, "ivs'tne c i-hw mints, <m cue tnut fi! «'-wi i ,! \l\ , H,dtvMemns sup 'mam.sh ,-b^ totu d.soise t.ars'rmLJ h\ ;,c\ m c,, 'v\L> Vmuman. Spoucd f t ye ,'a --casec eoUd-sm nlague so c,, rubornc p:ac.,k\ wv.mOme pm-gue's tularemia, brucellosis, acute enteritis, nongonococcal uremruiy lymphogranuloma venerium, trachoma, inclusion cimjunctivitis of the newborn, psittacosis, pseudomembranous colitis, gas gangrene, sente food poisoning, diarrhea, traveller’s diarrhea, diarrhea in infants, hemorrhagic colitis, hemolytic-uremic syndrome, bronchitis, listeriosis, anaerobic cellulitis, peptic ulcer, do 'ha to o «in* 5 o ·, ' o , s \ v, \ ·, r r, ,η1 u , fever, erysipelas, puerperal fever, necrotizing fasciites, nosocomial infections, pseudomonas h'teet'on, or e n o? r, , 0*0,00 |IW!76j in some embodiments, a composition described herein is administered to a patient with a bacterial infection before symptoms of the infection manifest or before symptoms of the hdVehon Kuomo vo· 0 s. before ths pasient n qiure\ uc.droom o! ho-'puah'.'.nmnt In some embodiments, a composition described herein is administered to a pat lent with a disease after 'iorVus of dv y-v s,\- m .'iif'.t ο? \«cu svnptoi w o* t'.> disease become sook' :e g. ado* 'be patient requires treatment or bospilaiication5. ji>91771 In some embodiments, a subject to be administered a composition described herein Is n as' ' , le , w ' ,. s ,,κ ο'»,'», sm, ineo ,n ,. ' id', t . mt r 1 canine. In certain embodiments, the animal is a feline. In certain embodiments, the animal Is a horse. Its certain embodiments, the animal is a cow. In certain embodiments, the animal is a mammal, e.g., a horse, swine, mouse, or primate, preferably a human, in some embodiments, the amoral 5s a pet or a iarm animal 10tM?8| l·' v, Oam entl'odmtenL·,, a sobtecf v b: ummmstesed „ u' t'posuim d.'wnlw tc'em is a human adult. In certain embodiments, a subject to be administered a composition described h-’*vn a hίί^ί'ϊ,ΐΐΐ <3oi,U mom dee* >0 ) earn oH K oemne embed mr nm, -a.,m m io !v administered a composition described herein is an elderly human subject. 100179) In certain embodiments, a subject to be administered a composition described herein is a premature human infant. In certain embodiments* a subject to be administered a composition described herein is a human toddler. In certain embodiments, a subject to be administered a composition described herein is a human child in certain embodiments, a subject to be , Vt,V u e'i' ios! ion d *.d hemm s us m laid h v. »\ \\\ \ \ e sup-eu to wf on i eemxwvjee oesm iK'-l Ivies m ad?r n ·»;*” m ns no; .m mmoi nf k *>\ n ar n months old. In a specific embodiment, a subject to be administered described herein is 2 years old or younger; )00180} k· yet other embodiments, a composition described herein may be administered to a patient who is at risk (e.g., at high risk) of developing a bacterial infection.. Patients that are at high risk of developing a bacterial infection include but are not limited to the elderly te.g., human elderly) and immunocompromised. In some embodiments, a composition described h-oe-rt <mmmiMereb to a panem -ο i sh <ri d· \>.: opine a beemt sal unecoom such o·, but not limited to an fmtnimosupprc>scd pariem. )e g„ as a result oi cancer treafmem or a franspianfuuon ov.o'* dfunru"ti J a p.em-uv "e ' " "-’ir - <~ν ex > kmm n upOivm v. *' d ri *es, a pot sot nsat >v,v. mth , .meet, -, p,Uu et >v«,' has \vo no u,\l wnh a eou v m haotown-·*» ' , \ ' -w \ ' \ \V s O'- "d , '0' ^ v »S1,\ so v embodiments* the compositions described herein may be administered prophylacticatly to patients who are at risk tor developing a bacterial infection. c.g., those with compromised immune systems due to. for example, ago, malnourtshmem, disease, chemotherapy, those who h. xeu ucak'd wu" a corny., of Had.K'U.ri nu.btol·, s, tnov v-'v p e > . '.vus\; ?e i. an open wound). In other embodiments* a patient at risk of developing a bacteria) infection is an 1IIV Λ!Γ*\ wc'seiU, , eaneer can, m, a p mem w.m I ,w m lov.v.e ,, 'T.m-phm' p oeeoe.e a patient with asthma {mg , severe asthma)* a drug user, a patient with a dermatologic conditions, a eeunns v tth viU \ u,,Hn. de\.', <c g , m nu>o «,vu a em) met h a 1 <.,rim , me wm-Vis or , pa:.sent who spends time in a confined iacuity (e.g... a prison inmate, a soldier, a patient in a longterm heal then re facility such as a nursing home, etc ). A patient to be administered a composition described herein may also be a patient with a chronic obstructive pulmonary dmo d 1 tropDj o" '"'n 'a <·»ι ere? r ' , ·> ir r ¢. > s bn \ ,i\ i' ew * v' O' ' {0018! j Ip u'5 5&amp;m cmlvdim.ots -s eorp’V^uM·'! dese* ,K’d herons admmvo'eb v a put5o'»' who has rot beers diagnosed with a viral infection (e.g., HI'V/AIDS), a fungal infection, or ars yeast infection, in certain embodiments, a composition described herein is administered to a patient who docs not belong to one or more of die following patient groups· an immunocompromised patient a cancer patient, an HIV/AfDS patient, a patient with asthma, 0 P OOiH who !. S * Ο,Χ'ν,,Μν ,s ,'}t 'MO. 00050. ,¾ p.Ok'iit W S' li.t" U' OHO .¾ v, k\M", . 0510 05'
Atpatlllt with:. a postil,. 11 dho epihodioidnt, tbe patient to li idniiiMped.:. 1- bompolWlS: devrr'x'd he*rn eoe''roi i ,»\c , vv>.mrd 50.¾., a omo^v J, 0 open wou^d di.r - g , v t .spgitrf or: lailoSall: haiiti j, |00182{ ir.etto’ oswd,m,"'t„, ϊ -oNo. 5 v 'v 00; m-moci ei a , 0 rpom v\ oom^s'w ; c' «0 is a subject with no 01 low level of expiession of one or more defensin peptides or a mufation/deietion in a gene or genes encoding one or more defensin peptides. In some ,5 - s'mo ϊ to odimmsiOO I 0 p> ^50,,¾ ,L Μ,ηΧν i'v" . sv.'i low or altered level of expression of one or more a-defenstns (e.g., DEFAI, DEFA1S. DEFA3, 1M Γ\: Ι;Γ, \\ΠΓΙ^οί ογπρκ*' 17-¾ ν,ο ΙΜΓΒί ΡΓ ΓΒ2 jf f R4, DEFBI 03 A, DEFBI04A, DEFBI05B, DHFB107B, DEFBI 088, DEFBI 10, DEFBI 12, DEFBI 14, DEFBI 18, DEFBI19, PEPS 123, DEFBI 24, DEFBI 25, DBFS 126, DEFBI 27, D1TBI28, DF.FHI29, DFFBI M, DLFBlook and or one or more b-defe πμι 5n K-g Old ΠΡ).
In: sime: imjFsHimedi. aMtjecf .1 &amp; te gdliimsf ifed «. ciinposlloi aAtih jeef with no or low or altered level of expression, of one or more of DEFA 1, DEFA 3. DEFAA DEFA5, DEFBI. DEFBI DEFBI 03 A, DEFBI04A, DEFBI OH 8, DFFBI 12, DEFBI! 4, DFFBI lb, DEFBI 19, DFFBI23, DEFBI24, DFFBI25, DEFBI26, DFFBI28. DEFBI29 and DEFB131. In certain embodiments, a subject to be administered a composition described herein is a subject with no or low or altered level of expression of one or more Toll receptors (e.g., TLR.1, TLR.2, TL.R3, TLR4, TI.R5, TI.R6, TI..R7,1I.RH, TI.R9. TLR 10. TLR i 1, and/or '-¾ " 1'' c 0' vo ' 0 ' s ' ' ' to , 0., η n -¾ 0 e -, ,\ -, . oo\ 000 , 5s .5 -itil' 'Λ I kit 5 55¾} 05 D\\ m oUC'> J l0\-' o| > \;ϊ' '\O05t 05 ,HJ0 05 050 0 ,78,-1 , ΐΤ U \M 5, SPAGILSIGIRR UU-bke receptor), IRAKI, IRAK2, 1RAK.4, TBKJ, TRAF6 and IKKE In ,vnr .mvXvvvv.;·, a oAmc-' ?<_> bo .dt-M^owe ,t O"f'!poo· ^n l-.ή Ά·; 0 Kv*cm 5-¾.. -mP i; -,¾ it' no or low or altered love! of expression of one or more of 1RAK2. SiGIRR, TLR I, TLR2, 71814, TER7. TLRK TLRfO and TRAF6. A low level of expression of a gene is a level that is lower v' g, ntoiv \ toG, 1 '' 'v. 1, ? fckt, ' > vM, ΐ foie -¾ " fold, 1 fob,, + > fo'o ^ tele 6 ibid, 7 fold, k fold, v fold, I0 fold lower) than the normal level of expression, wherein the not-nil eve* o.Aspvsw ! i\ du l .\ J , t , ^'-u ss-en -> ..cve-mu .d noonal m W„ we.es m a h <. the subject belongs by a skilled artisan and/or the level of expression in the majority of the subjects of the same species. An altered level of expression of a gene Is a level that differs (e.g., by more than. 20%, 25¾¾. 30%, 50%, 75%, 100%, 15072, 200%, 220%, 30072) from die normal L\A m evru^v'm, λ jo mo me mama. kwot oi evnes-vn .s the wt '.\pwion ss consul icd mv-mG m the s'vca - m whv1' d\ sub ce* tv -v g\ ", ., w, led mitsa : o cl o- t,v Όό of expression in the minority of the subjects of the same species. Wherein the ''norma Γ expression of one or more aefenski genes is: (i) the a verage expression level known to be found in sobjeeis noi display mg symptoms or not diagnosed with the disease or iafecuen to he treated', f imho average ev";,;f.smn L-vel detected m three, toe, er t-s-mU',, iwcmv bw, Oftv or moix vtbj'.i ts no, s.!spl,i\'ng s', Uiph'm,; ot vt deg'n-sob v, th the 0 va-vo; 0 t.p mm V j, et'd 0. ,mt the ie\el of vow -ν,ν deveted in a pat tr/to be ..dimmvered a c-.-rw-v' described herein before the onset of the disease or infection. 5-6 MiHteLjdL··^^ [00!§3| in certain embodiments, methods are described herein for treating or preventing a bacterial infection or a disease associated with a bacterial infection, wherein a composition ^^rvigosiug the sNAG uanotuvt' ts mpa-alg adrmmstotod 10 a patnm ns need o%ueh neatmem in so-tv 'UoK\hrno -its <t ;.NAt I « am-fib'-t oemfiwmmi -- tp'diea vmie.uK v O'sue or o'gvi which has an increased risk of a bacterial infection or disease.
[00184) In --0010 embodiments, an effechv ; amrnnu of die sNaG nnuniihers und-or a sNA6 nano; 1 her composition u> ndmmshered to 0 s-eyea [00185} Ir .some ..mboduioots, a eompAsmon iMmuMog iv s\Av> n.nt.Mdxis -, aem-uisemd mp s.Ob to the -me of the bo, lerscl ^ meet fee. t' a pmvm ,u to t-x see affeevd b' -, disease associated with bacterial infection. In yet other embodiments, a composition comprising d e sSAt' ΐ anofibev ts ubmrmamV n-p'-vd'v ό i t. vte a id word t| e ν'' o%iie Kvier,-, infection in a pm sent or to the site affected by a disease associated with bacteria! m lection hi yet other embodiments, a composition comprising sNAG nanofibers is applied in proximity to the site of the bacterial infection in a patient or in pros Irmly io the site affected by a disease i-. vanned "ViI’ i.it'k’f'fit] mfeet op in vet aned'or embud ment a rompo' nor ea-mp oamy to*. s\ V.' nammK, - is adrum omed xym.a o tom -me -.0 how ox. 0. > baeoxn-d ,o,octu.v }O0186| The sNAG nano fiber compositions described herein may hs administered by any of dv m,U", Ms l ,H>. ηκ\ί?ί·ί ,Ο ΙΟΌΟ s ,dO'?rOml 00 vine 5 ,Κ,' vet hi'OVO tO t SWO skilled <0 f"0 art. including but not limited to topically to the skin, topically to any other surface of the body (e.g,, mucosal surfaceg by inhalation,. intxanasaliy, vaginal!y. ree tally, hnccally, or sublingually. The mode of topical administration may vary depending upon the disease to be treated or prevented The shAG nonoftbet <xnonomnous an he fumotfeie-i for the v mmo-- woes ο·' n-ριο.ο admrmshatrot'k [60187} !r wse eunexOn? m, a e-sio-momm „en ^"ηγΤη*, \\-\G on o;v >,>i- a^pued to -he Am of a patient. For example, such eootpositton may be applied topically to the skin of a patient for treating and/or preventing 3 bacterial infection of the skin or a disease of the skin that is associated with a bacterial infection. (60188} In another embodiment, a composition described herein may be applied topically to 3 n Ui.oml o.ns ,·. 0 ^ a pnv n for w 'mp o, yarn ermpny't >m mav be a 'pu \i u> oe ' ίγ to oral n, icosn V' Iso-Yog and m pOscnOt'e ^ ,'uetuul mfectOn of the month m ptinwot a fevavot the mouth or gums that is associated with a bacterial infection |60!H6| In -,οον embodiment··,, a e<mg\>vmm - omprwms: -tmuddie-^ ^ applied to the wound m a '' idem t os ex nrpto mwo corepohiion r.uv ee appheu topic..id'·· >.n ., iK to vie of the wound or in proximity to the site oft.be wound of a patient for treating anchor preventing a bacterial infection of the wound or a disease associated with abacterial infection of the wound.
In one such embodiment, she wound is a bacterially infected wounds, for example, as diagnosed ' on. ot mo rsmboJ,,» ,< 5 1 d bnem f wo. ' , v ο* ' \ \s t wOi s di-scrib-ed herein, in yet other embodiments, a composition comprising sNAG nanoflhers Is not rtppl'oe 0' a wo.nui n a panor, o- w * m uppA'd so a h ,> tor 1 h n h < ,od w:\ Ά m a p etent }001 <16} In some embodiment*. a composition described herein may be applied topically to a eontti. erm.d w ,n,f smm.e area of s patient feu ems' pie, me t, omfe-suon sow, h, apphoo topically to genital, urinal or anal sunacc/area ibr treating and/or preventing germed, urinal or , ! 1 i.feOT J ! ' , 'O 0 V WO ' V s. t ' V-i v'' ' !k , \"\ 0«' w " ’ 0 v i t j * (00191} The above-haled methods for topical administration may me hide administration of the s\AG iraptTe 1 v d e tort.' m a os earn un ins.men., n eeh 1 loped ,. xv" w m, t 'dm a spray, a paste, a powder or any other formulation described herein or known in the an. The Ά \v 5e.,so'i 0'a\ also he applies n' a die-ving 0¾ a brne \' , \umyle m tieu? lee.t ιλϊ mfeetions/condtiions on the skin o?u patient. fftft!92{ in some embodiments, a composition described herein may be applied as a spray into the oral cavity and''or respiratory system of u patient. For example, such composition may be an] vc \.ispr.i\ ιν ίο aumt ,m,t o? pwemnO Iwora' u'ibehot of the me„,h 'v v, mmiv throa? or lungs os a disease condition 0) die mouth, nose, gums, throat or lungs that is associated n nh , baci.'!\d ΐ'ο',,ην^ hi 000 s,vh eril'vnmvm, the eo’-nposuta." tv\ he ^arohaie I to .v administered as an inhaler. |0019,1| ir, omoodmvnts, \ ^ or mocm eev'ibed ; os cm τλ oo a rede·.' ,m i se^'H'VM'iN t re eeOet', vet','., om ; r-.": !\>i o\v'!i --neb compo-usm ue\'v applied as a suppository for treating and/or preventing bacterial infection of the digestive tract, urn,i"\ η i't ν' r, ouses toe traet or „ ,ltv,'\, o! sue1' o-,m es tom is .ι-moo, -vd 0 v,h a KcWv.,» mfeefiom }60194j In another embodonenh a composition described heroin may be applied at the sm, oi' 8 surgical procedure. For example, such composition may be sprayed, applied as a cream, ointment, gel, membrane, or powder, or coated on the surface of the tissue or organ to be vsl'iu 4ed to κ * sn* ' ,h ρ"'Κ'.,ινο ot mv h ,s *\s. v, >vtef. to ds surge ,t! ot\".,v,n. In une embodiment, a composition described herein is applied at the site of the surgical incision, at the site of the excised tissue, or at the site of surgical stitches or sutures. Such administration of a composition described herein may prevent a post-surgical infection. For example, a composition described herein may be used during or after a surgical procedure which is known to pose high risk of a bacterial infection. Surgical procedures that are known to pose high risk of a bacterial infection include bowel resection, gastrointestinal surgical procedures, kidney surgery, etc. A composition described herein may be applied at the site of any of the above-listed or other s'urgl cal procedures. |00!9$! In yet other embodiments, « composition described herein may be coated on a device, for example an oral hygiene product, a catheter, a surgical instrument or another product, to be used in or Inserted into a patient, in order to prevent a bacterial infection in a patient. }60196} In .some embodiments, methods contemplated herein include a step that indudes deux non miioiiosK of bacterial infection n? a patient in sunn, cml-offmems, , Ο-, '0 , ' V ' ', , C-v -λ.-. tOU’Οί ΠΙΟΚ* b ν , ^ ' Οχ , biological sample of the nation!, in other embodiments diagnosis involves assessing whether the patient has one or more symptoms of a bacterial infection or a disease associated with a bacterial mfeehoo:.
