AU2019202473A1 - Methods And Compositions For Disrupting Biofilm Utilizing Chitosan-Derivative Compounds - Google Patents

Methods And Compositions For Disrupting Biofilm Utilizing Chitosan-Derivative Compounds Download PDF

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AU2019202473A1
AU2019202473A1 AU2019202473A AU2019202473A AU2019202473A1 AU 2019202473 A1 AU2019202473 A1 AU 2019202473A1 AU 2019202473 A AU2019202473 A AU 2019202473A AU 2019202473 A AU2019202473 A AU 2019202473A AU 2019202473 A1 AU2019202473 A1 AU 2019202473A1
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amino
substituents
group
chitosan
alkyl substituted
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AU2019202473A
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Shenda Baker
Stacy Townsend
William P. Wiesmann
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Synedgen Inc
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Synedgen Inc
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Priority claimed from AU2010289417A external-priority patent/AU2010289417B2/en
Priority claimed from AU2015255317A external-priority patent/AU2015255317B2/en
Application filed by Synedgen Inc filed Critical Synedgen Inc
Priority to AU2019202473A priority Critical patent/AU2019202473A1/en
Publication of AU2019202473A1 publication Critical patent/AU2019202473A1/en
Priority to AU2020277272A priority patent/AU2020277272A1/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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Abstract

Abstract Described herein are methods of disrupting (e.g., reducing the viscosity of, or dissolving) a preformed biofilm in a subject, the method comprising: administering to the subject an effective amount of a composition comprising a soluble chitosan or derivatized chitosan wherein the soluble chitosan or derivatized chitosan when administered contacts the preformed biofilm, thereby disrupting (e.g., reducing the viscosity of, or dissolving) the preformed biofilm.

Description

METHODS AND COMPOSITIONS FOR DISRUPTING BIOFILM UTILIZING CHITOSAN-DERIVATIVE COMPOUNDS
Cross Reference to Related Applications
This application is a divisional of Australian Application No. 2017203312, filed May 17, 2017. Australian Application No. 2017203312 is a divisional of Australian Application No. 2015255317, filed November 13, 2015. Australian Application No. 2015255317 is a divisional of Australian Application No. 2010289417, filed September 2, 2010. All of the above applications claim priority to US Application Serial No. 61/239,180, filed on September 2, 2009. All of the above applications are hereby incorporated by reference in their entirety.
Field of the Invention
The invention relates to soluble chitosans or derivatized chitosans and their use to disrupt or dissolve a biofilm in a subject.
Background
Microbial populations are present in body cavities including the mouth, ear, nose, throat, sinuses, respiratory tree including the lungs, gastrointestinal tract, skin, or wound. Unbalanced populations of bacteria can cause increases in particular microbial populations that are no longer controlled or in balance with the body. These conditions can occur through e.g., pathogenic infections, compromised immune system, and side effects from antibiotics. Pathogenic bacteria can adhere to a host or a surface, colonizing it to form biofilms.
Summary of the Invention
Compositions comprising soluble chitosans and derivatized chitosans (e.g., liquid, solid particulate and semisolid compositions) and related methods of use are described herein. In these embodiments, the derivatized chitosans are water soluble. Exemplary methods using the compositions described herein include, for example, methods of disrupting preformed biofilm in a subject; methods of preventing or delaying the time to the formation of biofilm in the mouth, ear, nose, throat, sinuses, eyes, respiratory system, and/or digestive tract of a subject; methods of treating or preventing complications of cystic fibrosis in a subject; methods of treating or preventing pulmonary infections in a subject; methods of treating or preventing gastrointestinal tract infections in a subject; and methods of treating a wound, e.g., treating or preventing wound infection in a subject. In some embodiments, a composition described herein can result in the clumping of bacteria, for example, to a
WO 2011/028967
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2019202473 09 Apr 2019 chitosan derivative. The clumped bacteria can be discarded, for example, expirated or expectorated by a subject, or can be ingested.
In one aspect, the invention features a method of disrupting (e.g., reducing the viscosity of, or dissolving) a preformed biofllm in a subject, the method comprising: administering to the subject an effective amount of a composition comprising a soluble derivatized chitosan wherein the soluble derivatized chitosan when administered contacts the preformed biofilm, thereby disrupting (e.g., reducing the viscosity of, or dissolving) the preformed biofilm.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99%, compared to the biofilm that has not been contacted with the composition.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 1, 2, 5, 10, 50, 100, 200, 500, or 1000 fold, compared to the biofilm that has not been contacted with the composition.
In one embodiment, the bio film is partially dissolved, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.999% of the biofilm is dissolved, compared to the biofilm that has not been contacted with the composition.
In one embodiment, the method further comprises administering an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic surfactant). In one embodiment, the second agent comprises another chitosan derivative, e.g., another chitosan derivative described herein.
In one embodiment, the second agent is administered in a dosage to achieve a synergistic effect.
In one embodiment, the second agent is administered together with the soluble derivatized chitosan (e.g., in the same composition or dosage form).
In one embodiment, the method further comprises administering an antibiotic, anti-inflammatory, or mucolytic (expectorant) compound to a subject in conjunction with, prior to or subsequent to the administration of the composition.
In one embodiment, the antibiotic, anti-inflammatory, or mucolytic (expectorant) compound is administered in a dosage to achieve a synergistic effect.
In one embodiment, the biofilm is in the respiratory system (e.g., airways, lung, throat, nose), ear, mouth (e.g., tooth, tongue, mucosal surface), digestive system (e.g., stomach, small intestine, large intestine, colon, bowel), skin, or wound.
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In one embodiment, the subject has a complication of cystic fibrosis (e.g., lung infection or respiratory congestion), pneumonia, oral disease (e.g., gingivitis, dental caries, or halitosis), ear infection, throat infection, sinusitis, a complication in immunocompromised patient (e.g., lung infection or respiratory congestion), gastrointestinal infection, enteritis, or a symptom thereof.
In one embodiment, the subject is infected with bacteria listed in Table 1,2, 3, or 4.
In one embodiment, the composition is administered topically, orally, or enterally.
In one embodiment, the composition is administered by inhalation (e.g., nebulizer, nasal spray, nasal swab, or sinus spray).
In one embodiment, the effective amount is therapeutically effective amount.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 5.0 to about pH 6.0, e.g., in wounds or duodenum.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 2.0 to about pH 4.0, e.g., in stomach.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 8.0 to about pH 8.5, e.g., in lower part of the gastrointestinal tract.
In one embodiment, the derivatized chitosan comprises a chitosan of the following formula (I):
OH / OH \ OH
H0''V~' Lo'^V'
HO-V r ho-V Λυ HO-V
NH NH/n NH I
R1 7 R1
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
WO 2011/028967
PCT/US2010/047758
Figure AU2019202473A1_D0001
2019202473 09 Apr 2019 formula (II) or Rl, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, C>-Cf, alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R* substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (II).
In one embodiment, between 4-30% of R1 substituents are a group of formula (II).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
In one embodiment, R2 is amino and R3 is an arginine side chain.
Figure AU2019202473A1_D0002
In one embodiment, R2 is amino and RJ is a lysine side chain.
In one embodiment, R1 is selected from one of the following:
WO 2011/028967
PCT/US2010/047758
Figure AU2019202473A1_D0003
2019202473 09 Apr 2019
Figure AU2019202473A1_D0004
In one embodiment, R2 is amino and R3 is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0005
k/UWV
Figure AU2019202473A1_D0006
Figure AU2019202473A1_D0007
In one embodiment, at least 1% of R1 substituents are selected from one of the
Figure AU2019202473A1_D0008
Figure AU2019202473A1_D0009
Figure AU2019202473A1_D0010
In one embodiment, R2 is amino and RJ is a substituted Ci-Q alkyl.
In one embodiment, R3 is C:-(% alkyl substituted with an amino group.
In one embodiment, RJ is C, alkyl substituted with an amino group.
In one embodiment, RJ is C? alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, RJ is C4 alkyl substituted with an amino group.
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In one embodiment, RJ is C5 alkyl substituted with an amino group.
In one embodiment, RJ is C6 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
2019202473 09 Apr 2019
Figure AU2019202473A1_D0011
Figure AU2019202473A1_D0012
Figure AU2019202473A1_D0013
Figure AU2019202473A1_D0014
In one embodiment, R is Cj-Ci alkyl substituted with a guanidino group.
In one embodiment, RJ is Ci alkyl substituted with a guanidino group. In one embodiment, R3 is C2 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, RJ is C4 alkyl substituted with a guanidino group. In one embodiment, R3 is C5 alkyl substituted with a guanidino group. In one embodiment, R3 is CR alkyl substituted with a guanidino group. In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0015
Figure AU2019202473A1_D0016
In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
In one embodiment, the nitrogen protecting group is Zert-butyloxycarbonyl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
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In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boe.
In one embodiment, R2 is amino.
In one embodiment, R2 is hydrogen and R3 is amino.
In one embodiment, R2 is hydrogen and R3 is guanidino.
In one embodiment, R2 is hydrogen and R3 is a substituted Cj-C6 alkyl.
In one embodiment, RJ is C|-C6 alkyl substituted with an amino group.
In one embodiment, RJ is Ci alkyl substituted with an amino group.
In one embodiment, RJ is C2 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
Figure AU2019202473A1_D0017
In one embodiment, R3 is Ci-Q alkyl substituted with a guanidino group. In one embodiment, R3 is C| alkyl substituted with a guanidino group.
In one embodiment, R3 is C2 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, R3 is C5 alkyl substituted with a guanidino group.
In one embodiment, R3 is C6 alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
WO 2011/028967
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Figure AU2019202473A1_D0018
Figure AU2019202473A1_D0019
Figure AU2019202473A1_D0020
Figure AU2019202473A1_D0021
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, tire molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and 30%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
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In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the functionalized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a method of preventing (e.g., reducing the degree of) the formation of a biofilm in the respiratory system of a subject, the method comprising: administering to the respiratory system of the subject an effective amount of a composition comprising a soluble derivatized chitosan, thereby preventing or treating the formation of a biofilm in the respiratory system of the subject.
In one embodiment, the method further comprises administering an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic surfactant).
In one embodiment, the second agent is administered in a dosage to achieve a synergistic effect.
In one embodiment, the method further comprises administering an antibiotic, anti-inflammatory, or mucolytic (expectorant) compound to a subject in conjunction with or subsequent to the administration of the composition.
In one embodiment, the method further comprises administering an antibiotic, anti-inflammatory, or mucolytic (expectorant) compound to a subject in conjunction with, prior to or subsequent to the administration of the composition.
In one embodiment, the subject is infected with planktonic bacteria or infected with bacteria listed in Table 3.
In one embodiment, the subject is diagnosed with bacterial pneumonia.
In one embodiment, the composition reduces the viability of planktonic bacteria.
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In one embodiment, the composition reduces colonization of the planktonic bacteria.
In one embodiment, the composition is administered by inhalation (e.g., nebulizer, nasal spray, or sinus spray).
In one embodiment, the effective amount is therapeutically effective amount.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan comprises a chitosan of the following formula (I):
OH / OH \ OH
. ^-0 _^-0
HO \ LO \ J-0 \
HO-V- V HO—-v- 7 ho-V
NH NH . 1 'Π NH I
R1 R1 R1
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
Figure AU2019202473A1_D0022
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidmo, C|-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
io
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In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (II).