[00197| The compositions described herein may exhibit sustained release progenies and/or n ,o be edn'wrwk'ied n > forimuatk« w\h eg η η «ηνκη ted tekvwe of sm h e-wrA'skaO·-- 1' some embodiments, the sNAG nano fibers biodegrade over time as described tit Section 5,3, s\v" ,, and these •vropoiik's o^vM \G ; anoduers nay I cue ό or euTUOunre to -a is tar -.. J sv -\n of the compositions described herein, in yet other embodiments, the compositions described herein tu hvn'i'i'Jed k'di\v.,3\ suvi<r ted ^ k'-^se-. n’M^’Skes'tvng, -s 'retb.vs kmo e t t’"e nr, T.„ <" v a \ Lsi kt v e1 i ' \ v V' v e so " tru v < ' ot nnn than about 6 hours, 12 hours. 18 hours, 2-1 hours {1 day), 2 days, 3 days, 5 days. 7 days (3 week), bO'sv ' 'w.vss ' -s,. \ \' ae ?u ost auk t i. he e "v mo' v patient, |Θ0198| ί onk'foi.ne.l ke.U'nentnnes me Jude a -u Ae >\>v rs-.i voe.e sop mane" e; a sNAG nanofibor composition tc.g,, of a cream, a membrane or a dressing], or a regiment of multiple druses or multiple applications of a sNAG nanafiber composition. A dose or an appl^otnm nan be adimmstered hose tv, -Gd>, wo-J· :\ ->r morUuK I m as a run so, -, oo'-e of a sNAG nanoftber composition may be administered once a day, twice a day, three times a day, f-n: 4ίι\>.i kn foe >!m,.s a-wo e^eg 3 Iucks. e\e-'-· ! usik ηο-'"-. l?tn"\, \-,t\ Ά \ή*ι %, every 48 hours, every1 72 hours, once a week, 2 times a week, 3 times a week, every other day, once in 2 weeks, once in 3 weeks, once in 4 weeks, or once a month. 1130344( \ sN AG oanoftbet cosnposthon oris he admins-eered nwa duration equal to or greater than 1 week, 2 weeks* 3 weeks, 1 month, 2 months, 3 months, 4 months* 5 months. 6 months. '3 months. 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 4 years, 5 years, 7 years, 10 years ηπτη-'κ ht one wsef- .'mbodunenL a A-\ti naeoilbes compo'dion does not /aum any .-,:3-: etfeets e-tuse- onh n dd moo effects dm mg die di-iartm oft t: irwouvm b, one such embodiment, a sNAG rtanofibcr composition does not lose its effective,ness or docs not cause generation of rcsisn-mr strains of bacteria in response to the treatment, in another embodiment, a s'NAG nanofiber composition does not cause irritation fe.g., moderate or severe irntaUon ? or allergy (e g.. moderate or severe allergy) [0020U) c o icenmr am ,vmne sN \G ndrkrhtxn > ' c mrevmvr p \,,'\ hi - ,m effect we amount oi the sNAG nanofiber is u*ed. Λ etYceiive amount may he an amount sufficient to achieve cue or more of the eifeeis described herein, for example at! amount effective to reduce or eradicate a bacterial infection, or reduce or eradicate one or more symptoms of a baUen.il snhemm Kw c\w' pic i eempO'-hon ovs c^emscae auotn U 7 to be ί of lac sNAG nano fibers per dosc/application of the composition in a form suitable tor topical delivery to a patient. In certain embodiments, a composition described herein composes about 0.25 to 20 mg csr\ bums 0 y \> 2p my , n abotu t -o 7p mu. s. n abetn i to tfop xm ebon1 I !o 12 oo i.i",, , ' to Is) j m cm dvut ' to g mg cm , Av U I tv y eg; > to , r'yyw 2 v k e e xw , \\\ \o ' ' ' be \ \, n, fv "be v ‘ ve wp , v e ,. " 'w form suitable for topical delivery to a patient, 5·? )00201) Gk'x "-G ' ' 1 ce 'V- , o'- ' c ,Jr» <>, me com vt o o^omer the: mtes \uJi -,s vtbMmno4· th a boon the smr me pvo>\, embaetet'u* agents xc e wt armn,''te\ d-Aus- ο pepu Je\ dev mm hw, y'ps>\"v ηι^ι ι,Ί\ν dv;,m> te *> ,w erelgosk", fever relief therapy, and/or other agents or drugs known to be effective against or commonly used for treatment and or prevention of bacterial infections or diseases associated with bacterial iwfeeisonx. |0Ο2Ο2| in some nbom nc* w, a sx'> " ,a o' com, bed ' au ' w whelms eve».1' ' m epoo v, dh ji? additional anfs-b», fenal agem for cs m piy m aiewsoue in ore ste a cmw' I mem a composition desen bed herein may be used to treat a bacterial infection or a disease associated with a bacterial infection in conjunction with a standard therapy commonly used to treat such bacteswh mbs mm 0¾ -nch A e am In o'v embodiment, s eompo-nm' desenbed herem : V\ be ad nmwtcteb v- patiom d tg vw .2 e oh '5 In^b; ύ s>ntfton',.> of a h eie* a 1 th esAu 05 a do,,'Use ,W"!,U'dsnu , haeknul mleeKm o.meemwmv s· a •amd.cdam A s.uv, P >ϊ;ν;ο (e,g., an antibiotic) known to be effective against such bacterial infection or such disease. |002PJ) in cesium emixidimcut.a, .a composition described herein is administered in conjunction with an antibiotic of one of the following classes of antibiotics; micro! ides !e,g., ocetbronwem, -mnhrerw,mm amiungpmvssoes ;,;.e „ xmukrjcm gent urncm, neme·,em, streplornyern), cephalosporins (e g., ceradroxib cefaclor, cefotaxime, eefcpimeh tluontquinolones {e,g„. ciprofloxacin, levofloxacin), penicillins (e.g , penicillin, ampicdlm. amoxicillin), toon eye imes fe.g , tetracycline, doxycy clinch tivtd carbapencms (e.g., mcTopcne-rs, imipenem), In some embodiments, a composition described herein is administered m C'N sii' \'ho , λ eh hu ago u m-s^ooe» ef.eon*o re i e,' <> pe\ 'v m u''' eon v uvV ' treat or prevent an S. auras infection, MRS A Infection, a PsemiouxmiLs infection, or a C, rfificuia infection. 100204} In a specific embodiment, a composition described herein is administered in conjunction with one or more of vancomycin, .sulfa drug (e.g.* co-trimoxa^oic/tritricihoprim-N'.iifnevOn'sa \d,'' ton 'Vnnetog, s, r\y e) - lev sv ehnd my "n evok'd bn-r*v , .. n'e*ol dh <. u . \ ' ten. op a,nr gm nip d n ea itpiM n(M.k.i. s *^ ,ν*"' » *'»' bacitracin, nitrofurantoin, hydrogen peroxide, novobiocin, netllmieio. methyiglyoxal. and bee defense!-! A composition described herein may also be administered in conjunction with a dressing comprising one or more of hydrogen peroxide, tobramycin, chlorhexidinc digluconatc, ehlorhexidinc gluconate, levofloxacm, and silver, in one embodiment, a composition dc.scribed j 'vC ' svV v.1 > thorn v" moK on ~ " v ν' , 'V' s infection, and particularly, an MRS A. infection. }0020fi} in some embodiments,, the compositions described herein are administered before {e.g., 1 minute, IS minutes. 30 minutes, 1 hour, 2 hours, 3 hours, h hours. 12 hours, 2-1 hours or more before* or arty time period in between), simultaneously with, or after i'e g., 1 minute* 15 minutes, 3d minutes, 1 hour, 2 hours. 3 hours, 6 hours, 12 hours, 24 hours or more after, or any time period, in between) administration of another therapy. For a example, such compositions muyh· udmmhdatvd helot e, stmuUurtv.ou’dy voh or a her adnursuArattou id an mm-huetepa: aeon; (e g.. an endbiotte). 11102061 in some of these embodiments, the compositions described herein may be administered to a patient to treat or prevent a bacterial infection or a disease associated with bucko a i'H.vu-v .ον» tk i\Uvs', bn. m d-yyue a i oe' so m t OvAnem <A the baemoa! '«V, ,mn ".'ith .motivt ..nuni-jcuniji spent ts χ m am, huvOo) In -wnc embed -men o· the cony\vd;<eiv Jev Hvd mu on ou\ be ,uemm\k\*xl V a p,u, o f wk- kv d.vompeA mswum e V cue <n tour, anu-bactcfta! agents (e.g, an antibiotic). In one embodiment. the compositions described herein may be administered to a patient who has undergone a course of treatment with an antibiotic (e.g., an antibiotic standardly used for the treatment of such bacterial infection! and developed resistance to such antibiotic.
[0020?! However* in certain embodiments, a sN AG nimotiber composition is administered alone. In one .such embodiment, a sNAG nano fiber composition is not administered with any other therapies, for example, it is not administered with an immunomodulator, an antibacterial agent te , ,m wnfovuet, , deferon pepab,, e kaVosm-hk^ popmk, u p w rehM'tho" m\ <0.;, an -m tfovsw h or α A', ,-· fohef ijeryn in one embodiment, t Ά s( < u.uiofdvt v. cmpo-mior =s nos adtc msm-xu m jiu.> itv wn \ *m * in cum 0 e nl\\i',ments, 0 et-Moe- compositions arc not administered in conjunction with an anti-vim I agent, an anti-fungal agent or an an-i -yeast agent:· ,:&amp;» Aits 100208} A pharmaceutical pack or kit which comprises any of the shove-described sNAG oompo-buons 0. d-o i.onlemphstec! I ir? pack or kit mm compose one or moo. containers ill Km with one or more ingredients comprising the compositions described herein. The composition is p/efernbly contained veidnn a scaled, wafer proof, sterile package which facilitates ronnwai of the composition without contamination. Materials (torn winch containers may be made include aluminum, toil, plastic, or another conventional material that is easily sterilized. The kit can contain material for a ample administration or multiple administrations of the composition, preferably wherein the material for each administration Is provided In a separate, waterproof* sterile; paekape 100200} In another embodiment, a container having dual compartments is provided. A first compartment contains any of the above--described sNAG compositions, while the second compartment contains another active agent such as another ami-bactcrial agent. In the field or the clinic, the composition in the first compartment can he readily combined with the agent in the second > ompunmom for sshwcqucn· admums-rution to a patient }002IO| \dbmonatb,, a kit designed for emergency or mifourv use can also centum dtspovahle pu "St; nh ed n sonmeut^ -,udi as soivim v scalpel, clump, tomunpen, cIums, m evLsm bandages, or the like.
[002111 Optionalh, av-oesak'd w.’h Ws n on pack , i" -. i rv'toc t" the torus pr wo>l· t K a governmental agency regulating the manufacture* use or sale of pharmaceuticals or biological prod5! wv vsjvpr oome vAx't-' appx>W by fo" agon,\ cAnucns’a one f.v or vie A human administration. For example, a kit can comprise a notice regarding FDA approval and/or tostructions for use.: 18821.2} The kits encompassed herein can be used in the above applications and methods.
6.. EXAMPLES fol Exnmok I: sNAG Nanahhm Fro os a Marine Diatom Promote Warmd liedfoe and IMgssULE&amp;m&amp;ste 1802IJ| I he-· eoorfoe da-mv «s'fntcx m u x \ -Us η, ηοΗνηχ pro no' 5 eu'.nvm. -> wowed heforg and expses-fon ofofotenswv and dun foe Akt= d tsl pfohwny pknx a seotrai rob' m the regulation of cutaneous wound healing by sNa.G nanaflbers. 8J.J Materials and Methods 1802141 sNAG/Talidcrm rumofibcrs arc produced and supplied by Marine Poly toes· Technologies and formed into suitable patches tor wound treatment. Wildtypc C3? Black and Akti null mice were housed at the Medical University of South Carolina animal facilities. Wildtypc and Aktl null mice, ages ranging from eight to 12 weeks., were anesthetized with 50% pure uv. eon and 5*5 fo ιχοΠηι anc e.ns InuneUt neb fo-foie w^undtng. Xtur I bur Re-not it l Chios': was applied to (heir dorsum to remove any unwanted hair. A dorsal 4mm circular area of skin was removed using an excision biopsy punch. Tnliderrn was placed onto each wound at das 0 or wounds were left untreated. At days 1,3.5, and 7 the wounds were photographed, measured, and excised using an K rum biopsy punch to ensure complete removal of the wound and .surrounding -An* Xsddiwvum xkJ uu I wean. s wst a v w\h\n Ld,deO< oe.n ve n iw\ end' .3-i.w p..,lion m -, p,net-on for HAT and n:ownx:Afowv0 ..v -. swear a [00215} Paraffin-embedded sections were sectioned and placed on microscope slides for staining. Slides were washed with xylenes to remove paraffin and rehydrated through a series of et.food skohoR Th- seenons w ere then inufoelen m 0 ' - in toe ·, fop fo peow. efo..i .n i-n Sections 'were incubated in a boiling Antigen Rctrival solution. 1% Annual scrum was used for t-fo- 1 uw bclore n-enKuw·; m jk- pi-mats ^nsfoedy e-dofc's'n ^ 5 4« )8 ode; s,>n Tl >. seetaui*- weie then wo ubated in tfo. prwuo anfovvi,. <weonght at h in a hutnfonv efowfowi \-i hn-.ui^flnw'esce·' eo , ' H xev . ' fo ' s fou e ,. ο. s V" e‘ m obi nuclear staining wail; TOPE 0-3 Images were captured using cortiocd rrheroaeopy. 180216| Hematoxylin ana cosin stammg was used to visualize basic structures such as the '.pidennts, damns. mw-eks and blood » esv.K and to dew-mew Hk onenbihon and appro im,Us focauon m the wound USill staining was ubo used to fo\-sn lo klwfo;K which cell types me annul Lied p> hah derm in ;m Mo binds, pendent manner.
[002Π[ Oihos- materials and method*· arc ikvrnb-.d ϊα ίίρυ: e des^ng: mns ·;.η·! m ill·.' results vcnon l\\''-'\, ud pet tot mod m uesen .me,, *\nh me m:lhod> Inown m the ust 6,.1,2: Results [002i8| \\ O'i k; / urm e?. !i". uo up-m ,* o ey,ifti,v Of'D-<‘ Figure 1 \ 'k-m-, a \\,'j F.or ueutwis of ''ho-yco Xk- m η -pen··,, x- \ \G a; i s\aG ΆνΆη vs" of settee sku\ v'O 1C Figure 1 B Gm'.', , R1 -FCR ,nmksss m i'C mfecOd cit'vi wnx · * snHc 1 ,outset o' \Ui -hRV\ i,m OMi'.l·'- ane vsessed *0' 1 λ! and s2o ,s , .0 .eey control, Figure 1C illustrates a signal transduction pathway transducing a signal from sNAG nemud'e-s 0' A\tl, ht\- „n,i D Annus [00210[ MeiMydiwodpd ·ΜΜΐΜ0: 0·ΑΜί wuliMMimiis w pp:mMiy· resmiM-Mslrniiderm wMd;iiJ treatment. Figure 2A shows representative image* of wounded and AKT1 null mice with and without treatment of Taiiderm. Figure 2B shows H&amp;E staining of representative mouse skis· sections from day 3 wounds. H&amp;H staining of wildiype and Akd wound exidsons indicate a Taiiderm ciepderidertt increase tn kerarmocYte proliferation and migration. The dashed lines indicate the area of keraiinoeyte proliferation u cross the wound margin. In both the wildly pc and XkF beAd woe ids , s', »\ un an c\ ,L U u, „s,v t s icet'Ch, hm* s'' t ^, · g , s, compared to the wildylpc and Akt I control This indicates that Taiiderm Increases kertainoctye Kcrnnmeut uniepdent of the Akt t pathwnx. Xhhongh Ί eiiderm uidnc- s a ,\>mpletc {crp'tHu'aU'Uj A the , ekvimis -w.C'vs Pu. mb mmum, fn„te w Adi ,v, ;stsn,u: hwk 0 rovasetdarization in the underlying tissue compared to tlte wildiype. This is evident by substantial hemorrhaging and infiltration of red blood cells in the Akt) utnimd.s, [00220I tNAG nano fibers stimulate cytokine and defmsin expedition in primary endotheUaf ,', ''s Figure 3 X •Ik'-w'·,'n'ntc'V'f's^ni b'n'm-* os i'!' festeb w F et '.vtoont s\ j,'
antibody directed against a-defensin. Figure 3B presents ELISA showing that nunoilber ne Revet of I Γ whs ns ihc s> >. tern· ϊ of «'ΆΆϊιμι^ * A
[00221 [ ,1 NAG nanofibers siinmiare defemin expression in primary endothelial 1¾% in an Akii dependent manner. Figures 4 A and 411 show qu.ustinnA 0 kT-Tf R an t lyses of sen: m v„ ieu t 1 tmaieu w P 05 without A v i, wult m s' n mu* ΠΉΜΡν t\)AP&amp; n Xd ίο,), Worirnunmn I Π3Κ inhibitor) or infected wuh a sm-n'nhf.d control or XMi shRNA lentlvintses and assessed for expression of the genes indicated. |00222| $NAC mittyfiben β-ξίφη^ΐη d expression in tnousa kentfhvKytes* figure 5A sUms WUK mof.a<Mes<.em Warning w m p-dekusm ' aee bison. cm. ao... )ocbes >η KgUtir embedded mou.se cutaneous wound sections irom WT and Akt I null animals on Day 3 A cuue icons 'ύϊη-.Λ ben he:/ model was des clops d m bolt; A I and /W1 mnl miec in a-ν'· s'- me A ets.g 1 ah h r" t'es \" w s *w hi ' v ow ' ' ^s " ^\c', '\sw n hthdoit'} no !\\1 "ona's n a,' Vstodernwg m mamse;· theamhn.'· l\>h h-m- to mewv.se deiensin expression in a healing wound has important implications tor treating arid conn-oiling womni mt- »umt Figure SB SV'Sl MO ' vO O 0 ' V V's s ' \ V , O 's S'o ' ' using NIHImaged software. Figure SC’ shows imrmmoftuoresccnt staining of WT and Akt( null . s.J. xi i o'k' x xs ' v \ '.}* ΌΡΚΟ ' \ * vo x' ^ o \o guci }k|>oto'knr * shamme us WT and Aka : t Aicieog mealed amines Π-e iemnuOdeorcwem labeling of wound sect inns illustrates that Taliderm treated wounds show an increase in B-defenxin 3 expression in an A.kti. dependent manner. Although the Akil treated wounds show a reasonable increase m B-defen.xin 3, the wild type treated wounds illustrate a more remarkable increase. Tins indicates that fwiefensin 3 expression is not only increased by application of the nunoithex hui A at least pan.tally dependent on the AkH pashwas (Tdeknsm .» expies-mm .-cents limited to die keratinocytes indicating this expression is keratinocyie specific (110223] -Us; «ι/'t j ..d; ? t ici-c; t pii >« dva.-r / A go g as s Figure 6 shows \> heimnie of Akt I dependent transcription factor binding sites. Using Gtmomaftx software. 500 bp upstream of the nance· muon slap site was wnw cod t\>r coos"x od \tx's on l·, e niRN A ui tiff , a, ,rd 5 lx 1.3 Conclusions (90224} The provided data show dud sNAG nanofiber stimulation of Etsi results from the activation of A.kti by these nanofibers. Nano fiber treatment resulted hi marked increases in die cow's' on of genes nxwKcc m it she uxx unieoin sj*> as II I u snowu I ts* targdh \ hi -I aed se^nal deiens· x, ,f\ uU ok and id' small anticiee'ohial eem *bw o.eeiE c s-wi ,¾¾ tn , as chentoudractunt-. Both tdwreucoUwaai mhibiuon ok the P13K Ak'· I padnvcv and \kt; knockdown using shRNAs resulted in decreased expression of these chernotactic factors. Akt i null mice exhibited a delayed wound healing phenotype dun is partially rescued by Taliderm nanofsberx. Taliderm treated wounds also showed an increase in dcdcusiti expression that is Akt i dependent [t>0225f 'Πκ sner-.'aof p-defen.\in 3 ',ΛρΓ'.'Ό'ΐοη end; v.-!Y;5wiueye- pr\d den users in \ nhderm treated wounds demonstrates the beneficial use ofTalidcmt ax an effective wound healing product. Taliderm acts to increase anti-microbial peptide expression in kerutinccytes in an Akt I dr-nond.' w oto ov* muo' -Osip .he οΊ O) \k. m t so fin > ί.-'η ΐ'Γ-Ν \{i ru.nof.lviv
This correlates with the results from other studies in the laboratory (Buff. Muise·Hebnerieks, " s, .e'i i Uisdsmo die lJ n v . ϋΐκ«ΙίΜ' s' s Sx \s results in decreased expression of these chcmotactic factors. |d022fe| Although the increased expression of β-deiensiri 3 is A.ktl -dependent. H&amp;E Λ sr.ne. s*t f .nil? wound esetM^os (Hgure 2H) .m-.eutev diu? i\MtX"m uct~ ttwp'iwvi of Asti in '=ui-0 r-ccr'tboheuoon Γλο?· fhossgk dv new r..t nnw> uv .vv;·· o;c on tire wound mat cm. dv nndeiKotv.; tsvne did not d. mon-tone the same ePnsishitson ?n wsv. aim i/oivsh Hus i?su:eat<> the that absence of Akt 1 is responsible for leaky blood vessels and the large amount of floating red blood cells in the dermis. This suggests that Taiiderm is dependent on the Akt I pathway for an increase in vascularization.
[00221} in «.on’nt'ny ιρ?-Λ.ϊ'\ο nanonbois ΓΓ-ΟκΙοϊόΠ mucuv wound heulinu v p o ly vesn Lo sc .nse, ''genesis, s nt sN '\A1 * he'"i;s..e. .T eedod'C'.nl >. f.s jU s d„ nu \ktl L*sl dependent pvhvav leading so changes in >-CiJ mobhr> and eysr-kes·; wrenon. < ύ\'> fuhderm 'snakd w'lmds show new-vf .'ww^n vt {'" \ nvs > !* u;i AvI < o* pendent n\o?ee!. o', si esimott of Aktl nd: n'n^iswith Knsdenw p n s I. som. ues Ik pi e. eA!\; letemg to wkedly ifyic.i o 1 sores mv\tv. pol.'e' noo onp' now ans’ <\ '*>?,> 'sv'nvivs n. ip.-its ' \hoit. - shut 1 In', is h?o'.s w,.*\> d os tne sNAH ae*'\e',e". nudw\kodseg so increased wound healing and cytokine secretion }0022Hj Taken kw'ssi.u do.*', tsoosog- ui.y.ysi a centra5 r.f. 'f t:i, \»tl *·Γ·Τ. o. she regulation of cutaneous wound healing by sNAG nanofihers and support the use uf these iSiOits Tv's .ο- .j, ss-we' .sod efso'lsve nves^d f>n . sihusn tng wonsw h. UiU-p | *10229 i This example desnonstrates that sN.AG nano fibers have a potent anti-bacterial effect »'λ: v lu< I dire. Aotd oe.O'i^*" J. vm, ΙίΙ'Λ,γ' rio a .so -w -¾ ih-’d sN At? n.mwhbe?;v son.c ovtuvess of defensir- .' s vtttX' so f. ·\ηη',.ν>", -- snd s i idv K UU if V U ' <r i ' * i ctl»'' Ό Λ 1 S i \k> , , \ W , ' ' ' " \ v , ' . til© ksBS-liCS af: W0;irnd ttas®;· 6.2,1 Materials and Met foods
jO023iij "''.',.\t ' Γ\......^ i.'Ά-ι 11,η<'αη vm uhCrd mad m to IV (Looenj were ra.ii;stained at 57' wbh 5 V C02 la endothelial basal medium 2 iLoeea)
Endothelial kwal medium 7 ιΙ'Τ-ίΜ.Ά wax vipplcm.mod ouh f{ growm me-Anm 2 Smg\'Guo;, a"' described by i mm a p^wduvs and IV nemcdmi m- plumy, m i bo. nrogen s Serum siat\aocm mas performed at 80-90% con fluency in ESM7 supplemented with 0.1% fetal cat f scrum (Valley Biomedical) for 24 hour** followed by .stimulation with highly purified pGIcNAc (50g.g.Tn.l * nano fibers (dN AG) in sterile- mater (provided by Murine Polymer Technologies, Jnc., Danvers, M !,\s ΓΝ \) i w Ά VN \e deitous.de:· < el onnebber’' u ,ed oi tt t - -study are s'"on biodegradable fibers derived from a longer form (NAG;, and nave an average length oi4-7i.tm >t i a pM\u - r ~iv\\ i. ,n weiyht of approx n’ ,\VN (O.oOODa i m mi Ί : urn <>s it* PDO^XOV (50μΜ) in worimannin ( lOOnM), ceils λ etc eve-treated for 45 minutes prior to 3 hour somt-hoon wuh .,N At 1ι A gig ml) 1002311 Statistical Analysis: Each quantitative experiment was performed at least in triplicate al leas! three independent rimes. All statistical analyses were performed using Microsoft Exceti ίο calculate means,, staiidard deviations and student t-test |00232j ,, , Miss o 5 sh‘iN A iemo t ,ouA ,, s \s, „e porches- s,, from Sigma A'-h ·,ί. 3 se-umlbed "EkO I ΑηΑΝΑ \ emo" wes •'urchosed from \ce y to 1 es.Av ,t„-s - άο e'ik'P'ugutej " 2'M'- Λ map tamed u DMfM supy! "octet, as abo\. t einiMt.d p'oducti.m .in ps'vhi'msM Mug w-h -\ s 2 md AfV G pi-'.'\..av-m seen vs purchased from Addgene using the protocol for producing lend viral par-ides from Addgene i:'or infection of target cells, 7 5 X 10' ceils were plated on 100 mm2 plates and allowed to incubate ox, ought TV w;v ,\ts e dk ,vm Uunwore t us ng a VuJ eonee* men-m V ’ og o\ p-.-hv,·-r·, and cubes scrambled eonnol or Aktl xbRVA lemu. u‘t;ses Altct tunsdn·, mm endothelial cells weie *> -η ο s'er-eo -uoe'ft" end stimulated wn i s\ At' fwH> nut fo' he-cts ^ i' reettoms were monitored for appropriate knockdown by RT-PCR. 11102331 , s1 r v; sc , i , v,' M pr D, RNA ,s ,, s ugw wtn RNAsrOi l ex
Inc,) following mam-fuemrer's Instructions, eDNA was sytnhestzed from 2 ,ug total ΕΝΛ with a Supotsv, ipt Ms'M Strme dj-unes s ku >, Imovgeny a<mg talmoi'cJb tollow'iAu th^ n amdlt tiros \ mstrtie'ums PCR racoons eennu χΌ λ ;? '.TtnmK tr ;!AA a \1 . l> t.M >m plrnsr pair St :Lol|&amp;,::;M£t*! iSAi :¾ tvs* isi /x'r^e: >stt 'ί/\ί f/^s2£ >
j0iO4} ( ^oh?H. condones vw ό ’M t so< 5 mm d'!-<'< < w les ol '>4 Γ uv 1 run, 'ΤΓ v eas.ee oe p !"u! I, ' "'-r 1 e\e ' PO 1 '>1' ' 3. \·' ' anevoo1 -J to * * t>eio number was empirically determined to be within the linear range of the essay id·· each primer |vtt used Mi screw useu-lve ΚΊ-Vi 'P ο u- pukmu. J ^ eh :1k* π »<wotnal p'utew subum! ede primers as internal controls Products were visualized on a Bio Rad Molecular Imaging System < 1 lereules t' \ 1 d'.! Real tune P( R v. as pet son red esmg a Bn she as ί 'VRR green MR< 'k ku m combination with an. MxdOOOP Real-Time PCR system both purchased from Stratagem:. Primers deu\ nag d e rdm-semef nbnmu S?f* were n ·<:.! e-< interna! eswmuks 10023${ Exchiomi Wound Heating Modeh Wild Type C57B1/6 and Aktl-/- [43) were used in all experiments. The Aktl null animals were created using an insenionai mutagenesis strategy at tl e UiUxIaooUwi styjt sue Uu* blocks e\ptesv'u>\ to'rv enow pioteu; Wound.ng ".as rcitetmei on oksIk'O eJ edt It .rale m^e M h 13 w·.. ,Λ.> ok' k»! o. J oi\ ks ess cutaneous wo mcR were created using a 4mm biopsy punch (MiRex), to e-eate two identical wounds on each. Hank V ' weto . .o^h u J us.i a, uo O. 1» aw v s tpo t ' y; e'WsihesU' r ^'V1: A e't ee'p hi. ί lyoTl ttrape.: wm iised:. M 4-!§® f mdnotibityPfh:: Ibr.. surgery:. Prior itQxgnrgerf: lair :sla$;ron3gSi0d ,ly depilation and the area was washed and sterilized using 70% ethanol. Wounds were cither treated o \ > s\ X'' nemh1' n e r v ve v ’ w > J \‘ lit „ " . te e ie*ί uiWe ued t" ' s ' < m , im t i wey o tu&amp;mirsd: alJ eitko tldb#pg;: the retTeupfcg:; skisr using:» ;j"(t o 'ps\ pus : iM' U'\* W "cod* v er bseO jn t'' oa^' tnmi Oe"\ee wo» ught at a N embed ded i n para Bln.... and sectiortesi for, anal y at s, [ftt>2?6| ' v , „ ό, ·<ί ί \Γ v, ' \ r~" , r
Histology Core Facilsiy at the Medical University of South Carolina, Department of Regenerative Medicine and Ceil Biology, Briefly, section* were cleared in xylene, rehydrated '•on v , ο ,\\v wnori (Ή,,ν 1 'ur<ws j I cJ uV core o, were then placed in ammonia water, ringed in ethanol and exposed to Eosin before dehydrating ,Ό ivtekohosa d,\ , v ' ' on u 'so e nonum1, w ' w·^ , s\t f R,el atd-AKar, C, lentoO 11,¾ ϊ sections w „;e x j -n.'h.vd nsmg an tt \ mens ΒΧΆ', n^u-sO'YC ; Ό objective hms, 0 13} and ew'Unmd name an Olympus t amera {Model ί>Ρ25* and DPC-RhA aegutsiEoft soflware.
[00237| Boutcnai inoculation, Tissue Gram Staining, CoBmy Forming Unit Quantitation', V,>k " , vivee vi'a, Ksoso'i'mwrd", wm , so v\\.e o on os id Staphyfococcm aureus (AT€€ 25023¾ were nicked and cultured overnight at 3Γ and adjusted to an ebaorbaocc of OD^nr 0.53. One mL of 5. aureus was spurs at HhOOOrpro. resuspended in sterile PBS, and lap! was used to Irmoeuiate each wound. sN.AG membranes were applied to the '!eavd eump rhs"y mimsles po-e moeuUunr xih c weie eumanved οα 1,,\ ' mo 5 poo wounding and wounds were harvested using an H rnrn biopsy punch One wound per animal was 0 \ o· 3 w , res chi in n p.naKss.naldOp.de at c C and the out” worn d w ,-- n tcrec and p.atee on LB media without antibiotic for bacteria! quantitation [see below). Wounds for tissue gram staining were embedded in paraffin and sectioned. Sections were cleared m s.yiene and rehydrated through a scries of alcohol and wore stained using a. tissue gram stain (Sigma-Alihu'h'b' puKCdmc^ JocrLd K 'ho. mum.tisfota; |d023H| Fra > y u ong λόιό wtvs'Mecp',^ d in ^ A d .'kVrsul 'rod's an m· toned o' 50 mm a, u7o< whik slul mg, ί. elonv unenng uwts iCH > λοο y U"ewt;„d v e:y - J'h turn series plated overnight at 'MX'. Number of colonies per platCper dilution were counted and CFU.'rn! were calculated.