In one embodiment, between 4-30% of R1 substituents are a group of formula (II).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
In one embodiment, R2 is amino and R3 is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0023
· · 3 ’
In one embodiment, R is amino and R is a lysine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0024
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0025
In one embodiment, at least 1% of R1 substituents are selected from one of the following:
II
WO 2011/028967
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Figure AU2019202473A1_D0026
Figure AU2019202473A1_D0027
In one embodiment, R2 is amino and R3 is a substituted Cj-C6 alkyl.
In one embodiment, RJ is C|-Cg alkyl substituted with an amino group. In one embodiment, R3 is C| alkyl substituted with an amino group.
In one embodiment, RJ is C? alkyl substituted with an amino group.
In one embodiment, RJ is C3 alkyl substituted with an amino group.
In one embodiment, RJ is C4 alkyl substituted with an amino group.
In one embodiment, RJ is C5 alkyl substituted with an amino group.
In one embodiment, R1 is C(, alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0028
Figure AU2019202473A1_D0029
nh2
Figure AU2019202473A1_D0030
Figure AU2019202473A1_D0031
Figure AU2019202473A1_D0032
In one embodiment, RJ is C|-C(, alkyl substituted with a guanidino group.
In one embodiment, R ’ is C i alkyl substituted with a guanidino group.
In one embodiment, RJ is C2 alkyl substituted with a guanidino group.
In one embodiment, R ’ is C3 alkyl substituted with a guanidino group.
In one embodiment, RJ is C4 alkyl substituted with a guanidino group.
In one embodiment, R’ is C> alkyl substituted with a guanidino group.
In one embodiment, R’ is C6 alkyl substituted with a guanidino group.
WO 2011/028967
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In one embodiment, R1 is selected from one of the following:
2019202473 09 Apr 2019
Figure AU2019202473A1_D0033
In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
In one embodiment, the nitrogen protecting group is Ze/7-butyloxycarbonyl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
In one embodiment, R is amino.
In one embodiment, R2 is hydrogen and R1 is amino.
In one embodiment, R2 is hydrogen and R° is guanidino.
In one embodiment, R2 is hydrogen and RJ is a substituted C|-C6 alkyl.
In one embodiment, R3 is C|-C6 alkyl substituted with an amino group.
In one embodiment, RJ is C| alkyl substituted with an amino group.
In one embodiment, RJ is C2 alkyl substituted with an amino group.
In one embodiment, R’ is C3 alkyl substituted with an amino group.
In one embodiment, RJ is C.i alkyl substituted with an amino group.
In one embodiment, R’ is C5 alkyl substituted with an amino group.
In one embodiment, RJ is C6 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
WO 2011/028967
PCI7US2010/047758
2019202473 09 Apr 2019
Figure AU2019202473A1_D0034
In one embodiment, R3 is Cj-C6 alkyl substituted with a guanidino group.
In one embodiment, RJ is C, alkyl substituted with a guanidino group.
In one embodiment, R1 is C- alkyl substituted with a guanidino group. In one embodiment, R’ is C- alkyl substituted with a guanidino group. In one embodiment, R3 is C4 alkyl substituted with a guanidino group. In one embodiment, R3 is C5 alkyl substituted with a guanidino group. In one embodiment, RJ is C6 alkyl substituted with a guanidino group. In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0035
Figure AU2019202473A1_D0036
In one embodiment, at least 25% of R! substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of fonnula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
WO 2011/028967
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In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment,'the chitosan is functionalized at between 20% and 30%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
In one embodiment, the poly dispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1 %, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a method of preventing (e.g., reducing the degree of) the formation of a biofilm in the mouth, nose, throat, ear, skin, wound, or digestive tract of a subject, the method comprising: administering to the mouth, nose, ear or digestive tract of the subject an effective amount of a composition comprising a soluble derivatized chitosan, thereby preventing the formation of a biofilm in the mouth, nose, throat, ear, skin, wound, or digestive tract of the subject.
In one embodiment, the method further comprises administering an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic
WO 2011/028967
PCT/US2010/047758
2019202473 09 Apr 2019 surfactant). In one embodiment, the second agent comprises another chitosan derivative, e.g., another chitosan derivative described herein.
In one embodiment, the second agent is administered in a dosage to achieve a synergistic effect.
In one embodiment, the method further comprises administering an antibiotic, anti-inflammatory, or mucolytic (expectorant) compound to a subject in conjunction with or subsequent to the administration of the composition.
In one embodiment, the method further comprises administering an antibiotic, anti-inflammatory, or mucolytic (expectorant) compound to a subject in conjunction with, prior to or subsequent to the administration of the composition.
In one embodiment, the subject is infected with planktonic bacteria.
In one embodiment, the subject is infected with bacteria listed in Tables 1 and/or 2.
In one embodiment, the composition reduces the viability of planktonic bacteria.
In one embodiment, the composition reduces colonization of the planktonic bacteria.
In one embodiment, the composition is administered topically, orally, enterally, or by inhalation (e.g., nebulizer, nasal spray, or sinus spray).
In one embodiment, the effective amount is therapeutically effective amount.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan comprises a chitosan of the following formula (I):
Figure AU2019202473A1_D0037
formula (I) wherein:
n is an integer between 20 and 6000; and
WO 2011/028967
PCT/US2010/047758 each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
2019202473 09 Apr 2019
Figure AU2019202473A1_D0038
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, C]-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (II).
In one embodiment, between 4-30% of R1 substituents are a group of formula (II).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
In one embodiment, R2 is amino and R3 is an arginine side chain.
Figure AU2019202473A1_D0039
In one embodiment, R2 is amino and RJ is a lysine side chain.
In one embodiment, R1 is selected from one of the following:
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Figure AU2019202473A1_D0040
2019202473 09 Apr 2019
Figure AU2019202473A1_D0041
In one embodiment, R2 is amino and R3 is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0042
Figure AU2019202473A1_D0043
In one embodiment, at least 1% of R1 substituents are selected from one of the
Figure AU2019202473A1_D0044
JWW
Figure AU2019202473A1_D0045
Figure AU2019202473A1_D0046
and at least 1 % of R1 substituents are selected from the following:
Figure AU2019202473A1_D0047
Figure AU2019202473A1_D0048
In one embodiment, R2 is amino and R3 is a substituted C|-C(, alkyl. In one embodiment, R3 is C|-C6 alkyl substituted with an amino group.
In one embodiment, RJ is Ci alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, R3 is Cj alkyl substituted with an amino group.
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In one embodiment, RJ is C5 alkyl substituted with an amino group.
In one embodiment, R3 is C6 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
2019202473 09 Apr 2019
Figure AU2019202473A1_D0049
In one embodiment, R3 is C|-C6 alkyl substituted with a guanidino group. In one embodiment, R3 is <% alkyl substituted with a guanidino group.
In one embodiment, R3 is C? alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, RJ is C5 alkyl substituted with a guanidino group.
In one embodiment, R3 is Cf, alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0050
In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
In one embodiment, the nitrogen protecting group is tert-butyloxycarbonyl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
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In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
In one embodiment, R2 is amino.
In one embodiment, R2 is hydrogen and R3 is amino.
In one embodiment, R2 is hydrogen and R3 is guanidino.
In one embodiment, R2 is hydrogen and R3 is a substituted Ci-C^ alkyl.
In one embodiment, R3 is Ci-Cg alkyl substituted with an amino group.
In one embodiment, R3 is Ci alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, RJ is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, R3 is C6 alkyl substituted with an amino group.
In one embodiment, R jwuv
Figure AU2019202473A1_D0051
Figure AU2019202473A1_D0052
is selected from one of the following:
Figure AU2019202473A1_D0053
In one embodiment, R3 is C|-C6 alkyl substituted with a guanidino group. In one embodiment, R’ is Cj alkyl substituted with a guanidino group.
In one embodiment, R3 is C2 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, RJ is C4 alkyl substituted with a guanidino group.
In one embodiment, R3 is C5 alkyl substituted with a guanidino group.
In one embodiment, RJ is C6 alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
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Figure AU2019202473A1_D0054
Figure AU2019202473A1_D0055
Figure AU2019202473A1_D0056
Figure AU2019202473A1_D0057
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and 30%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
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In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the functionalized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1 %, or is substantially free, of a chitosan polymer wherein one or more of the nitro gen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a method of treating or preventing (e.g., reducing the degree of) a complication of cystic fibrosis in a subject, the method comprising: administering to the subject an effective amount of a composition comprising a soluble chitosan or derivatized chitosan, thereby treating or preventing a complication of cystic fibrosis.
In one embodiment, the complication of cystic fibrosis is lung infection or respiratory congestion.
In one embodiment, the subject has a bacterial infection, e.g., bacteria list in Table 3, e.g., Pseudomonas aeruginosa.
In one embodiment, the subject comprises at least one biofilm.
In one embodiment, exopolysaccharides (EPS) of the biofilm comprises alginate and/or polysaccharide synthesis locus (Psi) (e.g., in Pseudomonas aeruginosa)·, acidic polysaccharide (e.g., in Burkholderia cepacia)·, collanic acid, poly-β-1,6-GlcNAc (PGA) or cellulose (e.g., in Escherichia coli): cellulose (e.g., in Salmonella): N-acetylglucosamine (GlcNAc), D-mannose, 6-deoxy-D-galactose and D-galactose (e.g., in Vibrio cholerae): polysaccharide intercellular adhesion (PIA) (e.g., in Staphylococcus): glucose and mannose rich component (e.g., in Bacillus subtilis): mannose polysaccharide (e.g., in Prevotella intermedia, Capnocytophaga ochracea, or Prevotella nigrescens).
In one embodiment, the biofilm is associated with actin and/or DNA released from bacteria or cells such as neutrophils.
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In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99%, compared to the biofilm that has not been contacted with the composition.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 1, 2, 5, 10, 50, 100, 200, 500, or 1000 fold, compared to the biofilm that has not been contacted with the composition.
In one embodiment, the bio film is partially dissolved, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.999% of the biofilm is dissolved, compared to the biofilm that has not been contacted with the composition.
In one embodiment, the method further comprises administering an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic surfactant). In one embodiment, the second agent comprises another chitosan derivative, e.g., another chitosan derivative described herein.
In one embodiment, the second agent is administered in a dosage to achieve a synergistic effect.
In one embodiment, the second agent is administered together with the soluble derivatized chitosan (e.g., in the same composition or dosage form).
In one embodiment, the method further comprises administering an antibiotic, anti-inflammatory, or mucolytic (expectorant) compound to a subject in conjunction with, prior to or subsequent to the administration of the composition.
In one embodiment, the antibiotic, anti-inflammatory, or mucolytic (expectorant) compound is administered in a dosage to achieve a synergistic effect.
In one embodiment, the biofilm is in the respiratory system (e.g., airways, lung, throat, nose).
In one embodiment, the subject is infected with planktonic bacteria.
In one embodiment, the composition reduces the viability of planktonic bacteria.
In one embodiment, the composition reduces colonization of the planktonic bacteria.
In one embodiment, the planktonic bacteria are clumped, e.g., to facilitate removal by expectoration, lavage, or chest percussion.
In one embodiment, the composition is administered by inhalation (e.g., nebulizer, nasal spray, nasal swab, or sinus spray).
In one embodiment, the effective amount is therapeutically effective amount.
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In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan comprises a chitosan of the
Figure AU2019202473A1_D0058
wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
Figure AU2019202473A1_D0059
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, C|-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (11).
In one embodiment, between 4-30% of R1 substituents are a group of formula (Π).