[00239} 1 o determine 1,. 11 ml from sNAiJ treated bacterial cub-mes F e.,.> eo’tua·" m sohCion O' ie h, atei w up v.oy ug 4 .ww'mmmws m ^ xw r U*ni ,me ?P ul of h, Ko p nd Ά At 0 to, Pm-,; hoi t, t. t. 1m os 'veto icon pic tod -C ^ td x FTnC vow -Liermmcd
|ft024U| v ' ' ' ' ' , , «.’·* ·, .axel! mow' UoM Λ ' M Ά M of biologically active human B-dciensin 3 pepiide {Pcpude Institute, Inc.) were tested tor their cl feet on bai.lew.d gmwth n- rise Inf. clou wound healing model dewnhed nbov·.'. F;k Is concentration negatively affected bacterial growth so the lowest concentration was chose?'! tor an-.4> tes \mi o.uh wound was nda.ted v, -hi o mot o, ICul or pepmk αϊί applied Aftet th?\c ο o n, wound." Vvov mu\ em'd, embedded Ά v kom κ and st upu u. '·. mature.' tei t'l l1 gnuonkme." "" ib'Cwb'jJ a?V"0 |Θ0241j β 5. siOiOoi-V A/m-Amc; Wild f> pe male o-ce were wounded and as leered w?th I4*’dot's ' e,"'!>" d.<>e-da;d ;d\'"0 \tV' u eeuhu.on one wound w.m-,eau.J vot’’ 0.2ug/?nL of β-dcicrtsin T antibody {Santa Cruz) while the other was treated with 0.2ug/rnL or normal vuut igG control armhmjy phanm O.nr/1. sVAG month} .mes were applied io el 1 or see a lies' antibody Irma merit on day 0. Antibody was applied every 24 hours. Mice were euthanized on day " and worm k wete hvs -..·>!„? e-!r g .n x mrr I'ayw nen.h Wee1 do wev Λ\ν o\oe\iy'* \r t' paraformaldehyde at I'd?, embedded in paraffin, seecbried, and analyzed using tissue graft! stain. CFU/mi quantitation w as performed from wounds harvested on day 2 a» described above |(H>242| !i!ϊίisi(f?ofhiofYSirrtC^.,. MkrD.W'pY: Pars Airs embedded tissue sections were rehydrated through xylene and a series of graded alcohols Sections were treated with 0,01¾¾ Triton-XlOO and subjected to umigeti retrieval using antigen unmasking solution {Vector laboratories) as a pressure cooker lor 5min and allowed to cool Skin sections were labeled w oh jVdefeaxiO e goo poly eh seal antibody (San in ( ru?A -nvolucnn rabbit polyclonal antibody t Santa ' \ nu Is MdkG 4-¢. J J' < Sk'ecu 4 w w i i <uw o ' i ukV ' ' " , ' ,'oe. overnight at 4" and appropriate secondary irnmunofiuoreseeni antibodies (invitrogert) tor 1 hour at room temperature. Control sections for each: antibody wore stained without primary antibody' Tissue sections were visualized using an Olympus fduroViow laser scanning eonibcal wiewv'CO'v i Mode! IX K,o am’ -.apm eo 5 eeo eat !empvs erne evag an Qe mpuv e cue a (Model FV5-ZM) and Pluovicw 5.0 acquisition software. All tissue sections were imaged using, •ho, oi immexkv Lns ptjymu^' Immeivea Ο.ΊΊ |0O243] HUVECs wee:.' either serum starved or treated with sNAG for 5 hours in culture and stained with antibodies directed against a-dsfensln 5 (FI TO. p-defensin 3 {'Texas Red), or 11 sPki)f Blue; hnapes wefe taken \s ag irnmmoMsmiracuv e u loscm.w u ed cukem defensin expression was visualized using a Zeiss Asiovert 100M con focal microscope and was - uptoted 'c ranl-lent :empe!Amttsmg w .m as the medmm, using I AM MO camera wVras Fluor o.M.W l 2A objective's. {$0244j <> , \?."J 'ifi'i. ϊ-αΈ- Fndothefial o®u οsow sursed enot to .oininiamf?! \\oh s\ s\ V*'* i nl· ' iV'COUiVU v s v" ' vOafs -> 0 0' Ο ' ~-Vi e o v i\ s a i v u' nod on * vv η - v, ' o wv v''' s' ·- v., at o* jt\1 pkK'hvs^ a? \' W utmh«ls tc'> h N on J"W; Ι-*\ h"»nvi>'''* 6,2.2 Results 6,2,.2,1, KenUwm'yh's md Emiutkeikti Ceils Express and Secrete Defenstns H hi m Simulated if hit s h 16' 100245} This example demonstrates that sNAG treatment modulates the expression of defensins, small anti-microbial peptides that are pan of the innate immune response. IS024H&amp;J ΐ o orv o^iso she AF ηνΰΚ Ή» tu ,ron'i-v ο*’ J >' pu ;mch v or?v\r\ human ηοΦΠν® vein ondothelui eel;s to euUnv v·,,.ο used bndoihelvl ,.cs - ο,\ρν~η both a type and β-type defensins when stimulated with sNAG. As shown in Figure ?A endothelial cells treated with sNAG show an up-regulation of β-defhnsin 3 and a-defensirt 1 mRNA 0|;pi:esstpp:'wi!hi:n:: I Mpr'Ofdi^ttWpS·.. iremmeni άj> hisses sod ulna eel show n} »'x-.<em·· pane nrr.o , tout.nurne o\ er 2 ' ddhorvih defensin genes were used to confirm the expression of the α- type defensins in primary rdoti sal v v van» Ik " ' \ i” ^ e \e v oos s\ V c rs, Mon of or Cot e ul celts was shown to increase foe expression specifically of α-dcfenains 1,4 and 5 and jkdefensm ' \chhoo t , sN \t; st'rn tlrtion of hunv.n keWiv,.\v-, νοον-Μ.J 0M\ev.t« " ο”'2 I'ke or vs, so' o' * e; which are hove tr Fabk1 1 1 w νχνν, ospgost i ,o ,n k\e4 *!>'> .t- iefenltft. genes .anti pAfeflnsiH: J: ht®: impressed M griimr® gpdglkpHai eelli and· mdiipisr ;P~ dek" sjit, usw a*c vpu "soc n> "ov, o\ k> νκαονn - so fes"vt. v to sN V; \M;oe orvi
'fable I: Geae arrav analysts reveals nutaeroas defenstn senes uaresttlaiecl b\ sNAG
[§02471 I .·ΐ icM w holder me '7··' Xt «-Apeedem defensm pr. -moo A-o ιλ , arsed an V-n proiem lex el, -A. xG Simulated endothelial edis x.,:re- su'cnecied η» nnmunoUnoresuenee ir-mg antiitodies direcied against both a and β defensins. As shown in Figure ?B. both (Vdefensin 3 and n~ {.miens in S are up-regulated upon. sNAG stimulation m ih.ix ceil type. However, stimulation of primary human keratioocytes (HaC'at) with sNAG did not cause increased expression of a- defensin but does cause an increase in the expression of IFderensin 3 (Figure 7€}. Takers together, these experiments suggest that sNAG stimulation results in an up-mpulmoi of Λ" msm ~t\V\ m wA5' s. unneey u - and pnn py cOolne! 0 -. old 6,2.2.2. aMAG~Depi!f(denf Defensin Expression Requires RRtl [002481 if 0·· mo-d·. 'mb tsK'd 0 iXi mow irat .A AG shrr.vkt.on m pi -o asv encotr.e IΊ cod0 v.st's rn .a-ei \-. -5 \ ao md \1 \P \ »n xo 0 \ O', a . 2§Oh i \ 0 K.s l\'' ' t ' -o*." . ' \k i. erdotV'ia! oiK and o' Λ . -J RR el a: l'\SLP< I A A. yy'T so '
To begin to determine ihe signaling pathway responsible idr the expression of deiensins, endothelial cells were serum starved and prc*treated with pharmacological inhibitors directed < i' mo t\ idr! M \1A v, r PDiUfd'· A mi/ χχ\\Ρ ' j\ L* on g real time PCR analysis shews that a-defensiu i mflNA levels ace greatly diminished after inhibition of either the PBK/Akt pathway or the MAP kinase pathway (Figure 8 A), FT-PCR. analysis of β-defensin 3 also shows that lev el- ace decreased by the inhibition of these pathway.-. ' --.-. (FiguscNBi Λ \b n , 1 rx'i'i oi ei’oti Ό s.'i ic - \ , -V' '\\.\w 0 phosphorylation of Akth a siandard indicator of its activation (figure 8(7(, To confirm that \IH is Indeed roquueb for defensin express wee lento -a: delharv of shRN.x directed -nuGroi \Ut w \ u\od OuamOnnve RT~Pi R of -.emm starved endothelial ceil· mfeeted wim seneuoled (SCR) control or Ahtl shRNA followed with sNAG treatment confirms that Akt I ex «press ion is iequi - f fo? s x xG-eepeneem -s-deflm- 5¾ expo -mo." {Figure 8ΓΜ S"ie> p-Jeteesom a<e x-t-'wt to be expressed m epithelial colls, lent! viral delivery of shRNA directed against Akt! wa» used in he5'e.-:-5 ker.t’if'oc\tes A:,Α, «π <N-\u 'i-ea'xvin o''sewn r m ox eJ I era; οο-λα- i 'footed vdf> scrambled 1<SCR> eonirol leads to a sig.niboam hwrea.se h> β-defcnsin 3 expmasion than is abrogated by Akt! knockdown (Figure SE), These results illustrate that sNAG treatment u $ \ PCs XG I t," emimoe ml ee K and '-a-mgR segue---·· f 5 0 s\ XG-oepeneent doteevn expression requires Akt! in both endorheliu.) cells and keratioocytes. 6,2.2.3, ί'./ν,Ί6’ Treatment *>f Cttuateeui: liimmis increase- Bejmshi Expretehn in Fm? 1001491 To von in the vV{\ nJeneo of Akt t Ά 'K\">|'UvM>"'i'. mAn-m,·- * -.-,',νή,Ηλ' :.55Ίd Aktl null animals w; used m an excAipr-al wound healing model. AI Mo u eh most irajm.rnaiiim leukocytes express mAiAsms (human.. rabbit, rat, and hamster), mouse leukocytes do not express rt-defensins. Therefore, pMoiensin expression in these mouse models was focused on. Treatment of cutaneous wounds with a dried form ofsNAG* a thin biodegradable n'cnhuik', an dm e d ,w wwehs m a Mmsi AM -i.uu.n, act m, .0 ,se n p-eeiensm t t '-p-- ,\um 50-,0 lUiOO 10-, '·! V dj ;-· pO OHM Figure 9 Ad I CO .< IK’Hi (Am 1 M,h>sc j I V, C\; 1 Vmshd f U AmpA ΐΟΟ,Άο \ut ns m,m„o au- w-ed to nark the k m, o-w\k- Al pw'w and she*Vi that the expression of β-delensm 3 is confined to die epidermal layer. To assess if sNAG· dovfAo'M , i 'Vn-nr ονΊΌν-'-.-η '\ Οο-νΆΊΐ via \k, , a »nw c ,w-v\ a p " T-nm a, u-.v .; an AUl mdl ammul model Vyoeod-, from Mol -mil nae d octet. v-oh A \v Oktnbtupcs shev t markedly reduced induction of p-defensm 3 expression (Figure 9A), To better visualize the epidermal layers that are expressing, fbddcntdn Ik Figure 9B shows a representative image of a wb , . o ' -ww do e \ Ά ,,,t ' < \vjsh a Ά induced p A'feuxm 3 expression mamh In the -.upiA -, A Lot r> cd’ Am iFigure 9Β»
Quantitative analyses shown in Figure 9C shows art approximate 5-tbki increase in p-defensio 3 'Or >V0U i? A \w treated w d-d w po an w.h - and 'hat ΑΆ *s se.pm ;d k'” 'has ms. re wo
h.2.2,4, x;Y.4£? Tremnmm increases fku Kfrtmics of Hon mi Omtu'e m iVI 4 a ba a/.% |0025O | Previous rests its have shown an increased kinetics of wound closure In diabetic mouse modeA n r·, won -e to A -\0 mee-meni A \A were KVed mr a -nrini.u off- , t in a dd type animals Excisions! wmmds were created in wild type animal* which were either treated with Me me obtain, door A A \t 1 c I ft usiL'e.ded 1 - wte soebot - v-ow't MmI, 3 end -,L\c-ped \ o , e, A h \ » \w\ - ' m htgme 10 Av e 0' ,ν,ο type wounds results In complete closure, as visualized by the solid line, at day 3 post wounding, flu's occurs two days earlier than m the control wounds. Akt! null animals display a delay in wound closure; these animals do not fully close the wound until 7 days post wounding. I'he 00! ' ' \0'\ , v n t ie All udl mm, K - ο-,,,οο w A \< i\\no , m m shown). These and mgs suggest shat A AG not only induces detenu in expression but also increases wound healing kinetics in wild type mire and may he a novel and effective iheruncuiie. 6,2.2.5, a WAG is an Effective. Aniimscrahmi Aguimt S.Aurctis [00251 [ DeienGo peptides a::c known to possess amirmerebwi properties that arc «olive agaln.M gram-positive and gram-negative bacteria. Since treatment of endothelial cells with s\ Us ti as. \ Oof.vvni eyves-mm ihrb v- .uG f-t\r 't m'd t;e urn. m G . an.-wous neon Is with sNAG dramatically increases β~ defenam 5 expression in vivo, the antimicrobial efficacy of ;>N ACi treatment in bactcrciHy infected wounds 'was assessed. |00252| To determine if sNAG decreases bacterial load in cutaneous wounds, wild type and -xkt 1 neb -ηΟ';..yum to ciouucou'- wound ho time, 0>i;o>cd by ublvAon with
x?.. ν' -, . . b fbeto I ά(\ ο.Α άοκ’ e/be? t-vuled w i- -Λ \G >n of. m ^rec.o. tv? J and 5 days post Infection As shown by the tissue gram staining In Figures 1 !A ami IIB. wild Ape an r «ο oc.aoo wwl -x \u show a s'i.r.ufiO.v * to iueivu o' - we. tV'"..\o won'. go. " ' o post wounding as compared with untreated wounds. In contrast, gram stained tissue derived from untreated wounds in Aku cub animals at S days post wounding show' «η accumulation of neutrophils which stain grant positive (figure 11B). indicating a potential lack of bacterial < ouran c s'! i\v ινρ.Αΐ! jtutv HV.vt v -Λ \v! Pw GJu-gj. sogC' -t that