WO 2011/028967
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In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
In one embodiment, R2 is amino and R’ is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
2019202473 09 Apr 2019
Figure AU2019202473A1_D0060
.ΛΛΛΛΖ
Figure AU2019202473A1_D0061
Figure AU2019202473A1_D0062
In one embodiment, R2 is amino and R3 is a lysine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0063
Figure AU2019202473A1_D0064
In one embodiment, R2 is amino and R3 is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
JWW JWVW
Figure AU2019202473A1_D0065
Figure AU2019202473A1_D0066
Figure AU2019202473A1_D0067
In one embodiment, at least 1% of R1 substituents are selected from one of the
Figure AU2019202473A1_D0068
AND at least 1% of R1 substituents are selected from the following:
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Figure AU2019202473A1_D0069
In one embodiment, R2 is amino and RJ is a substituted Cj-C6alkyl.
In one embodiment, RJ is Gj-C/alkyl substituted with an amino group.
In one embodiment, RJ is C; alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, R3 is C6 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0070
In one embodiment, IU is Ci-Ce alkyl substituted with a guanidino group. In one embodiment, RJ is C, alkyl substituted with a guanidino group. In one embodiment, RJ is Ch alkyl substituted with a guanidino group.
In one embodiment, RJ is C3 alkyl substituted with a guanidino group. In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, R3 is C5 alkyl substituted with a guanidino group. In one embodiment, R3 is Ci alkyl substimted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0071
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In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
In one embodiment, the nitrogen protecting group is /erZ-butyloxycarbonyl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
In one embodiment, R2 is amino.
In one embodiment, R2 is hydrogen and R3 is amino.
In one embodiment, R2 is hydrogen and R3 is guanidino.
In one embodiment, R2 is hydrogen and RJ is a substituted C|-C6 alkyl.
In one embodiment, R3 is C,-C6 alkyl substituted with an amino group.
In one embodiment, R3 is Ci alkyl substituted with an amino group.
In one embodiment, R1 is C2 alkyl substituted with an amino group.
In one embodiment, RJ is C3 alkyl substituted with an amino group.
In one embodiment, RJ is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, RJ is C6 alkyl substituted with an amino group.
Figure AU2019202473A1_D0072
In one embodiment, R3 is Cj-C6 alkyl substituted with a guanidino group.
In one embodiment, R3 is C| alkyl substituted with a guanidino group.
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In one embodiment, RJ is C2 alkyl substituted with a guanidino group. In one embodiment, R3 is C3 alkyl substituted with a guanidino group. In one embodiment, R3 is C4 alkyl substituted with a guanidino group. In one embodiment, R3 is C5 alkyl substituted with a guanidino group. In one embodiment, R1 is C6 alkyl substituted with a guanidino group. In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0073
Figure AU2019202473A1_D0074
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and
30%.
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In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1 %, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a method of treating or preventing (e.g.. reducing the degree of) a gastrointestinal tract infection in a subject, the method comprising: administering to the subject an effective amount of a composition comprising a soluble derivatized chitosan, thereby treating or preventing the gastrointestinal tract infection.
In one embodiment, the gastrointestinal tract infection is noninflammatory gastroenteritis, inflammatory gastroenteritis, invasive gastroenteritis, or nectrotic or necrotizing enteritis.
In one embodiment, the subject is infected with Staphylococcus aureus, Bacillus cereits, Clostridium perfringens, Clostridium botulinum, Vibrio cholerae, Escherichia coli, Clostridium difficile. Vibrio parahemolyticus, Bacillus anthracis, Shigella sp., Salmonella sp., Campylobacter jejuni, Vibrion vulnificus, Yersinia sp., Francisella tularensis, or Helicobacter pylori.
In one embodiment, the subject is infected with bacteria listed in Table 2.
In one embodiment, the subject comprises at least one biofilm.
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In one embodiment, exopolysaccharides (EPS) of the biolim comprises alginate and/or polysaccharide synthesis locus (Psi) (e.g., in Pseudomonas aeruginosa)', acidic polysaccharide (e.g., in Burkholderia cepacia)', collanic acid, ροΙγ-β-Ι,ό-GlcNAc (PGA) or cellulose (e.g., in Escherichia colt)', cellulose (e.g., in Salmonella)·, N-acetylglucosamine (GlcNAc), D-mannose, 6-deoxy-D-galactose and D-galactose (e.g., in Vibrio cholerae)·, polysaccharide intercellular adhesion (PIA) (e.g., in Staphylococcus)·, glucose and mannose rich component (e.g., in Bacillus subtilis)', mannose polysaccharide (e.g., in Prevotella intermedia, Capnocytophaga ochracea, or Prevotella nigrescens).
In one embodiment, the biofilm is associated with actin and/or DNA released from bacteria or cells such as neutrophils.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99%, compared to the bio film that has not been contacted with the composition.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 1, 2, 5, 10, 50, 100, 200, 500, or 1000 fold, compared to the biofilm that has not been contacted with the composition.
In one embodiment, the biofilm is partially dissolved, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.999% of the biofilm is dissolved, compared to the biofilm that has not been contacted with the composition.
In one embodiment, the method further comprises administering an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic surfactant). In one embodiment, the second agent comprises another chitosan derivative, e.g., another chitosan derivative described herein.
In one embodiment, the second agent is administered in a dosage to achieve a synergistic effect.
In one embodiment, the second agent is administered together with the soluble derivatized chitosan (e.g., in the same composition or dosage form).
In one embodiment, the method further comprises administering an antibiotic, anti-inflammatory, or mucolytic (expectorant) compound to a subject in conjunction with, prior to or subsequent to the administration of the composition.
In one embodiment, the antibiotic, anti-inflammatory, or mucolytic (expectorant) compound is administered in a dosage to achieve a synergistic effect.
In one embodiment, the biofilm is in the gastrointestinal tract.
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In one embodiment, the subject is infected with planktonic bacteria.
In one embodiment, the composition reduces the viability of planktonic bacteria.
In one embodiment, the composition reduces colonization of the planktonic bacteria.
In one embodiment, the planktonic bacteria are clumped.
In one embodiment, the composition is administered orally, or enterally.
In one embodiment, the effective amount is therapeutically effective amount.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 5.0 to about pH 6.0, e.g., in duodenum.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 2.0 to about pH 4.0, e.g., in stomach.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 8.0 to about pH 8.5, e.g., in lower part of the gastrointestinal tract.
In one embodiment, the composition is active below pH 6 (e.g., in stomach and duodenum) or near or above pH 8 (e.g., in colon).
In one embodiment, the composition is active between pH 6 and pH 8.
In one embodiment, the composition is delivered e.g., by capsule, time release capsule, pH release capsule, as a powder dissolved in the digestive tract, or lozenge.
In one embodiment, the composition is delivered by mixing in food, or dissolved in any liquid, e.g., in a liquid formulation, e.g., daily or multiple times daily.
In one embodiment, the derivatized chitosan comprises a chitosan of the following formula (I):
Figure AU2019202473A1_D0075
formula (I) wherein:
n is an integer between 20 and 6000; and
WO 2011/028967
PCT/US2010/047758 each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
2019202473 09 Apr 2019
Figure AU2019202473A1_D0076
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, C,-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (11).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (II).
In one embodiment, between 4-30% of R1 substituents are a group of formula (II).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
In one embodiment, R2 is amino and R3 is an arginine side chain.
Figure AU2019202473A1_D0077
In one embodiment, Rx is amino and R’ is a lysine side chain.
In one embodiment, R1 is selected from one of the following:
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Figure AU2019202473A1_D0078
In one embodiment, R2 is amino and R3 is a histidine side chain.
Figure AU2019202473A1_D0079
In one embodiment, at least 1% of R1 substituents are selected from one of the following:
Figure AU2019202473A1_D0080
Figure AU2019202473A1_D0081
AND at least 1% of R1 substituents are selected from the following:
Figure AU2019202473A1_D0082
Figure AU2019202473A1_D0083
In one embodiment, R2 is amino and R3 is a substituted Ci-C6 alkyl.
In one embodiment, R' is Ci-Cs alkyl substituted with an amino group.
In one embodiment, R3 is C| alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, RJ is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
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In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, RJ is C6 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
2019202473 09 Apr 2019
Figure AU2019202473A1_D0084
Figure AU2019202473A1_D0085
Figure AU2019202473A1_D0086
Figure AU2019202473A1_D0087
In one embodiment, R3 is CrC6 alkyl substituted with a guanidino group. In one embodiment, R3 is Ci alkyl substituted with a guanidino group.
In one embodiment, R3 is C? alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, RJ is C5 alkyl substituted with a guanidino group.
In one embodiment, R3 is C(, alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0088
In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
In one embodiment, the nitrogen protecting group is /erhbutyloxycarbonyl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
WO 2011/028967
PCT/US2010/047758
In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
2019202473 09 Apr 2019
In one embodiment, R7 is amino.
In one embodiment, R2 is hydrogen and RJ is amino.
In one embodiment, R2 is hydrogen and RJ is guanidino.
In one embodiment, R2 is hydrogen and R3 is a substituted Ci-C6 alkyl.
In one embodiment, R3 is Ci-Ce alkyl substituted with an amino group.
In one embodiment, R3 is C] alkyl substituted with an amino group.
In one embodiment, RJ is C2 alkyl substituted with an amino group.
In one embodiment, R' is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, R3 is C(, alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0089
In one embodiment, R3 is C|-C6 alkyl substituted with a guanidino group.
In one embodiment, R3 is Ci alkyl substituted with a guanidino group.
In one embodiment, R3 is C2 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, RJ is C4 alkyl substituted with a guanidino group.
In one embodiment, R3 is C5 alkyl substituted with a guanidino group.
In one embodiment, RJ is C6 alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
WO 2011/028967
PCT/US2010/047758
2019202473 09 Apr 2019
Figure AU2019202473A1_D0090
Figure AU2019202473A1_D0091
Figure AU2019202473A1_D0092
Figure AU2019202473A1_D0093
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and 30%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
WO 2011/028967
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2019202473 09 Apr 2019
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1 %, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a method of treating or preventing (e.g., reducing the degree of) a wound infection in a subject, the method comprising: administering to the wound of the subject an effective amount of a composition comprising a soluble derivatized chitosan, thereby treating or preventing the wound infection. Exemplary wounds include wounds on the skin of a subject, such as a wound that damages and/or penetrates the skin of a subject.
In one embodiment, the subject is infected with bacteria listed in Table 4.
In one embodiment, the wound of the subject comprises at least one biofilm.
In one embodiment, exopolysaccharides (EPS) of the biolim comprises alginate and/or polysaccharide synthesis locus (Psi) (e.g., in Pseudomonas aeruginosa); acidic polysaccharide (e.g., in Burkholderia cepacia): collanic acid, poly~P-l,6-GlcNAc (PGA) or cellulose (e.g., in Escherichia coli): cellulose (e.g., in Salmonella): N-acetylglucosamine (GlcNAc), D-mannose, 6-deoxy-D-galactose and D-galactose (e.g., in Vibrio cholerae): polysaccharide intercellular adhesion (PIA) (e.g., in Staphylococcus): glucose and mannose rich component (e.g., in Bacillus subtilis): mannose polysaccharide (e.g., in Prevotella intermedia, Capnocytophaga ochracect, or Prevotella nigrescens).
In one embodiment, the biofilm is associated with actin and/or DNA released from bacteria or cells such as neutrophils.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99%, compared to the biofilm that has not been contacted with the composition.
3/
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2019202473 09 Apr 2019
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 1, 2, 5, 10, 50, 100, 200, 500, or 1000 fold, compared to the bio film that has not been contacted with the composition.
In one embodiment, the biofilm is partially dissolved, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.999% of the biofilm is dissolved, compared to the biofilm that has not been contacted with the composition.