Aktl null animals have a defect in immune clearance mechanisms which is nor rescued by sNAG '.treatment;· jh02Mj lo p'onn.K·. \\ -G \n> euk’ hoew o' \ ob mgov in .ohm m τηην an of t \ $. mll-clcd wounds fiom both wild type and AkG null ηικχ enher Ά' f(,- neutvd ot uno eased wcuc h u'v- si- d and cultured \-- -.Gw\n so figure 11C. at £- n;\x post wounding bacterial mender ;.s eenki d \ seduced t If* mid) u wgg f\p> λ nsne.'. no: .ed with sM \v> Us.tuew''. mievue'. 'Jo number of bacteria detected in the Akii null animats is reduced tn comparison to wild type. isN AG treatment had a Hole effect on absolute bacterial number in the Aktl null animals At .1 days poM-infection (Figure IIB). there is a similar 10-fold decrease in CPU in sNAG treated will n oo wee .w eo-upaic I tv us'.se u.V - v'UtvG bp. \\ \U t:e .u J P'. oof . mm o ?>w\ a 2 -fold decrease in CPU as compared to untreated Aktl null animals, in general, the Aktl null animals have a lower bacterial load per wound vyhich may be reflective of an Akt:-dependent effect on other processes in addition to dcferisut expression. These findings suggest that sNAG treatment results in a marked reduction m bacterial load In infected cutaneous wounds in wild tyv Ui. e Go ?!v' vs \ko m.ii U"oe. sugcc-UiOg me pvsvbd.w d\ t ee envo- em \ veueesg s to anti-bacteriaI response. jft0£54j ί π shna nr he , o'.. tt (H'l A" ·. p , ,υί ' v ο ΛίΟ' h. rvi.VttVi'.iv !\uj i v e^" ot oo s.k \ ' \o s . . ' ' a. ' e^\ >**<' t\aiO i in solution with different amount of sNAG. for 3 hours and colony forming units were detw mmed V-. d\>w π in figure i 1 id s\ \< 1 he hie cm hud no duel '..feet an the mvo th m 5 aureus, indicating that sNAG is not directly inhibiting bacterial growth and may then be 'working em she up-regulation of defensina. £.2.2,6. Application »/DeJe»»m Peptide Mimics the %N>id Antihactcriai Effect [002551 To Adermnv ΆκΆη t.FOnon o.Gvensm pe-mde tan Nock 1><ku ,-nal mice non similarly to that shown for sNAG treatment, wiki type mice were wounded and inoculated with o. aureus as described above and then treated with biologically active human j>-defensin 3 peptide (1 .Oum) for throe days. Tissue biopsies 'were stained using a tissue gram stain and CPU was quantitated. Figure II F-G shows the results of these e>. peri merits. infected mice treated o nh O'deuvv;! ^'Vpnde mo* a J.\ .e sed h,a .omd ead, an .,rp o\t we, ' ^ foie \k\i> Uv t, \ ud''e bae: .tat t Figure 11G > - rmlur m tact Aeon sr w bd n c. owe deJ .-,1 * Ά ,s\ 'v i 10025$ | One of the mechanisms by which defenstn expression is induced is through simmlauon by bacterial LPS, possibly through the activation of'Fob like receptors. iSehtcd, M.E. and A J. Ouellette, 2003, Nat Immunol. 6(6):351 ··'?.} To test whether bacterial Infection a one κ tbG ;o mjt,> e {Wo’ensm .\m .Won n ilhin she fn'o ivnod" tested esp'O^uO of p defer oh v. u·, asses- .d ut in Ac ted sneT from wild Am, .msrruK .titer thw\ das \ pea kotn'dn'i \s shew'·! sr Figure i2Λ, Κκ"."Ά tn-eebou .none doe*· no- rudne·; the e\pim oo". m ' Ce o"s' oulu O.yo -, ''p, us sseownw " Ά, w- 1 o. \ * h'WO'.e 'vJu ^ v animals, sNAG treatment of infected wounds causes approximate 3·· to 5-fold increase in the exetO'S ,.T|Gl- f. tiss'5 w ohm . -ionf λϊ ,nue re-md 1 Figure !2B'* 1 tese fm.bng' 'tigg.M e,n sNAG treatment rapidly induces the expression ofdefensin expression resulting In marked ? UCi s G „ .it V , <. u'G h.lwo ids 6,2.2,7, Antibodies Directed Against $~Dejensw J Block the Antibacterial
Effect Me |00257| Ssnee defensins are secreted proteins, the inventors hypothesized that antibodies h'tried ngatust p' A f- tsvt 3 m o. be ode n, clock dr i U:b,k'n.na act". ·. * -¾ fe fits hypothesis, wounds were created, infected with ·$'. aureus end treated with sNAG as described
Λ'.. 1 ! C otiiii \ \ >. vtiies it,.' i A)'' , 0' ' , CO,' O . ' v ' .(. ''f'' Ot>A upphcuheu cedi His Omvc vL!\ λ \K υιιηύ ->C\;'Or-·· v- ere obum.-u and Gamed for p an? ρο-Ήΐ'. a bacteria. As shown in Figure I3A, seasons derived from wounds treated with P-defcnsm ' \ x ' v , ' s ' v e h h ^ ν' ,: ^ > f ' " \ ,ot to eoe e\ aoh section shown was derived from the wound area directly under the scab. Quantitation of CPU in these wounds shows that neutralization of β-defensin. 3 prior to sNAG treatment in S. aitreiu infected wounds results in a significant increase in bacteria. Animal a that were treated with an IgG isotype control show an approximate n-foid reduction in viable bacteria (Figure I3B). Taken together, these results suggest that sNAG treatment not only results in the increased kinetics of wound healing but also promotes an endogenous anti-bacterial response and supports the use of this nanoflher as novel therapy to enhance wound healing while concurrently do jvuvjv Ίΐ' old InUotS''! :6,2,3: : Conclusions 1002581 The findings presented here demonstrate that a marine diatom derived rumofiber, sNAG, may he used as a novel and effective method to enhance wound healing while V'X vt" Jem's eee e ismc wet s J .clect'on Tb<i data hemmi'daum m.u this TO A rrp -w cd material, which is presently used tor hemostasis, stimulates the expression of both α-typc and 13- type deletes ins in primary endothelial cells and an up-regulation of the p-type in primary .ketattnoeyies., |tHI259| IX'fensius arc an essential compoiKrd ot the innate immune .-v stem Those peptides jV-Vs, .tOtl-nOt ΓΟΌ.Ν .-' - 'rV'V'iC-, Jh'P Hi SC;", e g,tmst g”JOJ s' .-"d iVg.V !X O ' its'!!.! fungi, and many viruses. Detenstns arc small (3-4 kDa), cysteine-rich cationic peptides found in mammals., insects, and plants that are classified into different families in, p, and B) based on their p cts'ie >n d'snlt'i.L I'oidnc .'-.1. jcojuk me Bvugk to lv -pO' no. to neui'i'ph.G ate found j" very high concentrations (comprising approximately of the total cellular protein (Ganz, 17 and K 1 l Gm r, l^'>l. Gurr ^pm Immunol m-G ^s.i.Oy and .x veeree.J doting atu-rotembt si responses (Gan.c, T.: HAG, infect Immun. 55;3).563-711. U has also been shown that rabbit alveolar macrophages possess α-defertslns iw Krvrds comparable to rabbit neutrophils. (Ganz, T.. Of -J I'GA ) Immunol. '45.,-' ' 'Α,-ορ ) f. t eftsv w in, ,ouud m ep'dxd a' ceil JmVs >nG' jw ko-ai'i ο- \\\ o t.co^ai op 6 Gj d a, Is jlt.i.ji,! I,.'.. , M Sag." -\'5.-...G·" so' ottd t' ! h' , e*-j „uM, ‘ BsoG h v ' . Λ ",v otu ,, e '•.-aes ann <- 6 -em vm (Mathews. M . ot ah IG-H Infect Immun GTfn 37-10-56 and kidney whew they can he up- ' ^"Osi V Μ ( ii Ui “ v or ’ tan T-Hm-x xnnul K' , 1 <n >! P 1 vhtxr S,s ^nr
Opio Immunol, ote rte-wte. Human p-deiersssn 1 shDEJ-Bs) is one of she most important antimicrobial peptides in epithelial tissues. Defensin expression and section could be exsen . h ηηρηΐ,οη ste >. swmn s <mrd tbu.tpe.u - x 1 b emnnsewn; J nee K A b nwux *s considered pan of innate immunity and is non-specific and broad spectrum Therefore acquired temei m !esnunns\ ,w vui λ A the ex,‘new te.umbvnes, n t'.ot ,n :,xv,x 1002681 7 he data presented here also demonstrate that both ;>? vitro and in viva Akt l is wspmoe; for tUTonsu: o\r*ro.v,:>m ΛΑϋ b cssrnem docre'ixes Acme m-vx η·, inieeOon <ri\'iv,uvom wounds in wild type control animals but not in similarly treated Akt) null animals, if is also ,w - ' o f ok \ '\ ft. χί n v > e ' , v i i.^ χ o, A ·, j * me w kinetics of wound closin'·,·. Amiteds bled ade of Adekoxw revite m u. s'em-cuo·; nr the \\ AT antibacterial activity. Taken together these findings suggest a central role for Akti in the regulation of defensin expression that is responsible for the clearance of tecteri.nl infection arid, that xNAG treatment activates these pathways in wild type animals [002611 The tew drat mege-ss doit xls.Mb n carmen; of mfeemd wen-ids could Jrmpseuhx decrease bacterial load in patterns, at least in part, by the induction of defensm expression. Staphylaaorcm aureus is a bacterium frequently found coionwmg the skin and In the nose. It Is still a common cause of nosocomial infections, often causing postsurgicai wound infections. S aureus infections in hospitals have plagued healthcare workers for years and the widespread usage iri ,mt mmvs lo tmatruA- has lead to erb-uoue rexte.u! st-nos la, dm t moxcmev sw „ u μη w ό ,,,. ho , sf ‘i o' feekd wo, aw t A \\< dt r -ittcote vu„ v,e be Ivemsmi !o.so hot exempt,' the ;,tck of dark purp;e emm stmuwg sc the n- ,Ued AT race m figures ! IA and 118 indicates that the S. aureus infection has been cleared front these wounds. Both she in vitro and in vivo dam provides srsong evidence for the use of Talidetm-'sN'AO In she treatment of wounds to decrease bacteria! infection and therefore enhance wound healing. [110202} i otUrol xxfvruncnts mdn.<ne shut she amitectcnm eft cm aTVVAa1 is not du·- to a direct on, me bon of the snates-ui weh the Isreixoa but ?x due ίο doe, ηχην on a: teem xnco a,x the regulation of defensin? by Akt! activation, it is widely accepted that defettens are important pk s t u nrf’C n uv no v <mJ umebun m nrrme~te a wmHvx Most oriworev, vr their function is the direct killing of bacteria by in vitro mixing experiments with purified defersv-n peptides {Set-Hxh Μ K. and A. Goeibnc, ?.0xh\ Vh Immunol 6sbgff s ? j os so sum ar s.xpcrnvms as m Pigmy Π o'-m dne,t apphi'atuv" (" hv ρο-ν''d kmw"'" pm'o
Thedata here .show that an Induction of ddcnsir: expression in wiki type animals using a topical application of sNAG results in an antibacterial response, it has recently been shown that n'an.vg.mie mouse mod.'.K evpsvssnu: the human Oe:ensm 5 i.-en;. ,ec resistant ·ο λ p an infection that results in death of wild-type animals {Sa!zman< N.H., e.t at., 2003. Nature ' D ν''όϊ ίνα'η sn^e.'\Oia site m'pon nice of ton" sow n ne daiso· mf",' at: t i rn; c to b i a I response. pi02h3j it has been accepted that the «--subtype of deiensots art1 speed Hen lly expressed in neutrophils, whereas the jMype defensins arc epithelial In origin. β-typc defensin expression md'.uvd in o'-rorw A xa 5 m hteoan sc: -dwyK". both n eukum and m m. esnav aio who o healing inode! was detected The in vivo data illustrates that p-defensin 3 is mainly expressed in *".ο sup'tb is ill wc'-s .Jh\' ocat.u- 4¾ m -N \r I h;.> - .low-awcm a eh n vs was 0 r,, yriosj' kvJmeri ho nan p'detens'n 2 \> ,he m ons ,u 1 χ' nuhu ia\i m at' ,tv «hm o '"on x ,,4 ol , „V«H \ Mo* Pathol ' ΐ"" TO M Pev\ ' χ pc "s ^ cor ^Hh-u , \ -d ’ 2 π " tt:d -nnmue-s not s-n;y ,: v a rnechatutal earner hot ai->o rn j imams rbe ahhty ;o memo an ;nco v v.» e tun ate, son life,, ν'O'. , e ' iotv" s n u Pk cate '..vo'' " ^,>\\ mK riku re e m cutancoto moan: nnnnvt\ Π -v. e1·on tv ,111 d'.v,\ fo o s\ h? '.pee tie uh e-,ν .Tmev too e' pr.-wom .v \hn. o -hfferetn '",v'env"k a 3 \1 ''Λ m endothekf , oil·, Πί>< ;s risen.hy R.T-PCR, gone array analysis, immunofluorescence and ELISA (data not shown). The interaction between endothelial celts and leukocytes in tissue repair is one of the initial and most important steps in wound healing. The process of extravasation of leukocytes from the x, v,, ,. v χχ ns i Uud 'v > < kv,k1, f \ ' u V ' s o\ , .xWix" ’c todu-ed by '\Λ(ί and nxsv contribute hs Hv we ev-u> nenueph'd ondothcbal cellular interactions. More recently. It lias come to light that defensins exhibit biological activities beyond the inhibition of rniorobiui ceils, including their contribution to the adaptive immune response by exhibiting chemotaciie actri tty on dendritic (Hubert, P., d a!., 200’?, FASEB j. 2kl . ,n\i 1 oehv.n t,wv\ss\ md m.v tech sees sD-sr, u t K , e+J dOCl.iel fUe .'Cof 2t 2d Vf.4', j»,; .. ctauU"C\ tes ιΝη,ι.ο.ν.ϊΐΜ Γ., ct al, 200^, .1 Uivc-e Dermatol. 127(3>:ί·94-(χ04). Previous work shows tltat human beta defensins 1 and 2 have the ability to chemoaUraet inunature dcndrilie cells and Ύ cells tiuough the CC-chemokine receptor 6 (CCRti) (Y.Uig, l>. ct al . I Sucnc- 2nc; fTc^Vriff-Sh and that human be:a -ieiem-dn 2 cxn ho ioa ) ,κ 5 ΤΝΓυ ϊίν ·χχ] rseisruf h Κ me Ίν y C R6 .\\(,ρόγ f Nivonsahi, Γ ., !I 0*χν» , er ΰ I Nueno»,e, 20b4, irenxooiwy. s ’ F '' ^ ' m ) 1 Kenan s' Jekusm ' are 3 s\o ι «» h . ' sb,mn nnc , a to \ s ' '0 " ' w\' ' o' ' s '\\ ' ' . o" -· ^ s OsV'i uov unJ eeeoopm's jRohrb J , as as , 3010, 5 inuxum , ?0'0 > ΙοηΓνΆ-ν-χ no hue Αον that ^Oh nousmem utduee·» hah o xn-t p-defens-n ηχο-χΐοη m esmothelhd calx la ken hgxbex \ v x»oi\ busn mggess Hun drtfsss ns n ,o οηΜ.,ηο womb '"e bow ''os wth l \ men ami microhm! pioperiks, bus also by being themotaeac for other ceb types necessary tos proper V A ' x,*o oph ntm f ' non J d m-‘ so ‘ « . ns e»sO ^ n -. s msitr tcasa cos shea n> ooeK me, dsut vpxat apnlwmmn at a stwye eetenssr Joes not -aslant the cellular smoraettons required for increased chcmo attraction. cellular ixcru;snseut and wound diMtsfe. 100264} 1 he in »>m> data using bosh wild type and Aktl knockout animals confirms the s.xpnvoserf lor Alt i t -NAvix xho oe p-oetousO t, -. p»-, s-ooo f"X e ssu'ssv Seukoese’s »> ιΆ express a- defeosins like most other mammalian leukocytes s'Gan?, 2004, C R Biol, ef bp'- Ά ' 40i - ", , n-Jefeevn si 5Ό!"ρ of mlbsrw'ig mmiinv iobs x,?s na po"vb!e Treatment of airway epithelial ceils in vitm with alpha defensins 1 -3 causes a dose and time* dependent increased cell migration that requires activation of BI3K and MAPK pathways. iAeUisn» .1 , or al , 2004 xm 3 Respir ('ell Mw Bud io»xy»i v \\-\t j sbmuhlsos· «f endothelial cells has beers shown to result in the activation of MARK (Vournakss. j N , d al„ Ά?0.χ t \ ..