In one embodiment, the composition is a rinse, a leave-in rinse, a gel, a component of a dressing, or a time-release formulation.
In one embodiment, the method further comprises administering an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic surfactant).
In one embodiment, the second agent is administered in a dosage to achieve a synergistic effect.
In one embodiment, the second agent is administered together with the soluble derivatized chitosan (e.g., in the same composition or dosage form).
In one embodiment, the method further comprises administering an antibiotic, anti-inflammatory, or mucolytic (expectorant) compound to a subject in conjunction with, prior to or subsequent to the administration of the composition.
In one embodiment, the antibiotic, anti-inflammatoiy, or mucolytic (expectorant) compound is administered in a dosage to achieve a synergistic effect.
In one embodiment, the method further comprises rinsing the wound with a composition described herein.
In one embodiment, the subject is infected with planktonic bacteria.
In one embodiment, the composition reduces the viability of planktonic bacteria.
In one embodiment, the composition reduces colonization of the planktonic bacteria.
In one embodiment, the planktonic bacteria are clumped.
In one embodiment, the composition is administered topically.
In one embodiment, the effective amount is therapeutically effective amount.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
WO 2011/028967
PCT/US2010/047758
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 5.0 to about pH 6.0, e.g., in the wound.
In one embodiment, the derivatized chitosan comprises a chitosan of the following formula (I):
2019202473 09 Apr 2019
Figure AU2019202473A1_D0094
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
Figure AU2019202473A1_D0095
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, fonns a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, Ci-Q alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (II).
In one embodiment, between 4-30% of R1 substituents are a group of formula (II).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
WO 2011/028967
PCT/US2010/047758
In one embodiment, R2 is amino and R3 is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
2019202473 09 Apr 2019
Figure AU2019202473A1_D0096
Figure AU2019202473A1_D0097
In one embodiment, R2 is amino and R3 is a lysine side chain.
Figure AU2019202473A1_D0098
In one embodiment, R2 is amino and R3 is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
wuw
Figure AU2019202473A1_D0099
JMWV
Figure AU2019202473A1_D0100
Figure AU2019202473A1_D0101
In one embodiment, at least 1 % of R1 substituents are selected from one of the
Figure AU2019202473A1_D0102
Figure AU2019202473A1_D0103
and at least 1% of R1 substituents are selected from the following:
WO 2011/028967
PCT/US2010/047758
Figure AU2019202473A1_D0104
2019202473 09 Apr 2019
Figure AU2019202473A1_D0105
In one embodiment, R2 is amino and RJ is a substituted Cj-Cgalkyl. In one embodiment, R3 is Ci-Cg alkyl substituted with an amino group.
In one embodiment, R3 is Ci alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0106
In one embodiment, R3 is C]-Cg alkyl substituted with a guanidino group.
In one embodiment, R3 is Ci alkyl substituted with a guanidino group.
In one embodiment, R3 is C2 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0107
In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
In one embodiment, the nitrogen protecting group is Zert-butyloxycarbonyl (Boc).
WO 2011/028967
PCT/US2010/047758
2019202473 09 Apr 2019
In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
In one embodiment, R2 is amino.
In one embodiment, R2 is hydrogen and R3 is amino.
In one embodiment, R2 is hydrogen and R3 is guanidino.
In one embodiment, R2 is hydrogen and R3 is a substituted Ci-Cft alkyl.
In one embodiment, RJ is Ci-C6 alkyl substituted with an amino group.
In one embodiment, R3 is C, alkyl substituted with an amino group.
In one embodiment, R3 is C? alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
Figure AU2019202473A1_D0108
In one embodiment, R3 is Ci-C& alkyl substituted with a guanidino group. In one embodiment, R3 is C i alkyl substituted with a guanidino group.
In one embodiment, R3 is C2 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0109
Figure AU2019202473A1_D0110
WO 2011/028967
PCT/US2010/047758
2019202473 09 Apr 2019
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and 30%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In another aspect, the invention features a liquid composition comprising a soluble chitosan or derivatized chitosan described herein.
In one embodiment, the composition further comprises a thickening agent.
WO 2011/028967
PCT/US2010/047758
In one embodiment, the derivatized chitosan comprises a chitosan of the
2019202473 09 Apr 2019
Figure AU2019202473A1_D0111
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
Figure AU2019202473A1_D0112
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R“ is hydrogen or amino; and
R3 is amino, guanidino, C|-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R* substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of fonnula (II).
In one embodiment, between 4-30% of R1 substituents are a group of formula (II).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of fonnula (II).
In one embodiment, R2 is amino and R3 is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
WO 2011/028967
PCT/US2010/047758
2019202473 09 Apr 2019
Figure AU2019202473A1_D0113
2 and
ΟΧΛΛΛ/
Figure AU2019202473A1_D0114
Figure AU2019202473A1_D0115
Figure AU2019202473A1_D0116
In one embodiment, R2 is amino and R3 is a lysine side chain.
Figure AU2019202473A1_D0117
In one embodiment, R? is amino and R3 is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0118
Figure AU2019202473A1_D0119
Figure AU2019202473A1_D0120
In one embodiment, at least 1% of R1 substituents are selected from one of the
Figure AU2019202473A1_D0121
AND at least 1% of R1 substituents are selected from the following:
WO 2011/028967
PCT/US2010/047758
2019202473 09 Apr 2019
Figure AU2019202473A1_D0122
In one embodiment, R2 is amino and R3 is a substituted Ci-Ce alkyl.
In one embodiment, R3 is C,-C6 alkyl substituted with an amino group.
In one embodiment, R3 is C] alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, RJ is C5 alkyl substituted with an amino group.
In one embodiment, RJ is C6 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0123
In one embodiment, RJ is C1 -C6 alkyl substituted with a guanidino group. In one embodiment, RJ is Ci alkyl substituted with a guanidino group. In one embodiment, RJ is C2 alkyl substituted with a guanidino group. In one embodiment, RJ is C3 alkyl substituted with a guanidino group.
In one embodiment, RJ is C4 alkyl substituted with a guanidino group. In one embodiment, RJ is C5 alkyl substituted with a guanidino group. In one embodiment, RJ is Cft alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0124
WO 2011/028967
PCT/US2010/047758
In one embodiment, R“ is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
2019202473 09 Apr 2019
In one embodiment, the nitrogen protecting group is /m-butyloxycarbonyl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
In one embodiment, R2 is amino.
In one embodiment, R2 is hydrogen and R3 is amino.
In one embodiment, R2 is hydrogen and RJ is guanidino.
In one embodiment, R2 is hydrogen and R3 is a substituted C,-C6 alkyl.
In one embodiment, R3 is Ct-C6 alkyl substituted with an amino group.
In one embodiment, R3 is C| alkyl substituted with an amino group. In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, R’ is C3 alkyl substituted with an amino group.
In one embodiment, RJ is C4 alkyl substituted with an amino group. In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, R3 is C6 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
jvwv
Figure AU2019202473A1_D0125
nh2
Figure AU2019202473A1_D0126
WWW
Figure AU2019202473A1_D0127
In one embodiment, RJ is C|-C6 alkyl substituted with a guanidino group.
In one embodiment, RJ is C, alkyl substituted with a guanidino group.
WO 2011/028967
PCT/US2010/047758
2019202473 09 Apr 2019
In one embodiment, RJ is C2 alkyl substituted with a guanidino group. In one embodiment, RJ is C\ alkyl substituted with a guanidino group. In one embodiment, RJ is C4 alkyl substituted with a guanidino group. In one embodiment, RJ is C5 alkyl substituted with a guanidino group. In one embodiment, R3 is C6 alkyl substituted with a guanidino group. In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0128
Figure AU2019202473A1_D0129
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment embodiment, the chitosan is functionalized at between 20% and 30%.
WO 2011/028967
PCT/US2010/047758
2019202473 09 Apr 2019
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the composition is active below pH 6 (e.g., in stomach and duodenum) or near or above pH 8 (e.g., in colon).
In one embodiment, the composition is active between pH 6 and pH 8.
In one embodiment, the composition further comprises an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic surfactant). In one embodiment, the second agent comprises another chitosan derivative, e.g., another chitosan derivative described herein.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a solid particulate (e.g., solid lipid particulate) composition comprising a soluble derivatized chitosan described herein.
In one embodiment, the derivatized chitosan comprises a chitosan of the following formula (I):
OH / OH \ OH
^^O
ΗΟ^\~^ \ Lo \ \ >ο-\~
ΗΟ- ν—Ε ^Ύ HO—V- / HO-V
NH \ I NH/n NH |
R1 R1 R1
formula (I)
WO 2011/028967
PCT/US2010/047758 n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
σνννν
2019202473 09 Apr 2019 wherein:
Figure AU2019202473A1_D0130
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, Ci-C& alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (II).
In one embodiment, between 4-30% of R1 substituents are a group of formula (II).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
In one embodiment, R2 is amino and RJ is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0131
and
Figure AU2019202473A1_D0132
5(1
WO 2011/028967
PCT/US2010/047758
In one embodiment, R7 is amino and RJ is a lysine side chain.
In one embodiment, R1 is selected from one of the following:
2019202473 09 Apr 2019
Figure AU2019202473A1_D0133
Figure AU2019202473A1_D0134
In one embodiment, R' is amino and R3 is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0135
In one embodiment, at least 1% of R1 substituents are selected from one of the
Figure AU2019202473A1_D0136
and at least 1% of R1 substituents are selected from the following:
Figure AU2019202473A1_D0137
Figure AU2019202473A1_D0138
In one embodiment, R2
In one embodiment, RJ
In one embodiment, RJ
In one embodiment, RJ is is is
IS amino and RJ is a substituted C|-C6 alkyl. C|-C(, alkyl substituted with an amino group. C| alkyl substituted with an amino group. C2 alkyl substituted with an amino group.
WO 2011/028967
PCT/US2010/047758
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, R1 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, R3 is C6 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
2019202473 09 Apr 2019
Figure AU2019202473A1_D0139
Figure AU2019202473A1_D0140
Figure AU2019202473A1_D0141
Figure AU2019202473A1_D0142
In one embodiment, RJ is Cj-Q alkyl substituted with a guanidino group.
In one embodiment, R3 is C| alkyl substituted with a guanidino group.
In one embodiment, RJ is C2 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, RJ is C4 alkyl substituted with a guanidino group.
In one embodiment, R3 is C5 alkyl substituted with a guanidino group.
In one embodiment, R3 is C6 alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0143
In one embodiment, R“ is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
In one embodiment, the nitrogen protecting group is ter(-butyloxycarbonyl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
WO 2011/028967
PCT/US2010/047758
In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
2019202473 09 Apr 2019
In one embodiment, R2 is amino.
In one embodiment, R2 is hydrogen and R3 is amino.
In one embodiment, R2 is hydrogen and R3 is guanidino.
In one embodiment, R2 is hydrogen and R3 is a substituted Ci-C^alkyl. In one embodiment, R3 is C|-C6 alkyl substituted with an amino group.
In one embodiment, R3 is Cj alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, RJ is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group. In one embodiment, R3 is C6 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0144
In one embodiment, R3 is C1-C6 alkyl substituted with a guanidino group.
In one embodiment, R3 is C( alkyl substituted with a guanidino group.
In one embodiment, R3 is C? alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, R3 is C5 alkyl substituted with a guanidino group.
In one embodiment, RJ is C(, alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
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Figure AU2019202473A1_D0145
Figure AU2019202473A1_D0146
Figure AU2019202473A1_D0147
Figure AU2019202473A1_D0148
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and 30%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
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In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 5.0 to about pH 6.0, e.g., in wounds or duodenum.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 2.0 to about pH 4.0, e.g., in stomach.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 8.0 to about pH 8.5, e.g., in lower part of the gastrointestinal tract.