-v Rt·- -I χ x Λ'·''···ΡΓ i and so rah p- '-xeu q bo e, phe ma^ologiea* mhibmon of\x,k also inhibits Use expression of the defensive in i'iiro. These findings suggest that both pathways smpog. on 'he ioxn-w-x of eexmsu’ \χ .xsvsi by \\ \sj, hn\\. \> r, \Ut tb'a'Svn soseS'\ m u marked reduction of its expression both in vitro and in vivo. In myeloid cells, p-defetisin \ expsvxslon h controlled at she level of transcription, in part, by the Ets-family member f ϋ. I. xhax'Na s\ ol , Γ00ο, 1 urou' xr 'χ ιΚΑ>·Γ, rsl Mj h vj ass.; P l'S"p-s, h^'s Rsoi» ''Xs s\ , - newiMOa» ^ ' vs et^uCi χ 'ess ' , , ’Ott Hbe ( v "e !'^ 'v ' " o \ en b\l el a eel *» st ha' 1 v,en shov»n bus.. Akt' ' npsixxu' w Fssl bon " e- - mu; « »>' - j hm,; ,> A Orfobeal Je^eb'p"i-s' ij seeixAli ei A ,Ά'Α ΓΧχΓ'^ s ' '»» x 1' y-sP ; Ax-sonajvtmn ol endotbeoul sJb x 'e b m ex' ,o-ed es,\ess.» " ,e Us’. x xvaebb letatyh \s' 1 s w ' ν Λ\ Ο ”? ho ^ Η' θ’ e’kVHV <. Ο \ ,Λν>ι.' 'S \ Ο , ΌΛΝ \ ,Ν '4&amp;{άν.££4ι“®·1Τ · }00265s) Thus far. x.\!AG treatment Has resulted if) a secies os'down stream activities; ho'iK'stas -y .01 rngrutk^ ^ > M ptoHetuttn'i nukused sr'mo * loon-', rnd .w Cev tew V",, οκοϋ Oon of Or. iin-'s urnra-uv tov'onso i.'kbing m urn ivieieu.u futO'ous )βί^2ΐ>6) Giser t! -. 0 at'} 'tjc jvc so ofdtabet',' '»,uk"it\ v. d t}. ,tv |\"p itais'o \wv pvsam with chrome wounds and .onmlkaumi:·, due to wound intVeuoo. new ellnLai treatment.*· are in ' " i emar-d H> , run v voo ·. w nacotib.t4* or ih>i . , ' "o' ·. e -Ό e kinetics of wound Healing but act to stimulate innate irrun unity thus providing anti-bacterial acuvity. The obvious importance of these observations' is the application to nosocomial infections. Of the nosocomial iniection.se surgical wound infections predominate; with statistics showing up to 8% of all surgical patients. The direct cost c-i these types of infections is approximately 4 5 billion dollars per year Clive?} that rietersGns are part of the innate immune system, activation of these pathways will preclude the generation of resistant organisms an well ;j« allow- for the uTUibiotic-indepcndeui eleunme.. ofhuue? uu w ivumi· G’w ofsN'AG in ;j ho-'pda! shin 0, to t·. too much vi the s om une o„nk> J<\ reduce the pmdue'nm el u.n'aio'.t'. ?o\ \te"' 'V' o\ t \o vge^^'C1 iwwe ^0000, \;cce nr ' ' ' ee * a <Ό\ \ namoOvrs w? 11» highly ·»··'ϊν*\ sal n dte < lm?r,k ,t,ona 6,3 Esample3; sNAC» h an OTcctbc AiUmngrnbial Aasdnst Pn'advmmtii’'xcmumtKtx |tH$26?j This csun:plo rams; sates that s\ '-G sumoudvrs h>o „ an onn-akk'tenui cHer-
Hi vivo,. |00268| Afatesiah and Method*: Wild Type C57B1/6 mule mice between 8-12 weeks old wow' wikuch J create 1 * sate, a twm Ρ'ορ\- punch s.\hbe\\ to c-wne two kw ι·.,η wot. \G m v ' \ Viv.oa-v. , es 0.,.00 v '^ώΐίΟ ^o'ju .or , \ .> - - ut, see (VetEquip. Inc.) Isofluraoe was used at 4% for induction; 2¾. for surgery. Prior to surgery hair was removed by Pseudomonas deruginnsd were picked and cultured overnight at 37* and adjtsss^d :(? an ui-sorbasuw ru OO.;o Q fi.t. Fuch wound w'as inoculated with 1.5x10'* ein wound ο- i\ a. i.'iCin-w.f Aiks Hs tnuiun·'· non inoculation, wounds wets' ouhu coated with -ΛAt.; rt'ord'inueniotsiC'fied V’-t'h dtsitHed oa." nest v. otip a- A; or eti t-'"iea>'r taonil c?a!:n r* η» On day 3 animals were euthanized and entire wounds «-etc harvested including the surrounding Akin using an 8 mm brer sc ranch Aldtev* One woynd per am mo I was t:\cd O', e might in t'b. p •u'iifonwddehs de :u 4 Cl embedded in ρ.ηαηηη. ,eid sctbcmod ιο; ;n':.ihsi-’., -md ihe other svouud --0:0-: euhurcd and plated on LB media v ilhoui snub-otic foi bauenai ijuantkaiaow bar caitufmg. wound sections were· placed in 0.5ml bacterial media an incubated for 30 min at 3/4/ w hile shaking. Colony forming units (CPU) were quantitated using a dilution aeries plated overnight tu 37C Number of colonies per piate/per dilution were counted and CFU/ml were calculated. [0026^} v , 1 re e .wo o \X Ui no ament of w msu.-s. n.eetee \vn.t www > ne - esse bacteria was assessed. As shown in Figure 14, at 3 days post infection bacterial number is *" rwed\ teduieo it-n>r> thus ' ’bid' u a nm; b- l-vured wm -Λ-uj m ,wmearw-'i to us’4retned unnnah Πκ"<· finding-. suggest that -A \ίΰ treatmettt Jesuits m a mailed reduction m lucieMal load of gram negative bacteria, and specifically P. ue>mighutyj< in infected cutaneous wounds vtn addition to n duel ion nt l-aotcrial load ofgmm positive bacteria shown In f .·, no pic 2¾ IMEmMMlkML· ' ' ' 1 }60276j This example demonstrates that a number of derensinx and TedMike receptors are unregulated by sN'AG nvufrnem of human endothelial cdte.
}002"Ί| ' v -, . ϊ lb mm t'lim ' o, e·- w rr,"‘’Jo> crow s w T> \M ms ibc.J ino'j-u'. bet- -,, t i't atm letev^wtm X \te ranofthois 'XX ό 'Mm h<wi'\ R\\-'ea ,ewU R\f l-o , * e w ' ,, v , v ,-- ' '0' s ' - rtnu-ti Π - \' s. --X % Π eR\ \ a up !~w .6 >- np/s and labeled, The slides wote prepared idr h}hrickzauon with uRNA 1/ soak mg in blocking volution iSw,vna hw-buffered sob no pH Ari, m K;O0ml aH/f Γ- BX-V.\ ,. \a\. to tbi/A-i a? R.T x \. ibeu ru.sed and e-wee. /rook's - m tem.ng „uvkd . - ge- aRX \ from -Λ vix-e m-,1 e/X ".etc rybr,ti"„d with -dmes tfoul si 1;, benut. .e< at-A r A* e mm. h->om·\ν. An A hoses η -'V ii- ^ lASDSued \ .\S(An,l i". Rv U and diye-l Hw \|<do-wese scanned and hybridization detected using Perkin-Elmcr Scan Array equipment and SeanAnay Express software Veil, updated. To idenrify up-regulated genes, mtcroarray data was analyzed using Agilent GcneSpiang G MX, 11 Btomionrurion Dsu Analysis }002"2| G - ?· 'C'Hri.i. u \J u 1, Cbw/aM3, \ΤΛν 1 / de ensu's. </Di T V'wi , etcr-e \ /. - - h lol we e\ s 4 d|< JR <. <n mje sHl·1
oXiwd receptor-, .aid 1'RAFo fi'Xf mceptm assoc Lit ed fuero; 6> fOsiso·. eonnols t A-e/ v 1 '.rosrseXo vnw s Jiogewase t-- p;ogh,m ^ u'onobyjww'.euase w t turn ptoiee i. liAPH tg;yvtKk'h>dew~pt*ONphav d 'hydrogen :t\e*. RIM ί 3't HMKo-om;.! pnMe*n s ikh 5 IK <(t d'-Minim Ο, AC HM Achn R'= |O02?3| Re.Mtiis: Results of the microarr&amp;y gene chip analyses and Q--PCR validation of rmcroarray results are presented in Tables li-Vt below. Using a cu&amp;tont perse chip it was deiem-otcd thata. nemk.r of deion-uns and Foll-hhe receptor' are ep-regulued h> sN V 1 treatment of human endothelial cells }00274} l'41'hko u<.esvns Hi KM it'. Hgl'k > es-Vived κ, epto*s ny u\^ m. v. so^ottie o'ok'st tat puttotf'N oft' i' \"t ,t eei'ijOfvt’is leaning to u;y w.Oo's of ts’o tn or'Ti mu I'ttetOM m,\ 'vi Red ϋ id.O'.oe mm me-y v.ef\ out me sfd leMsUrtto no. toi o' it:f ehmts. !.1η·1·.τ. j L. and 1 \ Hoffnnsrsn, 2000, (Fun Opm Nh-'uHmi, 3(7 5'1n02 I 'Thtsltnv: k'fet v t' .hetevtho an stoats .nanno, 11 that pem tees pOteeno-·. p-. .y, ndw' up fn. antimicrobial peptides dToll and I R-wheeler which are induced by TLRs. (Lemaitrs, 8., et a 1., '-T t. od Nose'.41 cue W .that w, Μ 1 ct .n . \ I MHO 1 tot no »* 2P-\ '> Roeeni work has also linked human deiensin expression to TLR activation. Human p-deiensin 2 was s'vw r ό K' mum oO * i a rwa'v -mhoha·-..Η'ν sr >s i t-R-^ eopenoen. rs',smvr Π5'!+' kJ eta’ 200¾. 3 htssmstsoi 1" t!2t p ον20ό \ iohi'C V'emo - h ** I „„v. snowe v ned sate loot au p-detenion 2 induedons m respond to Cniumydu pneumonia ei monocytes, (Romano 3 trr rtoUL k et a; , 2*504 M-MS Isnsustsml Med M;-.ruhsoI >2V I ir*H4 ) hnpoh.uuly the R!3K'\k· g ’."v'.tv s t.' v C't upo tend .n * LR μ*,ί, . n\ f'<H'.e'*0'\ . o'ihohetg . .',n " coo non \< v, x 'Λ Knir, „nd Μ Γ) s , ' Kh3, V i R1 as rs ΐ \ ' v,"p‘ ' ' "
Since it is known that stirnulation of TLRs cars lead to increased deh.usMn w mitosis, fhi- woK suggests the potential for sNAG as a stimulator of instate immunity and bacterial clearance via
Tahir* 111 I ,kS uf <smf tui-rnmihiiad In rr\s3>nrs.w ίο *s\ AG Nidrsulmhm
Table III: [Vileroarray Ceise Expression {HIJVE'C Response to sNAG lOug/sn! 5 hours)
Table IV; DEFCB3 Mieroarray Gene Validation (ΛΒ Prism n&amp;h sNAG (Ithig/ml), 11 1 A BT for 5 h)
Table V: TfdM ikp
•Vtleraarrisv Gem1 FAnre^Gars
6,5 Aanndc .5; sNAG and Lorn; Fiber NAG Differ in Their (i arise Esorossion Profiles. HMlJAj Hus ovm pi' teomosnaies thn A xh met-'·,K"s d t'f> foon long ro.de\ xe dlvis in their effect on gene expression, ana specifically in their effect on expression of scare of the deferixin.s and Toll-tike receptors. 1002?6| Muterinis ami Methods: Human Defensin Chip probes {concentration: 20uM, quomux S Ά A'-em SA law* spen ng coax * \se*\ vne Urn οροχχ s sees evoe \una;,oe techniques. HIJVI-C arid HaCa» cells wore cultured as described m section 6 2, and treated with
Co Os\ oners, i \ xA o' A Xc ' ϊ X s , s\ v ' ' ' v v '!l A 's s extracted w--ith RNAsol (Tcltest, Inn,) following manufacturer's instmetions, ami am pi (lied using Amino Aliys Message A. MP! M II sR'NA amplificaiion kit (Applied BlosyatesnA Daring RMA unAseeatvn,„P\ 'X Am ee is o vied o d I \ V,l one ,dA x A'", r, is 'e e,e,e f ν,,Ρ "· s "*t A u l"* o a , e ' \\,” e ^ , s s 'sc v. ee " si' Oi ,,e 'vi < eRK '\ S v,!\ V' ο,\Ά<'\' 'V Ά ο ΙτΛ'Άν ’ e po's Ο, η ‘000ml j!1 A'*, 5 dv\v- \, NuV,o 0 ΑΆΆ, Ki nemo nee diyed ΑνΆ eri'U.isr nv e-onl io\>, ro.dk <dc\v a w aR s \ iron"· I \ Α οη,ΐ ΑΛΑ ere red
Ah were nutted. hybndte .d with dr.- slides {theI slide: demoned al AT t«r 0 nun, hybruihed A ay i cons λ d A' π 0' he SDS amd e X Sm ' -mo r - UW o xnsed on,, c"' T I he \* loo tng exemplary graphs in Table VII illustrate experiments! sot up:
[002^ fh GnVv OK' WiO,’i and ^f IV11.' OOP deH'C ' F, Ο-'ηΌ Pa'Xir "irx'r Έχ ! λιτ,ο cgosnnssui and bean \ru> l spxv, \-Giv-.na Y3 0, updated, to- e-vh slide, C' 5, tv t and c<* r^xoo f uot'ev.eme -,- I···' auahrod lu do n A la- Veounej ana a a..,.-.- 0.-0s oed ona-vu"" tv Ja :.i; a a - asxK w' nxng \gdent xanaX a''! a.. - 1\ \ 1 \ BioiofounaiHO D a a \aaa; vs-. Genes of interest analyzed; DEFA 1, DEf An, DEFA4, DEFA5, DEFA6, DEFER DEFBO13A, DEFB! 04 A, DEEB! 05B. DEFB108B, DEΓ B1J 2, DEFB 114, DEFB i j 8, DEFB119, DEf: B12E DEFB!24, DEFB 125, Di:HD>, DEFB;27. Dl:EBI2K IFF·8129, L>l; hBEG, DEFB-< 'DEFA" ηι-ΚοΚη-οπ. and 'Όι.ΕΒ" pKesbnMS'K Π F :. Π RiO, Π 2 ΤΪ R3 Ή R4, Π R5 TLR6, TLR7 and TLRb { TER^-Tol! receptor}; S1G1RR (Single IG 1L-1-related teeeproi); IR-¾I- "'tH OvO' os ' 'W ο e ; ΒΙΟίΆ Ρ-ϋοιοονη; loot Γ3107Λ tP-deienvu KG; Aagasx a ,Λ-aavl·· sPa.r s ητΡηο ,a p-sena* - ; K \ G Positive controls: 1433Z {TymsineaGmonohydrogenase/tryptophan 5 monohydrogenase actition pri'-asaF GAP!) pcJ^e-Tabi'lx J, s-ApKvsrbatt deh'.axvgXiM \ ΚΛΜ,Ϊ 7 \ iRilwuxF 'Mown L13a); UBC (Ubiquitio €}; AOTB fActin B). |00278| Remits: Results or the microarray gene chip analyses are presented in T&amp;f>fc$ VSII it'd IX belox T able· \ ΟΙ A-*", - t;;x expxvno:! ; \ hooiea e-nbiheU voce andKb^Fa' ed.s f I'P.ATGafter 2h ;·; 24 b eKposute to cither 1.. MAG fibers or AG tnntoEbcrv Table 1\ shows «ene expression In human keraiinocyie cell line (HaC&amp;t) after 2h or 34 h exposure to either IN AG fibers or sNAG oanoflbers. The results demonstrate that gene expression profile induced b> long pop,-N --aeetylGiieesanune fibers Ί NAG 3 iKifers From ihe gens as pres1; a as " ο P ncH eeu i \ -\ Xs- a so » x \v Ά u '' v ' e v IWii 'G \\ v j ei let ntuau effect, on expression, of derenstn gen.es and Toll receptor genes.
Table IX; Mkrearray Defetssin Gem Expression in Human Keraiinoeyte Ceil Line (HaCat), Fold Change
6*6 {1)02^91 : life fs-deCfbci f&amp;W$i n u" vui-yd p>. iU'N \o ftk r- r-tvdneed as pvs -ass \ ,ΐοοο shed svv Vu'jrraL.x,.: i \ Fida'il
Nii> LsOL'-V 5; ae-i .vO': η n she eomom of o-i-ch of άinch .:- nvorposvtod hesvn K ^'..see-v se n\ oaor- n ' F-'R'sh- n voakiar -\- ¢- enihn-J -n w \pu Lane -¾ so; -.vs \ *Osts s-s -sg a k'fr -V .grdwihiimellla.· ioltaeing: ite isiymldfimidpalgab Irons Mgfeleoffig ®lf grek ffiep^-eri. jsQhjrp-i \ as -s s’k'p'-i i.:-c ---j-vas;--o and pnri-K'n-os-i groeev swlsss^g ϊϊ^ ban Or-' v'nure sib--:s so -pooded s.! tin sn c-dvss t -\ so hiUn--w'e tosso-sias.---; .m-s s aleho-s :'s . os.-, - -'aasa-e .a J o\in >' s ng and λ -tc p and st Ή \\t fs u\n ο\ί in m e s.m f- i;v' d ο onions average 20~5dnm x l-2nm x -· ΙΟΟμιη. Batches of fibers were individually quality controlled using chemical and physical test parameters, and each hatch met strict purity criteria prior to release. Final batches were required to be substantially free of proteins, metal ions, and other components. The fibers were then shortened by irradiation to produce sNAG membranes. Briefly, the starting material combined 60 g of pGk'NAc slurry al a eoneuiuration of I mg/mL. The concentration of the pGIcNAc slurry was confirmed by filtering 5 mL into a 0.2 urn filter.
I ό I i.»f pGh \A. sfuemtkimnye t ' g pCII- \ \i wav filleted tour kamaooe of ; wet -.:he I .v wake .eke w .sv *h„r> kn.sfeued nto .5 tod poiei. <vhvb >s . ivr.nid ' ib^tsm somtvxbk container, and subjected to 200 kGy gamma radiation. Other irradiation conditions were tested s-'i *h. n s *Y.‘. .s ee ptbk'N -\e eo upovhe w, ,i> s, decVd n Figure HA 1002^01 v , . . \ , ’ λ ^ \ " "v e nc moieudat wemlu of pGk N Ac in ad union dfd nor disturb the nucrosirueinrc of the libers pGIcNAc was irradiated under different conditions: as a dry, iyophiJized material;. as a dry I'Vmh.me as t eoei e us ne,1 s unv χ'Ό o weight m volume'* arc ts . bib \ skm\ :e no r ' Λ vtn. i Ί )>Ί. ,',d,o wesg;* 'ebnen.n oo ,i mm- ade* we*' n n* HihbPO-’ .OihGibO oalvt s> w o achieved at an nradiation dose of 1,000 kgy for dry polynu:.u, and 200 fgy for iv,l polymer (Figure t5A). M1028II The chemical and physical structure of the fibers was maintained throughout u.uc.Jitod cs ' coded hs mbmed vlFt S'\ ei urn i Figtii* 15B)„ e cose nJ asow, a\d vnixoq. < lee trim rmerest opes tSe Ms} analysts Mseroeenf le ohservanon oi ητ,κ-edod libers knowed c -decrease in the particle length (Figures I5C and 150K The majority of the fibers are less than about ;5 //m in length, with an .average length of about 4 nm, 6.7 F cam pie ?: sNAG Nanaflbcrs and 1-imt* Form j>--C>i.eN Ac Fibers Differ In Their Effects on Metabolic Rate ami Scrum Depmatton el Umbilical Curd Vein Ewclothdial Cells |Μ62Η2{ ΙΛ.'ϊ ' Horn,.' Pooled I'nib^'lo'divvi Karan vt.mdem cowl\, n endothelial cells {EO Cmnhres t were mmne.nned at X·' <" oath b;k- <'0. in endothelial basal meJuno 2 iF ex. t w ' -sepph nemeo w eb I r p -"n Cieditru 2 N,ne,',et>„,so as des,--\!vd 1 \ t. ' o' o v, V”vi ο - < \ v \ ' ', ' xti-χΐ' cm' e ' ' ilFM hwd sofpl- 'ne Vi,;..' \v th i“, {,",U t.a ; --,-a.bo'.o BR1 1 sv 21 b fe loo>.b e\ shrmlatb’e wnd \ r,Gf If ' CO s'-; ml, R&amp;D fysK'mM ,-¾r -,-. nd twghw pu; uled pGk Vo u.mofvw at s\ \G n.ino;U'e;s m ---A-nT watCi tmer ide-l I \ Mas use PuA :vci I'eehnoiogkw, hu , Danvet -, X Vs , l F X' -' Ts h> * > < ηχ * d η s w - o x '"s'' ο- ' o 1 '
Ux-O-xW, Vs 2 diPcvs,* ' ,S\,W-i WOW U-Vu '5*> ixUi blue ,,Χ,',ηχΚνΊ b> -h'<„, , i ,'J Ο-',,Οίχ U'Uu a he n,vsv-m»u" e d aa Ml \ ,μ I 0 b n\Mvi'i' a,set „\ l Co- d.Tne-w.vuvoh jr oto mex assay in procedures described by the marejicctere;: {Promega}, \mnA} v-, ' \- 100284} pGk NAi, did *#>? affeei menthol k rate. As shown in Figure 16, pGlcNAe did not result hi a higher metabolic rale as measured by MTF assays, indicating that this polymeric materia! was not causing marked increases In cellular proliferation. {0028?{ v X ' , , , ^ ' r X , ' \ . 0 , ' tOV' * pldeN Ac suvrx bad a due-i odds s on Vi \ serum warx-ed Ft cc!K were nv,ue-l web Vlid' or with different concentrations of pGlcNAc fibers. As shown in Figure 1? at 4k h and ?2 h after ''' non 0-10-.,101-0, ·,,χ - wnp,n, d wnhdv h-mhsvnmu ot ,-sds pence *-v non Vi., r, wax ahem ?-fold reduction :n the numbm ot cob·, a I Ice 48 h οι 72 h, Λ- 4K h. dns drereaxu in cell numbet was 5 --xuec b\ the add* son of VI GP .v w On , ο I '-on m prdeA X- Sloes s U ewv ed or PM go w As 72 h, the decrease in cell number was rescued by the addition of VEGF or largely rescued by the addition of pGlcNAc fibers at 100 tsg/mb These results Indicated that like VEGF, pGkNAe fiber treatment prevented cell death: induced by scrum deprivation.
MH>286{ Vwl- -W 10028?| *NiG Induced Mio'kc-Ί Iru'reuse m hietabdic Rate. As measured by N4T ϊ assays, sN AG at 50, 100 or 200 gg'mi resulted in a higher metabolic - ate of bC than VEGF {Figure 18), I0028H} s\. P> JiJ m>t pi\>h'c? Lf 0>>m ,ν.Ο oVosb Gee; a cl be s'crnm TmV atoa To test ir x\ xG tYocw hat a wavs- etVeet on l 0, serem starred IT relk wo\ t e, AM w *h X EGf m >ηη different concentration.- of xNAG fibers. As shown :n Figure 19, ai. 48 h after serum starvation, as conipared with the total number of ceils plated {control}, there was about 2-fold reduction in d i wneivi- w , oH- Hv\ deeiC.me - , 1' nnnh" i wa- vx^ m\i tw is, ,dd tiou ot VTaIF aot by the addition of sNAG fibers at 5(K 100 or 200 pg/ml. These results indicated that not like X Gt χ\ >vs c, 'cu J>0 no: me Iwb i m lu i. - \e , ' cnvi n>' {00289} V V ' \ \\S Ov s k’f' Uikt UK ' \\ IT li ks .'X .' V Λ \ the incunoi'C ' »'ϊ 'Ot utu sieved : i'n u Μ Π svsuv ηκ, eo.>, not xsCk' upopuva. of serum-starved EC in u trypan blue exclusion test. tfcfy Test Arli.de 100290} ' , x Λν 0,0 s' \ s Ϊ s \ \s.. >\\vs ' , S ,\ ' OOM ϊ ΛΌ ii X > H ' 1 ' ' mtpm. was utilized. The lest article was supplied sterile fey Marine Polymer Technologies, Inc. 6.8.2 }0St29I{ BiOvOtnpudbjhty of the lest eruele w ux ¢- χη\Ι ui roox->o fibrobldsi 1.929 mammalian ids \o utdoevei vu. n^U\ ,Oru-k'0'.aus observe i !*·!?''1 92‘i .yIN e *S rx s, p.'vs exposure to the test article. The observed cellular response obtained from the positive control article (Grade 4) and negative control article (Grade 0 } confirmed the suitability of the test system. Based on the criteria of the protocol* the test article Is considered oon-tossc and meets she repuuooK'iUs s.»r she Flube-a fes·. Inremaouniil Orgaru/ener: for Standaidilation (ISO ϊ 1G99.T5 guidelines. See Table X below.
6.8.3 Intramuscular Implantation Test - ISO - 4 Week Implantation 6.8.3.1. Materials and Methods [00292] To evaluate the potential of the test article to induce local toxic effects, the Intramuscular Implantation Test - ISO - 4 Week Implantation (“Intramuscular Implantation Test”) was used. Briefly, the test article was implanted in the paravertebral muscle tissue of New Zealand White rabbits for a period of 4 weeks. The test article was then evaluated separately using two control articles: positive control Surgicel (Johnson and Johnson, NJ) and negative control High Density Polyethylene (Negative Control Plastic).
[00293] Preparation of Test and Control Articles. The test article measured approximately 1 mm to in width and 10 mm in length. The two control articles were prepared. The positive control, Surgicel (Cl), measured approximately 1 mm in width by 10 mm in length and was received sterile. Negative Control Plastic (C2), measured approximately 1 mm in width by 10 mm in length and was sterilized by dipping in 70% ethanol. )80294) ·'' ' ,,, Γ,π*'," rJ , v ''>' η imp t<n O'' , >’ o! the test,, the dorsal skies of the aniro&amp;Is were clipped kecof fur and loose hair was removed by n:e'-noco'a \mcuum L»i·,.h anim d vre, .ipmonuateK tnesUmtued Prnn to mg-hun-mem, bs·; arc., was swabbed with a surgical, preparation solution. |80295| Dose s-itlmimstraifon. Four test article strips were surgically implanted into each of the paravertebral muscles of each rabbit., approximately 2.5 cm from the rrudlinc and parallel to the spinal cob me and approximately 2.5 cm from each other The tost article strips were implanted on one side of the spine. In a similar fashion, positive control article ships (Sutgkel! were implanted in the contralateral muscle of each animal Two negative control setups vbee-nwc y oiu ol t'L me' we?o noplnn^d cats fd p'-'on^ 'he lu t to no test mt-wo uv to 0 xmmo m'dant sne-- o* e ti ,i ssdo m bo s'nv mu it of "»" \t.\ps' \ ,,a a cn ,n h a\t χί' article strips and eight of each control article strips arc required for evaluation. 1882961 Post-Daw Ptwetittrex. The animals were maintained for a period of 4 weeks. The animals were observed daily tor this period to ensure proper healing of the implant sites and tor ο n mol <nm.s ή k'M. os * wxv,uious mO mLd .ill ehmeul msmskwtaKws \t die end of the obsorv at:on pored, 'he <mma ,> wo ό weig r f finch a nm;J λ .w '.tcrtlR'V e\ >ut "ge'"a rle barbiturate. Sufficient time was allowed to elapse for the tissue to be cot without bleeding. 1882971 fiiw.vi Gbscnwrivas. The paravertebral muscles itt winch the test or comvo! articles v *u Ί f, '\ Λ\\ Λν ν' t> worn each , ίΓ«< 1 she ruwc! w w - rc v- so> ew 'fnil\ s u'u j a.onm, tlv vnpl ,m vtos a uh a vatp> a' Λ Lomi. out''hi t'S\ue V- e e^emd implant tissues were examined grossly, but without using excessive invasive procedures that o mif h we dumps J the n"tepw'\ of c,w uv-ue tin h}siO"',nho',og„\U mb'eamm The h\s,.ev were placed in properly labeled containers containing ΙΟΊ» neutral buffered formalin. 188298) Hisiopaihoiogv. Following fixation in formalin, each of sire implant sites was excised fu'm me I eg,.'" mas* ,n trvu,> fhe imp! tm v,e, emu.am 'g n <„ upkme t m,no"i,!L w w examined maeroscopically. Each site was examined for signs of inflammution, encapsulations hr ni'”h sgn p, stockists, ,iu,l dtwolo amm own; no n; ovn<; ,wu r ΰ.....“Normal 1 -Mi id 3:::: Marked oisf: 11¾ ill-llM &amp;&amp;$ % :S!ifc:Cjf tis?Sa®: containing the implant site sv&amp;s processed. Histologic slides of hematoxylin and eosin stained sections were mepared by Toxikon The slides were esak-ated and eroded by tight microscopic exaroihstiop.: |0029¥| \dv'y\T " o'.t dvdo'' Γ:,'-loKowr'i; o ttextoues 6i0i:Q|t:dal:dtaetip«; w&amp;m msmmi; if: ssrlerdiidpii dlJserya||o:a:#f edelt intpaM sitt t 1. Inflammatory Responses: a. Polymorphonuclear leukocytes b. Lymphocytes c. Eosinophils d . Plasma ceils e v M aerop hapes E Giant cells rgs .Ifeerostl. h iX'siCi is ration 2. Healing Responses: a Fibiosis b Pau\ Intdware |§i30i| Each category ot r-mpoos·. wa-*· ^ladcd asin^ she tbih^-iou Svak. § :::: Momts.i :liJ '"d¥dry'SI|g|t,. 1 - M i ld 2 :::: Moderate :¾ ^::M:altedr RKBhS j v e w > n rw-ί < \ >. e s ·, w s v ^ ' s ' m tiO-n the nopUm u-suo s neriree m jiujd.:.ieJ mots o rer -, mo chore.tesrose-' o'' tioona <nt (ίπρ ' -v v Re ,<iso c X' < lyo t o'* v o es· \
Sbalo; 0 =- RmoivMosde: 0,5 :::: up to 0,5 roro, Very flight 1 ::: 0.6 - 1,0 mrn„ Void 2 ~ I.! 2.0 mm. Moderate 5 ::: > 2.0 mm. Marked [003021 The intramuscular implantation Test was conducted based upon the following rdlferarteev;. 1. IRG ΗΗ'Ηό, ΗΉ Bmiogieal EvaE-dtio·; of VledieH Devices Γ as c I es^lor f nea> /Effects-Itipldotation ' HO 0'"' O' 2v 0¾ * vnnmvot Med ,a \v * Γ, i 2 ,G , e
Preparation and Reference Material's. - M fvt s 'S + " ' v 0. f Tra u so "nV'v·'' ' v " o -, e
Biornateriab tor Surgical implants with Respect to Effect of Materials on Muscle end Bone. * \M M r ' ' ,ΛΜ ^ -!,0 d ‘Mw'dre u<r ^"mt To ?r, V\ ,. s oH,r~dc'
Materials^ 5. 150/fEC 17025, 2005, General Requirements tor the Competence of Testing and { shbramm ; ahoraaak's [003031 The results of the Intramuscular implantation Test were evaluated based upon, the folkm $η^ ci uen..i 1 Caioulutod Kvmg. hot each imp!anted site, t total score is determined, t ho average .seesra ;pf Ihe: Mt0silps;§sr eab|; gmHmlsts -pompMed; fp :i fe of t&amp;vBnf std /sldR for f fed: , <r, 0 ' "o v 'ee v ,o v·' , ^ o e unt ol v os , <t ! non w \wRidm o u <.
IbeTdi/f/df Sldrddeivif y; /11# fsgdi sssigMdras /fellow# 0 · 1,5 No Reaction* > 1:,5: - T,5 Mild Reaction ' ^ Wee , ve so t >:. 0.0; M arked: Reaction * \ ncg-Wo caleuhumn mpmvd ts^erolUt 2 Mod in vmn o> ' V Rating Hi w- v l'v v% vs m οΛ dikd v% u Dor·,αοονν Bused on the observations of sh iuaom ;r e , vlnora m,\\ pudem m re~pmv·,, η fierrmvora s rose ,u «nt me p,uheioc> 'W .-s\ν' v \ revw d? Knee wov u\ dnung ja4OeV$on ,ot *K emm-warnm m He ,mnv o pms; ded m ne neeato u wpvt, t \ desv urnra narrative report regarding the biocompatibility of the test material is provided by the pathology observer). 6.8.3.2. Results [00304] The results indicated that the test article was non-reactive when implanted for 4 weeks (Bioreactivity Rating of 0.2) when compared to positive control Surgicel; and nonreactive (Bioreactivity Rating of 0.0) when compared to negative control High Density Polyethylene (Negative Control Plastic).
[00305] Clinical observation. Table XI below shows results of the macroscopic evaluation of the test article and control implant sites indicated no significant signs of inflammation, encapsulation, hemorrhage, necrosis, or discoloration at the 4 week time period. Some test sites and the majority of the positive control, Surgicel, were not seen macroscopic ally and serial sections were submitted for microscopic evaluation.
Table XI:
3>s itxssw $saj*«s«s£aiivis s-KSSSKirs-ws: Ssc iiMsisssss} C; * s'.-iewsv's.ssi ί Ϊ '· i'cXSX ίίίκ'ί·". X'Ss«5-,v!fi.s: i
v: ·> SKSSS&amp;8B. .5 » St* S-ftJSiKi» [SSF fi<5 Site F>XS8iS 5 ' siSiiisSs siSs":*» ο: -Λ » Jiss: ,45¾¾¾¾¾1 [00306] Implantation Site Observations (Microscopic). Table XII below shows results of the microscopic evaluation of the test article implant sites indicated no significant signs of inflammation, fibrosis, hemorrhage, necrosis, or degeneration as compared to each of the control article sites. The Bioreactivity Rating for the 4 week time period (average of three animals) was 0.2, (Cl - Surgicel) and 0.0 (C2 - Negative Control Plastic) indicating no reaction as compared to either of the control implant sites. The pathologist noted there was a moderate polymorphic and histiocytic (macrophages) infiltrate around the in situ test article that was not unexpected given the nature of the test material.