In one embodiment, the composition further comprises an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic surfactant). In one embodiment, the second agent comprises another chitosan derivative, e.g., another chitosan derivative described herein.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1 %, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a semisolid (e.g., ointment, cream, gel, paste, or liniment) composition comprising a soluble derivatized chitosan described herein.
In one embodiment, the derivatized chitosan comprises a chitosan of the following formula (I):
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Figure AU2019202473A1_D0149
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
Figure AU2019202473A1_D0150
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, C1-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (Π).
In one embodiment, between 4-30% of R1 substituents are a group of formula (II).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
In one embodiment, R is amino and R is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
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Figure AU2019202473A1_D0151
In one embodiment, R? is amino and R3 is a lysine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0152
Figure AU2019202473A1_D0153
In one embodiment, is amino and R3 is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0154
In one embodiment, at least 1 % of R1 substituents are selected from one of the following:
Figure AU2019202473A1_D0155
and at least 1% of R1 substituents are selected from the following:
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Figure AU2019202473A1_D0156
In one embodiment, R° is amino and R3 is a substituted C1-C6 alkyl.
In one embodiment, R3 is Cj-Cf, alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group. In one embodiment, R3 is C3 alkyl substituted with an amino group. In one embodiment, R3 is C4 alkyl substituted with an amino group. In one embodiment, R3 is C5 alkyl substituted with an amino group. In one embodiment, R3 is C(, alkyl substituted with an amino group. In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0157
Figure AU2019202473A1_D0158
Figure AU2019202473A1_D0159
Figure AU2019202473A1_D0160
In one embodiment, R3 is C|-C6 alkyl substituted with a guanidino group.
In one embodiment, R3 is C1 alkyl substituted with a guanidino group.
In one embodiment, R3 is C2 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, R3 is C5 alkyl substituted with a guanidino group.
In one embodiment, R3 is Cf, alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0161
WO 2011/028967
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2019202473 09 Apr 2019 . 7 . . .
In one embodiment, R' is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
In one embodiment, the nitrogen protecting group is te/7-butyloxycarbonyl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
In one embodiment, R2 is amino.
In one embodiment, R2 is hydrogen and R3 is amino.
In one embodiment, R~ is hydrogen and R is guanidino.
In one embodiment, R* is hydrogen and R is a substituted C|-Cft alkyl.
In one embodiment, R is C|-C6 alkyl substituted with an amino group.
In one embodiment, R’ is Cj alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, RJ is C3 alkyl substituted with an amino group.
In one embodiment, RJ is C4 alkyl substituted with an amino group.
In one embodiment, RJ is Cs alkyl substituted with an amino group.
In one embodiment, RJ is Cf, alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0162
NH2 >
NH2
In one embodiment, R3 is Cj-C& alkyl substituted with a guanidino group. In one embodiment, R3 is Ci alkyl substituted with a guanidino group.
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In one embodiment, R3 In one embodiment, RJ In one embodiment, RJ
In one embodiment, R3 In one embodiment, RJ
In one embodiment, R1 is C2 alkyl substituted with a guanidino group, is C3 alkyl substituted with a guanidino group, is C4 alkyl substituted with a guanidino group, is C5 alkyl substituted with a guanidino group, is C(, alkyl substituted with a guanidino group, is selected from one of the following:
Figure AU2019202473A1_D0163
Figure AU2019202473A1_D0164
Figure AU2019202473A1_D0165
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and 30%.
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In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 5.0 to about pH 6.0, e.g., in wounds.
In one embodiment, the composition further comprises an additional agent, e.g., a pharmaceutical agent, or a non-phannaceutical agent (e.g., a non-toxic surfactant). In one embodiment, the second agent comprises another chitosan derivative, e.g., another chitosan derivative described herein.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a dressing or an ointment comprising soluble derivatized chitosan described herein, wherein the dressing or ointment is fonnulated such that the soluble chitosan or derivatized chitosan described herein is diffusible into a subject upon administration to the subject.
In one embodiment, the composition of the derivatized chitosan was autoclaved before use in the body.
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In one embodiment, the derivatized chitosan comprises a chitosan of the
2019202473 09 Apr 2019
Figure AU2019202473A1_D0166
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
wvw
Figure AU2019202473A1_D0167
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R is hydrogen or amino; and
R3 is annno, guanidino, Ci-C6 alkyl substituted with an ammo or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (II).
In one embodiment, between 4-30% of R1 substituents are a group of formula (II).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
In one embodiment, R2 is amino and RJ is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
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Figure AU2019202473A1_D0168
In one embodiment, R2 is amino and R3 is a lysine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0169
Figure AU2019202473A1_D0170
In one embodiment, R2 is amino and R3 is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
MWW wvw
Figure AU2019202473A1_D0171
In one embodiment, at least 1% of R1 substituents are selected from one of the following:
Figure AU2019202473A1_D0172
Figure AU2019202473A1_D0173
and at least 1% of R1 substituents are selected from the following:
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Figure AU2019202473A1_D0174
In one embodiment, R2 is amino and R3 is a substituted C]-C6 alkyl.
In one embodiment, R3 is Cj-Cg alkyl substituted with an amino group.
In one embodiment, R3 is C i alkyl substituted with an amino group.
In one embodiment, RJ is C-> alkyl substituted with an amino group.
In one embodiment, RJ is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C6 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0175
In one embodiment, R3 is C|-Cg alkyl substituted with a guanidino group. In one embodiment, R3 is C, alkyl substituted with a guanidino group.
In one embodiment, R3 is C2 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group. In one embodiment, R ’ is C4 alkyl substituted with a guanidino group.
In one embodiment, R3 is C5 alkyl substituted with a guanidino group. In one embodiment, R3 is C6 alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0176
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In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
2019202473 09 Apr 2019
In one embodiment, the nitrogen protecting group is /er/-butyloxycarbonyl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
In one embodiment, R7 is amino.
In one embodiment, R2 is hydrogen and R3 is amino.
In one embodiment, R2 is hydrogen and R1 is guanidino.
In one embodiment, R2 is hydrogen and R3 is a substituted Ci-Cg alkyl.
In one embodiment, R3 is C|-C&alkyl substituted with an amino group.
In one embodiment, R3 is Ci alkyl substituted with an amino group.
In one embodiment, R3 is C? alkyl substituted with an amino group. In one embodiment, R3 is C3 alkyl substituted with an amino group. In one embodiment, R3 is C4 alkyl substituted with an amino group. In one embodiment, R3 is C5 alkyl substituted with an amino group. In one embodiment, R3 is C6 alkyl substituted with an amino group. In one embodiment, R1 is selected from one of the following:
VWW
Figure AU2019202473A1_D0177
nh2 jww nh2
In one embodiment, RJ is C1-C6 alkyl substituted with a guanidino group.
In one embodiment, R3 is Cj alkyl substituted with a guanidino group.
Figure AU2019202473A1_D0178
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In one embodiment, R3 is C2 alkyl substituted with a guanidino group. In one embodiment, R3 is C3 alkyl substituted with a guanidino group. In one embodiment, R3 is C4 alkyl substituted with a guanidino group. In one embodiment, R3 is C5 alkyl substituted with a guanidino group. In one embodiment, R3 is Cg alkyl substituted with a guanidino group. In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0179
Figure AU2019202473A1_D0180
Figure AU2019202473A1_D0181
Figure AU2019202473A1_D0182
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and 30%.
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In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 5.0 to about pH 6.0, e.g., in wounds.
In one embodiment, the dressing or ointment further comprises an additional agent, e.g., a pharmaceutical agent, or a non-phannaceutical agent (e.g., a non-toxic surfactant). In one embodiment, the second agent comprises another chitosan derivative, e.g., another chitosan derivative described herein.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the dressing or ointment has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a kit comprising:
a soluble derivatized chitosan described herein; and instructions to disrupt a performed biofilm in a subject.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99%, compared to the biofilm that has not been contacted with the derivatized chitosan.
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In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 1,2, 5, 10, 50, 100, 200, 500, or 1000 fold, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the biofilm is partially dissolved, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.999% of the biofilm is dissolved, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the kit further comprises an additional agent, e.g., a pharmaceutical agent, or a non-phamiaceutical agent (e.g., a non-toxic surfactant). In one embodiment, the second agent comprises another chitosan derivative, e.g., another chitosan derivative described herein.
In one embodiment, the second agent is administered in a dosage to achieve a synergistic effect.
In one embodiment, the second agent is administered together with the soluble derivatized chitosan (e.g., in the same derivatized chitosan or dosage form).
In one embodiment, the kit further comprises an antibiotic, anti-inflammatory, or mucolytic (expectorant) compound to a subject in conjunction with, prior to or subsequent to the administration of the derivatized chitosan.
In one embodiment, the antibiotic, anti-inflammatory, or mucolytic (expectorant) compound is administered in a dosage to achieve a synergistic effect.
In one embodiment, the biofilm is in the respiratory system (e.g., airways, lung, throat, nose), ear, eyes, mouth (e.g., tooth, tongue, mucosal surface), digestive system (e.g., stomach, small intestine, large intestine, colon, bowel), skin, or wound.
In one embodiment, the subject has a complication of cystic fibrosis (e.g., lung infection or respiratory congestion), pneumonia, dental plaque, oral disease (e.g., gingivitis, dental caries, or halitosis), ear infection, throat infection, eye infection (e.g., conjunctivitis), a complication in immunocompromised patient (e.g., lung infection or respiratory congestion), gastrointestinal infection, enteritis, or a symptom thereof.
In one embodiment, the subject is infected with bacteria listed in Table 1,2, 3, or 4.
In one embodiment, the derivatized chitosan is administered topically, orally, or enterally.
In one embodiment, the derivatized chitosan is administered by inhalation (e.g., nebulizer, nasal spray, nasal swab, or sinus spray).
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In one embodiment, the effective amount is therapeutically effective amount.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 5.0 to about pH 6.0, e.g., in wounds or duodenum.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 2.0 to about pH 4.0, e.g., in stomach.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 8.0 to about pH 8.5, e.g., in lower part of the gastrointestinal tract.
In one embodiment, the derivatized chitosan comprises a chitosan of the following formula (I):
Figure AU2019202473A1_D0183
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
A/VVV
Figure AU2019202473A1_D0184
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R2 is amino, guanidino, C, -C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
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In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (Π).
In one embodiment, between 4-30% of R1 substituents are a group of formula (II).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
In one embodiment, R2 is amino and R~ is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0185
Figure AU2019202473A1_D0186
In one embodiment, R° is amino and R3 is a lysine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0187
Figure AU2019202473A1_D0188
In one embodiment, R? is amino and R'1 is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0189
Figure AU2019202473A1_D0190
Figure AU2019202473A1_D0191
In one embodiment, at least 1% of R1 substituents are selected from one of the following:
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Figure AU2019202473A1_D0192
Figure AU2019202473A1_D0193
Figure AU2019202473A1_D0194
In one embodiment, R2 is amino and R3 is a substituted C|-C6 alkyl.
In one embodiment, RJ is Cj-C6 alkyl substituted with an amino group.
In one embodiment, R3 is C, alkyl substituted with an amino group. In one embodiment, RJ is C? alkyl substituted with an amino group.
In one embodiment, RJ is C3 alkyl substituted with an amino group.
In one embodiment, RJ is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, R3 is C6 alkyl substituted with an ammo group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0195
In one embodiment, R3 is C1-C6 alkyl substituted with a guanidino group. In one embodiment, RJ is Ci alkyl substituted with a guanidino group.
In one embodiment, R1 is Cb alkyl substituted with a guanidino group. In one embodiment, RJ is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C4 alkyl substituted with a guanidino group. In one embodiment, R3 is C5 alkyl substituted with a guanidino group.
In one embodiment, R3 is C6 alkyl substituted with a guanidino group.
WO 2011/028967
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In one embodiment, R1 is selected from one of the following:
2019202473 09 Apr 2019
Figure AU2019202473A1_D0196
In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
In one embodiment, the nitrogen protecting group is tert-butyloxycarbonyl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
In one embodiment, R2 is amino.
In one embodiment, R2 is hydrogen and RJ is amino.
In one embodiment, R2 is hydrogen and R3 is guanidino.
In one embodiment, R2 is hydrogen and R3 is a substituted Ci-C6 alkyl.
In one embodiment, R3 is Cj-C6 alkyl substituted with an amino group.
In one embodiment, R3 is C| alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, RJ is C ; alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, R3 is C6 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
WO 2011/028967
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Figure AU2019202473A1_D0197
2019202473 09 Apr 2019
Figure AU2019202473A1_D0198
In one embodiment, R3 is C1-C6 alkyl substituted with a guanidino group.
In one embodiment, R3 is Ci alkyl substituted with a guanidino group.
In one embodiment, R3 is C? alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, R3 is C5 alkyl substituted with a guanidino group.
In one embodiment, R3 is C<-. alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0199
Figure AU2019202473A1_D0200
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
WO 2011/028967
PCT/US2010/047758
2019202473 09 Apr 2019
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and 30%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the functionalized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the kit has less than about 20%, 15%, 10%, 5%, 2%, or %, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, tnargine, etc).
In one embodiment, the kit has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a kit comprising:
a soluble derivatized chitosan described herein; and instructions to prevent (e.g., reduce the degree of) the formation of a biofilm in the respiratory system of a subject.
In one embodiment, the kit further comprises an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic surfactant). In one embodiment, the second agent comprises another chitosan derivative, e.g., another chitosan derivative described herein.
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In one embodiment, the second agent is administered in a dosage to achieve a synergistic effect.
In one embodiment, the kit further comprises an antibiotic, anti-inflammatory, or mucolytic (expectorant) compound to a subject in conjunction with or subsequent to the administration of the derivatized chitosan.
In one embodiment, the kit further comprises an antibiotic, anti-inflammatory, or mucolytic (expectorant) compound to a subject in conjunction with, prior to or subsequent to the administration of the derivatized chitosan.
In one embodiment, the subject is infected with planktonic bacteria or infected with bacteria listed in Table 3.
In one embodiment, the subject is diagnosed with bacterial pneumonia.
In one embodiment, the derivatized chitosan reduces the viability of planktonic bacteria.
In one embodiment, the derivatized chitosan reduces colonization of the planktonic bacteria.
In one embodiment, the derivatized chitosan is administered by inhalation (e.g., nebulizer, nasal spray, or sinus spray).
In one embodiment, the kit comprises an effective amount (e.g., therapeutically effective amount) of derivatized chitosan.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan comprises a chitosan of the following formula (I):
Figure AU2019202473A1_D0201
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
WO 2011/028967
PCT/US2010/047758
2019202473 09 Apr 2019 jww
Figure AU2019202473A1_D0202
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
RJ is amino, guanidino, C1-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (Π).
In one embodiment, between 4-30% of R1 substituents are a group of formula (II).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
In one embodiment, R2 is amino and R is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0203
Figure AU2019202473A1_D0204
□ 3
In one embodiment, R“ is amino and R is a lysine side chain.
In one embodiment, R! is selected from one of the following:
PCT/US2010/047758
Figure AU2019202473A1_D0205
WO 2011/028967
2019202473 09 Apr 2019
Figure AU2019202473A1_D0206
In one embodiment, R2 is amino and R3 is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0207
In one embodiment, at least 1% of R1 substituents are selected from one of the following:
Figure AU2019202473A1_D0208
and at least I % of R1 substituents are selected from the following:
Figure AU2019202473A1_D0209
In one embodiment, R2 is amino and R3 is a substituted Ci-C6 alkyl. In one embodiment, R3 is C, -C6 alkyl substituted with an amino group.
In one embodiment, RJ is Ci alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, RJ is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
WO 2011/028967
PCT/US2010/047758
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, R3 is C6 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
2019202473 09 Apr 2019
Figure AU2019202473A1_D0210
Figure AU2019202473A1_D0211
Figure AU2019202473A1_D0212
Figure AU2019202473A1_D0213
Figure AU2019202473A1_D0214
Figure AU2019202473A1_D0215
Figure AU2019202473A1_D0216
In one embodiment, R3 is C|-Cg alkyl substituted with a guanidino group. In one embodiment, R3 is Cj alkyl substituted with a guanidino group.
In one embodiment, R3 is C2 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group. In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, R’ is C5 alkyl substituted with a guanidino group. In one embodiment, R3 is C6 alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0217
In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
In one embodiment, the nitrogen protecting group is Zeri-butyloxycarbonyl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
WO 2011/028967
PCT/US2010/047758
2019202473 09 Apr 2019
In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boe.
In one embodiment, R2 is amino.
In one embodiment, R2 is hydrogen and R3 is amino.
In one embodiment, R2 is hydrogen and R3 is guanidino.
In one embodiment, R‘ is hydrogen and R is a substituted Cj-C6 alkyl.
In one embodiment, R3 is Cj-C6 alkyl substituted with an amino group.
In one embodiment, R3 is Cj alkyl substituted with an amino group.
In one embodiment, R3 is C? alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C'4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, R3 is C/ alkyl substituted with an amino group.
Figure AU2019202473A1_D0218
In one embodiment, R3 is C1-C6 alkyl substituted with a guanidino group. In one embodiment, RJ is C, alkyl substituted with a guanidino group.
In one embodiment, R3 is C? alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C6 alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
WO 2011/028967
PCT/US2010/047758
2019202473 09 Apr 2019
Figure AU2019202473A1_D0219
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and 30%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
so
WO 2011/028967
PCT/US2010/047758
2019202473 09 Apr 2019
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the kit has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the kit has less than about 20%, 15%, 10%, 5%, 2%, or 1 %, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a kit comprising:
a soluble derivatized chitosan described herein; and instructions to prevent or treat a complication of cystic fibrosis in a subject.
In one embodiment, the complication of cystic fibrosis is lung infection or respiratory congestion.
In one embodiment, the subject has a bacterial infection, e.g., bacteria list in Table 3, e.g., Pseudomonas aeruginosa.
In one embodiment, the subject comprises at least one biofilm.
In one embodiment, exopolysaccharides (EPS) of the biolim comprises alginate and/or polysaccharide synthesis locus (Psi) (e.g., in Pseudomonas aeruginosa)·, acidic polysaccharide (e.g., in Burkholderia cepacia)·, collanic acid, ροΙγ-β-Ι,ό-GlcNAc (PGA) or cellulose (e.g., in Escherichia coli); cellulose (e.g., in Salmonella)·, N-acetylglucosamine (GlcNAc), D-mannose, 6-deoxy-D-galactose and D-galactose (e.g., in Vibrio cholerae)·, polysaccharide intercellular adhesion (PIA) (e.g., in Staphylococcus)·, glucose and mannose rich component (e.g., in Bacillus subtilis)·, mannose polysaccharide (e.g., in Prevotella intermedia, Capnocvtophaga ochracea, or Prevotella nigrescens).
In one embodiment, the biofilm is associated with actin and/or DNA released from bacteria or cells such as neutrophils.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99%, compared to the biofilm that has not been contacted with the derivatized chitosan.
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In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 1, 2, 5, 10, 50, 100, 200, 500, or 1000 fold, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the biofilm is partially dissolved, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.999% of the biofilm is dissolved, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the kit further comprises an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic surfactant). In one embodiment, the second agent comprises another chitosan derivative, e.g., another chitosan derivative described herein.
In one embodiment, the second agent is administered in a dosage to achieve a synergistic effect.
In one embodiment, the second agent is administered together with the soluble derivatized chitosan (e.g., in the same derivatized chitosan or dosage form).
In one embodiment, the kit further comprises administering an antibiotic, antiinflammatory, or mucolytic (expectorant) compound to a subject in conjunction with, prior to or subsequent to the administration of the derivatized chitosan.
In one embodiment, the antibiotic, anti-inflammatory, or mucolytic (expectorant) compound is administered in a dosage to achieve a synergistic effect.
In one embodiment, the biofilm is in the respiratory system (e.g., airways, lung, throat, nose).
In one embodiment, the subject is infected with planktonic bacteria.
In one embodiment, the derivatized chitosan reduces the viability of planktonic bacteria.
In one embodiment, the derivatized chitosan reduces colonization of the planktonic bacteria.
In one embodiment, the planktonic bacteria are clumped, e.g., to facilitate removal by expectoration, lavage, or chest percussion.
In one embodiment, the derivatized chitosan is administered by inhalation (e.g., nebulizer, nasal spray, nasal swab, or sinus spray).
In one embodiment, the kit comprises an effective amount (e.g., therapeutically effective amount) of derivatized chitosan.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
WO 2011/028967
PCT/US2010/047758
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan comprises a chitosan of the following formula (I):
2019202473 09 Apr 2019
Figure AU2019202473A1_D0220
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
Figure AU2019202473A1_D0221
R3 fonnula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, C]-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (II).
In one embodiment, between 4-30% of R1 substituents are a group of fonnula (II).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
S3
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3
In one embodiment, R is amino and R is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0222
and
Figure AU2019202473A1_D0223
In one embodiment, R2 is amino and R3 is a lysine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0224
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0225
Figure AU2019202473A1_D0226
Figure AU2019202473A1_D0227
In one embodiment, at least 1% of R1 substituents are selected from one of the
Figure AU2019202473A1_D0228
and at least 1% of R1 substituents are selected from the following:
WO 2011/028967
PCT/US2010/047758
Figure AU2019202473A1_D0229
2019202473 09 Apr 2019
Figure AU2019202473A1_D0230
In one embodiment, R2 is amino and R3 is a substituted Ci-C6 alkyl.
In one embodiment, R3 is C|-C6 alkyl substituted with an amino group.
In one embodiment, R3 is C| alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group. In one embodiment, R3 is C3 alkyl substituted with an amino group. In one embodiment, R3 is C4 alkyl substituted with an amino group. In one embodiment, R3 is C5 alkyl substituted with an amino group. In one embodiment, R3 is C6 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0231
Figure AU2019202473A1_D0232
Hih2
Figure AU2019202473A1_D0233
Figure AU2019202473A1_D0234
Figure AU2019202473A1_D0235
In one embodiment, R3 is C|-C6 alkyl substituted with a guanidino group.
In one embodiment, R3 is Cj alkyl substituted with a guanidino group.
In one embodiment, R3 is C2 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, R3 is C5 alkyl substituted with a guanidino group.
In one embodiment, R3 is C6 alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0236
WO 2011/028967
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2019202473 09 Apr 2019
In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
In one embodiment, the nitrogen protecting group is iert-butyloxycarbonyl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
In one embodiment, R2 is amino.
In one embodiment, R2 is hydrogen and R3 is amino.
In one embodiment, R2 is hydrogen and R3 is guanidino.
In one embodiment, R2 is hydrogen and RJ is a substituted Ci-Cg alkyl.
In one embodiment, R3 is C|-C(, alkyl substituted with an amino group.
In one embodiment, R3 is C| alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, R3 is C6 alkyl substituted with an amino group.