Table XII: 3 V. ίΐίί.
I ” A?»
K \ “VSMK'iS .’’•'Ness&amp;'.iK ·«*! Λ rtyfc A&amp;s&amp;a: 5iv;s< 3.0
AmsttaKi :'svs*· ^Ss.wsr*<“ Ό
Viifc&amp;si V. -«: .V «i.»·'·'"'s 4
Vs.iia.iS ArasT· ίΛΐΐ'! IV-v A'«f'. Avw.igy .$ V.®r¥)" &amp;S
Assift:4 S«sw i Arwage ΐί-ίί Sttere - A rai ses <'i « i> 3 ♦ t&amp;sssl
Table XII: 4 W«?k fca^festsskis
Ϊ » r**s Sss C i " Sis^scal <"{:VviS5'.< C««&amp; . ^ >
AtSSXi: Τί'5 ν·.ί»ίΑ'Λ>·.·χ^νϊ'»: : S ΛϊϊϋΛϋΐ ί". i Vis? >Λ' «"ijsr4^ * -'..' . X s>:i'.'.cy ( .: V;» i. -'_\SS'>>“ ': -·-' .' > .ViHSSAi Visf< (Awssgi r^Vsis -Λν«ϊ»^ί. i Swire}»· ·&amp;*
Aasssszl Vviv· f -¾ «s'*^ issi Vi.tf - jft'f 2 Si's*4) ' < Wd Si x&amp;v.bssx·, <S B;i«'s-;’S'-~“ *«** *' .¾ MS:' 1Β.Ϊ.Β Si 72 < i- : i 2-i
Table XII: i^AsaVt: 4 Wtt&amp;
T'V?K'! S:i¥
Ci. Λ is*i$SS:??J
< '. * ''«*$fcA\i &amp;£&amp;3>ίκ>χ?. K\rtV‘VM****j'vi>· * V. X* % S‘\^J
c^smsJ ;>v 3 A
Aw»x\X' · v $.$. Α*&amp;$$ ¢3 &amp;^fc&amp;x«rag$*** <· .4
^«axS Vw'*·! A\V.·-.*§·»· iW:χήίΥ λίνιγϋ \\i«S>.' vJ \ίΧο·<·: As>ι .<£>· Γ*·ϊ ^ίχν·. Aoxx^Cl&amp;'x-ix-it·'·' ·ν:ϊ
* cfS^X^>A*)XV *' Α’<>χ:&amp; ΑχήΆ ^Χχ-υ α cs ΑίΧίννίί ΧχΥ XV.Υ>'00 ΐ·.ν' ίΧ Λϊ^ΐΟΧ ^ ·*Α .«··?
,ΧκϊΧ ^s >* ΧΧ'Ϊ " &amp;S X I ·* V ίνϊ«4Μ« y^f«c¥LShi5 Sa&amp;ss " * ϊ ν "V·.. K<o*rS3«ss 6.8.4 Intracutaneous Injection Test - ISO 10993-10 [00307] USP 0.9% Sodium Chloride for Injection (NaCl) and Cottonseed Oil (CSO) extracts of the test article were evaluated for their potential to produce irritation after intracutaneous injection in New Zealand White rabbits. The test article sites did not show a significantly greater biological reaction than the sites injected with the control article. Based on the criteria of the protocol, the test article is considered a negligible irritant and meets the requirements of the ISO 10993-10 guidelines. Results are shown below in Table XIII.
Table X!ft: l«fraeura«e»iis Test Skit? Reset ion Scores NaO Extrae* '' ψ 1 :τίϊ.::.V SOr :.,-1-::. --i i.:;:::-:: Uv: O-.:ΐίκήϊίι.
SiKiSS: ίΐ'Γ TjSSV^fUcfsS -Sift: i&amp;pgmi&amp;fearHetne. l! %«ιΛ ·. . ' ll I II: 1 V * 1, 1. x 1. t ' , ' CSO F.Uraei
- ιΟΟ^ΐίξΡ*»::»!, iKif li$$d!V>:
iHi:l!sseS.3isia OliS ί Μνχΐα Ο::::'::·,: :V>ί (:>;;;;.ιγ··ί ί; '> 'Λ>Κ'!»ί Η 0 ! „,S! VlMi' i>:> < ' " Λ ! ' 1 ' < "V ίί Υ. V >»* ‘1 it' ϊ:3Υ--Οί^5&amp;:0«0: ΟΟΥ-Υν:':':;:;. T : Ο::::.: C - 0::1:::-.::. Ssg®· Λ' 'i>wa^Sil.; MisS«. ϊ§^ε®:-'':: s>^'i&amp;!ssisa.: |35 iws :is4eits3s: ^a-e;«>s:-^:!v«l!s^ :fey;: '12:. ., , p. sriss^tsi'S:: 3' 6.8,5 RTsuman Msxnidzatkm Test - ISO 10993-10 |808ftt| * >Ps C ^ VxlinnC"* ~ c {o 1 - e e - ,\ ' ! >,a < 0- d < " «€""*'< .- . ."'the ΐΟΌ eHO k' I, K'itOCi 00 ' nuOOu'i,' u\t. 5 >'0 Οί tbit 0} O, S pn., ..} di.' . oefooye iU°o sensitization).. following an Induction phase, Therefore. as defined by the sett ing system of Kh-ofTirt·:, fid" i- a (bade ! ren* non and die test nriuOe s'->.1 i.wf:.\1 ,s\ h<onii. -\e,il dk'fgen:'. poienhak Bo-ed on the evdeos of du. pro locok <>ade i .-.on-hi .onion one i- not considered 'i^n'vunt arvHh' * -, .ηηΆ' ovct-'hc K'pu" 'oven- ,d the ISO I'W: r g.niVe'V·· ? eir* are .shown bdow in Table XIV. 1 able \iV-
Skis? Examination Data
Tte Siist (ijiseita .«si ttempmsd- basest supea tiw piaoiff-a·®. «$η·*ι»«»ί*>ΐί··Φίχ^Λ· 7- j ¢)03091 AH publications, patents and patent applications cited in this specification are herein incorporated by re science as si each individual pidnieetioo or paiesn application were specifically ss'd ndn so: fl\ seer.etch Ό tv 'hvorponoeb e> s c-e'e-'e.. eh hoi eh she Mfeymog o\ emo-a··. been described in some detail by way of Illustration and example for purposes of clarity of understanding, it voll be readily apparent to those of ordinary ski!; in the art sir hgh- of the teachings of this invention that certain changes and modifications may he made thereto without departing from the spirit or scope of the appended claims.
[00310] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated feature but not to preclude the presence or addition of further features in various embodiments of the invention.
[00311] It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

Claims (44)

  1. The claims defining the invention are as follows:
    1. A method for treating a disease or a condition associated with a bacterial imbalance in a subject in need thereof, comprising topically administering a composition comprising shortened fibers of poly-β-Ι—►4-N-acetylglucosamine (“sNAG nanofibers”) to a subject, wherein the sNAG nanofibers are less than 10 μηι in length, wherein the sNAG nanofibers comprise 70% or more than 70% of N-acetylglucosamine monosaccharides, and wherein the sNAG nanofibers do not have an effect, or substantially have no effect, on bacterial growth or survival of Staphylococcus aureus bacterial cultures in vitro.
  2. 2. A method for treating a disease or a condition associated with a bacterial imbalance in a subject in need thereof, comprising topically administering a composition comprising shortened fibers of poly-β-Ι —>4-N-acetylglucosamine (“sNAG nanofibers”) to a subject, wherein the sNAG nanofibers are from about 1 μηι to less than 10 μηι in length, wherein the sNAG nanofibers comprise 70% or more than 70% of the N-acetylglucosamine monosaccharides, and wherein the sNAG nanofibers do not have an effect, or substantially have no effect, on bacterial growth or survival of Staphylococcus aureus bacterial cultures in vitro.
  3. 3. Use of a composition comprising shortened fibers of poly-β-Ι—►4-N-acetylglucosamine (“sNAG nanofibers”) in the manufacture of a medicament for treating a disease or a condition associated with a bacterial imbalance in a subject in need thereof, said treating comprising topically administering the medicament to the subject, wherein the sNAG nanofibers are less than 10 μηι in length, wherein the sNAG nanofibers comprise 70% or more than 70% of N-acetylglucosamine monosaccharides, and wherein the sNAG nanofibers do not have an effect, or substantially have no effect, on bacterial growth or survival of Staphylococcus aureus bacterial cultures in vitro.
  4. 4. Use of a composition comprising shortened fibers of poly-β-Ι—►4-N-acetylglucosamine (“sNAG nanofibers”) in the manufacture of a medicament for treating a disease or a condition associated with a bacterial imbalance in a subject in need thereof, said treating comprising topically administering the medicament to the subject, wherein the sNAG nanofibers are from about 1 μηι to less than 10 μηι in length, wherein the sNAG nanofibers comprise 70% or more than 70% of the N-acetylglucosamine monosaccharides, and wherein the sNAG nanofibers do not have an effect, or substantially have no effect, on bacterial growth or survival of Staphylococcus aureus bacterial cultures in vitro.
  5. 5. The method or use of any one of claims 1 to 4, wherein the sNAG nanofibers have the microstructure of non-irradiated poly-β-Ι —>4-N-acctylglucosaminc fibers that are about 100 μ m in length.
  6. 6. The method or use of any one of claims 1 to 4, wherein the sNAG nanofibers have the microstructure of non-irradiated microalgal poly-β-Ι—>-4-N-acetylglucosamine fibers.
  7. 7. The method or use of any one of claims 1 to 4, wherein the infrared spectrum of the sNAG nanofibers is equivalent or substantially equivalent to that of non-irradiated poly-β-1—>-4-N-acetylglucosamine fibers that are: (i) about 100 μ m in length, and/or (ii) derived from micro algae.
  8. 8. The method or use of any one of claims 1 to 4, wherein the chemical and physical structure of the sNAG nanofibers is the same or substantially the same as the chemical and physical structure of non-irradiated poly-β-Ι —>-4-N-acetylglucosamine fibers as determined by infrared spectrum, elemental assay and scanning electron microscopic analyses, wherein the non-irradiated poly-β-Ι —>-4-N-acetylglucosamine fibers are: (i) about 100 μm in length, and/or (ii) derived from microalgae.
  9. 9. The method or use of any one of claims 1 to 8, wherein the subject is a subject diagnosed with the disease or condition, or displaying one or more symptoms of the disease or condition.
  10. 10. The method or use of any one of claims 1 to 9, wherein the bacterial imbalance is an imbalance in bacterial microbiota.
  11. 11. The method or use of any one of claims 1 to 9, wherein the bacterial imbalance is an abnormal or altered bacterial microbiota.
  12. 12. The method or use of any one of claims 1 to 11, wherein disease or condition is an intestinal condition.
  13. 13. The method or use of claim 12, wherein the subject has an intestinal bacterial microbiota that differs from intestinal bacterial microbiota in a subject or subjects with no symptoms of the intestinal condition.
  14. 14. The method or use of any one of claims 1 to 11, wherein disease or condition is a skin condition.
  15. 15. The method or use of claim 14, wherein the subject has a skin bacterial microbiota that differs from skin bacterial microbiota in a subject or subjects with no symptoms of the skin condition.
  16. 16. The method or use of any one of claims 1 to 15, wherein the sNAG nanofibers are in an amount effective to achieve one or more of the following: (i) reduce the severity of the disease or condition, or one or more symptoms thereof, (ii) reduce the duration of the disease or condition, one or more symptoms thereof, and (iii) prevent the progression of the disease or condition, or one or more symptoms thereof.
  17. 17. The method of any one of claims 1 to 15, wherein the sNAG nanofibers are in an amount effective to achieve one or more of the following: (i) reduce or eliminate a bacterial cell population, and (ii) prevent an increase in the number of bacteria in a bacterial cell population.
  18. 18. The method or use of any one of claims 1 to 17, wherein the bacterial imbalance is associated with a bacteria resistant to one or more antibiotics.
  19. 19. The method or use of any one of claims 1 to 18, wherein the subject is a human.
  20. 20. The method or use of any one of claims 1 to 19, wherein the sNAG nanofibers fibers are less than 10 μηι in average length.
  21. 21. The method or use of any one of claims 1 to 19, wherein the sNAG nanofibers fibers are less than 8 μηι in average length.
  22. 22. The method or use of any one of claims 1 to 19, wherein the sNAG nanofibers fibers are between 4 μηι and 7 μηι in average length.
  23. 23. The method or use of any one of claims 1 to 22, wherein the majority of the sNAG nanofibers fibers are at least 1 μηι in length.
  24. 24. The method or use of any one of claims 1 to 23, wherein the length of the fibers is determined by scanning electron microscopic (SEM) analysis.
  25. 25. The method or use of any one of claims 1 to 24, wherein the poly-β-Ι—>-4-N-acetylglucosamine is a microalgal poly-β-Ι—>-4-N-acetylglucosamine.
  26. 26. The method or use of any one of claims 1 to 24, wherein the poly-β-Ι—>-4-N-acetylglucosamine is not a crustacean poly-β-Ι —>-4-N-acetylglucosamine.
  27. 27. The method or use of any one of claims 1 to 26, wherein the sNAG nanofibers comprise more than 80% of the N-acetylglucosamine monosaccharides.
  28. 28. The method or use of any one of claims 1 to 26, wherein the sNAG nanofibers comprise more than 90% of the N-acetylglucosamine monosaccharides.
  29. 29. The method or use of any one of claims 1 to 26, wherein the sNAG nanofibers comprise 100% of the N-acetylglucosamine monosaccharides.
  30. 30. The method or use of any one of claims 1 to 29, wherein the sNAG nanofibers are non-reactive when tested in an elution test, an intramuscular implantation test, an intracutaneous test, and/or a systemic test.
  31. 31. The method or use of any one of claims 1 to 30, wherein the composition does not comprise an additional anti-bacterial agent.
  32. 32. The method or use of any one of claims 1 to 31, wherein the composition is not administered in conjunction with an anti-bacterial agent.
  33. 33. The method or use of any one of claims 1 to 32, wherein the composition is not administered with an immunomodulator.
  34. 34. The method or use of any one of claims 1 to 33, wherein the composition does not comprise an additional therapy which is encapsulated, immobilized or formulated in the sNAG nanofibers.
  35. 35. The method or use of any one of claims 1 to 34, wherein the composition does not comprise an additional active ingredient.
  36. 36. The method or use of any one of claims 1 to 35, wherein the composition is not administered in conjunction with another therapy.
  37. 37. The method or use of any one of claims 1 to 36, wherein the composition is formulated as a cream, a gel, a liquid, a suspension, an ointment, a membrane, a powder, a spray, or a suppository.
  38. 38. The method or use of claim 12, wherein the composition is formulated as a suppository.
  39. 39. The method or use of any one of claims 1 to 38, wherein the sNAG nanofibers have been produced by irradiation of the poly-β-Ι—►4-N-acetylglucosamine fibers.
  40. 40. The method or use of claim 39, wherein (i) the poly-N-acetylglucosamine fibers have been irradiated in the form of dry fibers, a dry fiber membrane or a dry lyophilized material at 500-2,000 kgy, or (ii) the poly-N-acetylglucosamine fibers have been irradiated in the form of a suspension, a slurry or a wet cake at 100-500 kgy.
  41. 41. The method or use of claim 39, wherein (i) the poly-N-acetylglucosamine fibers have been irradiated by gamma irradiation in the form of dry fibers, a dry fiber membrane or a dry lyophilized material at 750-1,250 kgy, or (ii) the poly-N-acetylglucosamine fibers have been irradiated by gamma irradiation in the form of a suspension, a slurry or a wet cake at 150-250 kgy.
  42. 42. The method or use of any one of claims 39 to 41, wherein the irradiation is gamma irradiation.
  43. 43. The method or use of any one of claims 1 to 42, wherein the composition comprises 0.2 to 20 mg/cm2 of the sNAG nanofibers per dose or application of the composition.
  44. 44. The method or use of any one of claims 1 to 43, wherein the composition increases metabolic rate of serum-starved human umbilical cord vein endothelial cells in a MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and/or does not rescue apoptosis of serum-starved human umbilical cord vein endothelial cells in a trypan blue exclusion test.
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