Figure AU2019202473A1_D0237
In one embodiment, R3 is C|-C6alkyl substituted with a guanidino group.
In one embodiment, R3 is Ci alkyl substituted with a guanidino group.
WO 2011/028967
PCT/US2010/047758
2019202473 09 Apr 2019
In one embodiment, R3 is C2 alkyl substituted with a guanidino group. In one embodiment, R3 is C3 alkyl substituted with a guanidino group. In one embodiment, R3 is C4 alkyl substituted with a guanidino group. In one embodiment, R3 is C5 alkyl substituted with a guanidino group. In one embodiment, R3 is C6 alkyl substituted with a guanidino group. In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0238
Figure AU2019202473A1_D0239
Figure AU2019202473A1_D0240
Figure AU2019202473A1_D0241
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and 30%.
WO 2011/028967
PCT/US2010/047758
2019202473 09 Apr 2019
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the kit has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the kit has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a kit comprising:
a soluble derivatized chitosan described herein; and instructions to prevent or treat a gastrointestinal tract infection in a subject.
In one embodiment, the gastrointestinal tract infection is noninflammatory gastroenteritis, inflammatory gastroenteritis, invasive gastroenteritis, or nectrotic or necrotizing enteritis.
In one embodiment, the subject is infected with Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Clostridium botulinum, Vibrio cholerae, Escherichia coli, Clostridium difficile, Vibrio parahemolyticus, Bacillus anthracis, Shigella sp., Salmonella sp., Campylobacter jejuni, Vibrion vulnificus, Yersinia sp., Francisella tularensis, or Helicobacter pylori.
In one embodiment, the subject is infected with bacteria listed in Table 2.
In one embodiment, the subject comprises at least one biofilm.
In one embodiment, exopolysaccharides (EPS) of the biolim comprises alginate and/or polysaccharide synthesis locus (Psi) (e.g., in Pseudomonas aeruginosa); acidic polysaccharide (e.g., in Burkholderia cepacia); collanic acid,
WO 2011/028967
PCT/US2010/047758
2019202473 09 Apr 2019 poly-P-l,6-GlcNAc (PGA) or cellulose (e.g., in Escherichia coli): cellulose (e.g., in Salmonella): N-acetylglucosamine (GlcNAc), D-mannose, 6-deoxy-D-galactose and D-galactose (e.g., in Vibrio cholerae): polysaccharide intercellular adhesion (PIA) (e.g., in Staphylococcus): glucose and mannose rich component (e.g., in Bacillus subtilis): mannose polysaccharide (e.g., in Prevotella intermedia, Capnocytophaga ochracea, or Prevotella nigrescens).
In one embodiment, the biofilm is associated with actin and/or DNA released from bacteria or cells such as neutrophils.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99%, compared to the biofilm that has not been contacted with the drivatized chitosan.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 1, 2, 5, 10, 50, 100, 200, 500, or 1000 fold, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the biofilm is partially dissolved, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.999% of the biofilm is dissolved, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the kit further comprises an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic surfactant). In one embodiment, the second agent comprises another chitosan derivative, e.g., another chitosan derivative described herein.
In one embodiment, the second agent is administered in a dosage to achieve a synergistic effect.
In one embodiment, the second agent is administered together with the soluble derivatized chitosan (e.g., in the same derivatized chitosan or dosage form).
In one embodiment, the kit further comprises an antibiotic, anti-inflammatory, or mucolytic (expectorant) compound to a subject in conjunction with, prior to or subsequent to the administration of the derivatized chitosan.
In one embodiment, the antibiotic, anti-inflammatory, or mucolytic (expectorant) compound is administered in a dosage to achieve a synergistic effect.
In one embodiment, the biofilm is in the gastrointestinal tract.
In one embodiment, the subject is infected with planktonic bacteria.
In one embodiment, the derivatized chitosan reduces the viability of planktonic bacteria.
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In one embodiment, the derivatized chitosan reduces colonization of the planktonic bacteria.
In one embodiment, the planktonic bacteria are clumped.
In one embodiment, the derivatized chitosan is administered orally, or ent erally.
In one embodiment, the kit comprises an effective amount (e.g., therapeutically effective amount) of derivatized chitosan.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 5.0 to about pH 6.0, e.g., in duodenum.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 2.0 to about pH 4.0, e.g., in stomach.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 8.0 to about pH 8.5, e.g., in lower part of the gastrointestinal tract.
In one embodiment, the derivatized chitosan is active below pH 6 (e.g., in stomach and duodenum) or near or above pH 8 (e.g., in colon).
In one embodiment, the derivatized chitosan is active between pH 6 and pH 8.
In one embodiment, the derivatized chitosan is delivered e.g., by capsule, time release capsule, pH release capsule, as a powder dissolved in the digestive tract, or lozenge.
In one embodiment, the derivatized chitosan is delivered by mixing in food, or dissolved in any liquid, e.g., in a liquid fonnulation, e.g., daily or multiple times daily.
In one embodiment, the derivatized chitosan comprises a chitosan of the following formula (I):
Figure AU2019202473A1_D0242
formula (I) wherein:
n is an integer between 20 and 6000; and
WO 2011/028967
PCT/US2010/047758 each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
2019202473 09 Apr 2019
7WW
Figure AU2019202473A1_D0243
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, C1-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (II).
In one embodiment, between 4-30% of R1 substituents are a group of formula (II).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
In one embodiment, R is amino and R is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0244
In one embodiment, R2 is amino and R’ is a lysine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0245
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Figure AU2019202473A1_D0246
In one embodiment, R is amino and R3 is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0247
In one embodiment, at least 1% of R1 substituents are selected from one of the following:
Figure AU2019202473A1_D0248
and at least 1% of R1 substituents are selected from the following:
Figure AU2019202473A1_D0249
Figure AU2019202473A1_D0250
In one embodiment, R* is amino and R3 is a substituted Ci-C& alkyl. In one embodiment, R3 is C|-C6 alkyl substituted with an amino group.
In one embodiment, RJ is C| alkyl substituted with an amino group.
In one embodiment, RJ is C2 alkyl substituted with an amino group.
In one embodiment, R’ is C3 alkyl substituted with an amino group.
In one embodiment, RJ is C4 alkyl substituted with an amino group.
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In one embodiment, RJ is C5 alkyl substituted with an amino group.
In one embodiment, RJ is C(, alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
2019202473 09 Apr 2019
Figure AU2019202473A1_D0251
Figure AU2019202473A1_D0252
Figure AU2019202473A1_D0253
Figure AU2019202473A1_D0254
In one embodiment, RJ is C i -C6 alkyl substituted with a guanidino group. In one embodiment, RJ is C, alkyl substituted with a guanidino group. In one embodiment, RJ is C2 alkyl substituted with a guanidino group. In one embodiment, R3 is Ci alkyl substituted with a guanidino group. In one embodiment, RJ is C4 alkyl substituted with a guanidino group. In one embodiment, R3 is C5 alkyl substituted with a guanidino group. In one embodiment, RJ is C6 alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0255
In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
In one embodiment, the nitrogen protecting group is /erNbutyloxycarbonyl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
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In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
2019202473 09 Apr 2019
In one embodiment, R? is amino.
In one embodiment, R? is hydrogen and R3 is amino.
In one embodiment, R2 is hydrogen and R3 is guanidino.
In one embodiment, R2 is hydrogen and R3 is a substituted Cj-C6alkyl.
In one embodiment, R3 is C|-C6 alkyl substituted with an amino group.
In one embodiment, R3 is C| alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, R3 is Ci alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
WWW
Figure AU2019202473A1_D0256
nh2 jVUW J“JWV nh2
Figure AU2019202473A1_D0257
In one embodiment, R3 is C|-Cf, alkyl substituted with a guanidino group. In one embodiment, R3 is Cj alkyl substituted with a guanidino group.
In one embodiment, R3 is C2 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group. In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, R3 is C5 alkyl substituted with a guanidino group. In one embodiment, R3 is C(, alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
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Figure AU2019202473A1_D0258
Figure AU2019202473A1_D0259
Figure AU2019202473A1_D0260
Figure AU2019202473A1_D0261
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and 30%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
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In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the kit has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the kit has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a medical device constructed to fit into the respiratory system of a subject, e.g., to contact nose or throat, the medical device comprising a soluble derivatized chitosan described herein. In one embodiment, the surface of the device is coated with a soluble derivatzed chitosan.
In one embodiment, the subject has a bacterial infection, e.g., bacteria list in Table 3, e.g., Pseudomonas aeruginosa.
In one embodiment, the subject comprises at least one biofilm.
In one embodiment, exopolysaccharides (EPS) of the biolim comprises alginate and/or polysaccharide synthesis locus (Psi) (e.g., in Pseudomonas aeruginosa); acidic polysaccharide (e.g., in Burkholderia cepacia); collanic acid, poly-P~l,6-GlcNAc (PGA) or cellulose (e.g., in Escherichia coli); cellulose (e.g., in Salmonella); N-acetylglucosamine (GlcNAc), D-mannose, 6-deoxy-D-galactose and D-galactose (e.g., in Vibrio cholerae); polysaccharide intercellular adhesion (PIA) (e.g., in Staphylococcus); glucose and mannose rich component (e.g., in Bacillus subtilis); mannose polysaccharide (e.g., in Prevotella intermedia, Capnocytophaga ochracect, or Prevotella nigrescens).
In one embodiment, the biofilm is associated with actin and/or DNA released from bacteria or cells such as neutrophils.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99%, compared to the biofilm that has not been contacted with the derivatized chitosan.
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In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 1,2, 5, 10, 50, 100, 200, 500, or 1000 fold, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the biofilm is partially dissolved, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.999% of the biofilm is dissolved, compared to tire biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the medical device further comprises an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic surfactant). In one embodiment, the second agent comprises another chitosan derivative, e.g., another chitosan derivative described herein.
In one embodiment, the second agent is administered in a dosage to achieve a synergistic effect.
In one embodiment, the second agent is administered together with the soluble derivatized chitosan (e.g., in the same derivatized chitosan or dosage form).
In one embodiment, the medical device further comprises an antibiotic, antiinflammatory, or mucolytic (expectorant) compound to a subject in conjunction with, prior to or subsequent to the administration of the derivatized chitosan.
In one embodiment, the antibiotic, anti-inflammatory, or mucolytic (expectorant) compound is administered in a dosage to achieve a synergistic effect.
In one embodiment, the biofilm is in the respiratory system (e.g., airways, lung, throat, nose).
In one embodiment, the subject is infected with planktonic bacteria.
In one embodiment, the derivatized chitosan reduces the viability of planktonic bacteria.
In one embodiment, the derivatized chitosan reduces colonization of the planktonic bacteria.
In one embodiment, the planktonic bacteria arc clumped, e.g., to facilitate removal by expectoration, lavage, or chest percussion.
In one embodiment, the derivatized chitosan is administered by inhalation (e.g., nebulizer, nasal spray, nasal swab, or sinus spray).
In one embodiment, the medical device comprises an effective amount (e.g., therapeutically effective amount) of derivatized chitosan.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
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In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan comprises a chitosan of the following formula (I):
2019202473 09 Apr 2019
Figure AU2019202473A1_D0262
fonnula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of fonnula (II):
Figure AU2019202473A1_D0263
R3 fonnula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, Cj-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of fonnula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (II).
In one embodiment, between 4-30% of R1 substituents are a group of formula (Π).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
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In one embodiment, R9 is amino and R3 is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
2019202473 09 Apr 2019 »ΛΛ/νν
Figure AU2019202473A1_D0264
Figure AU2019202473A1_D0265
In one embodiment, R2 is amino and R3 is a lysine side chain.
Figure AU2019202473A1_D0266
In one embodiment, R2 is amino and R3 is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0267
.Λ/WV
Figure AU2019202473A1_D0268
Figure AU2019202473A1_D0269
In one embodiment, at least 1 % of R1 substituents are selected from one of the
Figure AU2019202473A1_D0270
uww
Figure AU2019202473A1_D0271
Figure AU2019202473A1_D0272
Figure AU2019202473A1_D0273
and at least 1% of R1 substituents are selected from the following:
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Figure AU2019202473A1_D0274
In one embodiment, R2 is amino and R3 is a substituted Ct-C6 alkyl. In one embodiment, R3 is C1-C6 alkyl substituted with an amino group.
In one embodiment, R3 is Ci alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, R3 is C6 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0275
In one embodiment, RJ is Ct-C6 alkyl substituted with a guanidino group.
In one embodiment, R3 is C| alkyl substituted with a guanidino group.
In one embodiment, R3 is C2 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, R3 is C5 alkyl substituted with a guanidino group.
In one embodiment, RJ is C6 alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0276
100
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In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
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In one embodiment, the nitrogen protecting group is tert-butyloxycarbonyl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
In one embodiment, R? is amino.
In one embodiment, R2 is hydrogen and R3 is amino.
In one embodiment, R2 is hydrogen and R3 is guanidino.
In one embodiment, R2 is hydrogen and R3 is a substituted CrC6 alkyl.
In one embodiment, R3 is C|-C6 alkyl substituted with an amino group.
In one embodiment, R3 is C| alkyl substituted with an amino group. In one embodiment, RJ is C2 alkyl substituted with an amino group. In one embodiment, R3 is C3 alkyl substituted with an amino group. In one embodiment, RJ is C4 alkyl substituted with an amino group. In one embodiment, R3 is C5 alkyl substituted with an amino group. In one embodiment, R3 is C6 alkyl substituted with an amino group. In one embodiment, R1 is selected from one of the following:
wvw
Figure AU2019202473A1_D0277
nh2
Figure AU2019202473A1_D0278
•JWW
Figure AU2019202473A1_D0279
In one embodiment, RJ is C|-C6 alkyl substituted with a guanidino group.
In one embodiment, R3 is C i alkyl substituted with a guanidino group.
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In one embodiment, R3 is C? alkyl substituted with a guanidino group.
In one embodiment, RJ is C3 alkyl substituted with a guanidino group. In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, R3 is C5 alkyl substituted with a guanidino group. In one embodiment, R3 is C6 alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0280
NH2
In one embodiment, at least 25% of Rl substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment claim, the chitosan is functionalized at between 20% and 30%.
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In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the medical device has less than about 20%, 15%, 10%, 5%, 2%, or 1 %, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the medical device has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a nebulizer comprising a solution or powder of soluble derivatized chitosan described herein.
In one embodiment, the subject has a bacterial infection, e.g., bacteria list in Table 3, e.g., Pseudomonas aeruginosa.
In one embodiment, the subject comprises at least one biofilm.
In one embodiment, exopolysaccharides (EPS) of the biolim comprises alginate and/or polysaccharide synthesis locus (Psi) (e.g., in Pseudomonas aeruginosa)·, acidic polysaccharide (e.g., in Burkholderia cepacia)·, collanic acid, poly-P-l,6-GlcNAc (PGA) or cellulose (e.g., in Escherichia coli); cellulose (e.g., in Salmonella)·, N-acetylglucosamine (GlcNAc), D-mannose, 6-deoxy-D-galactose and D-galactose (e.g., in Vibrio cholerae)·, polysaccharide intercellular adhesion (PIA) (e.g., in Staphylococcus)·, glucose and mannose rich component (e.g., in Bacillus subtilis)·, mannose polysaccharide (e.g., in Prevotella intermedia, Capnocytophaga ochracea, or Prevotella nigrescens).
In one embodiment, the biofilm is associated with actin and/or DNA released from bacteria or cells such as neutrophils.
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In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 10, 20, 30,40, 50,60, 70, 80, 90, 95, or 99%, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 1, 2, 5, 10, 50, 100, 200, 500, or 1000 fold, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the biofilm is partially dissolved, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.999% of the biofilm is dissolved, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the nebulizer further comprises an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic surfactant).
In one embodiment, the second agent is administered in a dosage to achieve a synergistic effect.
In one embodiment, the second agent is administered together with the soluble derivatized chitosan (e.g., in the same derivatized chitosan or dosage form).
In one embodiment, the nebulizer further comprises an antibiotic, antiinflammatory, or mucolytic (expectorant) compound to a subject in conjunction with, prior to or subsequent to the administration of the derivatized chitosan.
In one embodiment, the antibiotic, anti-inflammatory, or mucolytic (expectorant) compound is administered in a dosage to achieve a synergistic effect.
In one embodiment, the biofilm is in the respiratory system (e.g., airways, lung, throat, nose).
In one embodiment, the subject is infected with planktonic bacteria.
In one embodiment, the denvatized chitosan reduces the viability of planktonic bacteria.
In one embodiment, the derivatized chitosan reduces colonization of the planktonic bacteria.
In one embodiment, the planktonic bacteria are clumped, e.g., to facilitate removal by expectoration, lavage, or chest percussion.
In one embodiment, the derivatized chitosan is administered by inhalation (e.g., nebulizer, nasal spray, nasal swab, or sinus spray).
In one embodiment, the nebulizer comprises an effective amount (e.g., therapeutically effective amount) of derivatized chitosan.
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In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan comprises a chitosan of the following formula (I):
Figure AU2019202473A1_D0281
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
Figure AU2019202473A1_D0282
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, Ci-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (Π).
In one embodiment, between 4-30% of R1 substituents are a group of formula (II).
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In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
In one embodiment, R2 is amino and R’ is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
2019202473 09 Apr 2019
Figure AU2019202473A1_D0283
Figure AU2019202473A1_D0284
In one embodiment, R2 is amino and R' is a lysine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0285
Figure AU2019202473A1_D0286
In one embodiment, R' is amino and RJ is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
<ZWW JUWJ
Figure AU2019202473A1_D0287
In one embodiment, at least 1% of R1 substituents are selected from one of the
Figure AU2019202473A1_D0288
and at least 1% of R1 substituents are selected from the following:
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Figure AU2019202473A1_D0289
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Figure AU2019202473A1_D0290
In one embodiment, R? is amino and R3 is a substituted C,-C6 alkyl.
In one embodiment, R is C|-C6 alkyl substituted with an amino group.
In one embodiment, RJ is C| alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, R3 is Cf, alkyl substituted with an amino group. In one embodiment, R1 is selected from one of the following:
JVVW .ΛΛΛΛΖ JWW .ΛΛΛΛΤ TyXW
Figure AU2019202473A1_D0291
In one embodiment, R3 is C|-C6 alkyl substituted with a guanidino group. In one embodiment, RJ is C, alkyl substituted with a guanidino group. In one embodiment, RJ is C-> alkyl substituted with a guanidino group. In one embodiment, R3 is C3 alkyl substituted with a guanidino group. In one embodiment, R3 is C4 alkyl substituted with a guanidino group. In one embodiment, R3 is C'j alkyl substituted with a guanidino group. In one embodiment, R3 is C2) alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0292
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In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
In one embodiment, the nitrogen protecting group is terf-butyloxycarbonyl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
In one embodiment, R2 is amino.
In one embodiment, R2 is hydrogen and R’ is amino.
In one embodiment, R2 is hydrogen and R3 is guanidino.
In one embodiment, R2 is hydrogen and R3 is a substituted C|-C6 alkyl.
In one embodiment, R3 is C i -C6 alkyl substituted with an amino group.
In one embodiment, R3 is Ct alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an ammo group.
In one embodiment, R' is C3 alkyl substituted with an ammo group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, RJ is ϋβ alkyl substituted with an amino group.
Figure AU2019202473A1_D0293
In one embodiment, RJ is CrC6 alkyl substituted with a guanidino group.
In one embodiment, RJ is C, alkyl substituted with a guanidino group.
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In one embodiment, RJ is C2 alkyl substituted with a guanidino group. In one embodiment, R3 is C3 alkyl substituted with a guanidino group. In one embodiment, R3 is C4 alkyl substituted with a guanidino group. In one embodiment, R3 is C5 alkyl substituted with a guanidino group. In one embodiment, R3 is C6 alkyl substituted with a guanidino group. In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0294
Figure AU2019202473A1_D0295
Figure AU2019202473A1_D0296
Figure AU2019202473A1_D0297
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and 30%.
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In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the nebulizer has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the nebulizer has less than about 20%, 15%, 10%, 5%, 2%, or 1 %, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a medical device constructed to contact a wound of a subject, the medical device comprising a soluble derivatized chitosan described herein. In one embodiment, the surface of the device is coated with a soluble derivatzed chitosan. Exemplary wounds include wounds on the skin of a subject, such as a wound that damages and/or penetrates the skin of a subject.
In one embodiment, the subject has a bacterial infection, e.g., bacteria list in Table 4.
In one embodiment, the subject comprises at least one biofilm.
In one embodiment, exopolysaccharides (EPS) of the biolim comprises alginate and/or polysaccharide synthesis locus (Psi) (e.g., in Pseudomonas aeruginosa); acidic polysaccharide (e.g., in Burkholderia cepacia); collanic acid, ροΙγ-β-Ι,ό-GlcNAc (PGA) or cellulose (e.g., in Escherichia coli); cellulose (e.g., in Salmonella); N-acetylglucosamine (GlcNAc), D-mannose, 6-deoxy-D-galactose and D-galactose (e.g., in Vibrio cholerae); polysaccharide intercellular adhesion (PIA) (e.g., in Staphylococcus); glucose and mannose rich component (e.g., in Bacillus
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In one embodiment, the biofilm is associated with actin and/or DNA released from bacteria or cells such as neutrophils.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99%, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 1,2, 5,10, 50, 100, 200, 500, or 1000 fold, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the biofilm is partially dissolved, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.999% of the biofilm is dissolved, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the medical device further comprises an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic surfactant).
In one embodiment, the second agent is administered in a dosage to achieve a synergistic effect.
In one embodiment, the second agent is administered together with the soluble derivatized chitosan (e.g., in the same derivatized chitosan or dosage form).
In one embodiment, the medical device further comprises an antibiotic, antiinflammatory, or mucolytic (expectorant) compound to a subject in conjunction with, prior to or subsequent to the administration of the derivatized chitosan.
In one embodiment, the antibiotic, anti-inflammatory, or mucolytic (expectorant) compound is administered in a dosage to achieve a synergistic effect.
In one embodiment, the biofilm is in the wound.
In one embodiment, the subject is infected with planktonic bacteria.
In one embodiment, the derivatized chitosan reduces the viability of planktonic bacteria.
In one embodiment, the derivatized chitosan reduces colonization of the planktonic bacteria.
In one embodiment, the derivatized chitosan is administered topically.
In one embodiment, the medical device comprises an effective amount (e.g., therapeutically effective amount) of derivatized chitosan.
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In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 5.0 to about pH 6.0, e.g., in the wound.
In one embodiment, the derivatized chitosan comprises a chitosan of the following formula (I):
Figure AU2019202473A1_D0298
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
Figure AU2019202473A1_D0299
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, fonns a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, C|-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of fonnula (II).
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In one embodiment, between 4-30% of R1 substituents are a group of formula
2019202473 09 Apr 2019 (Π).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
In one embodiment, R2 is amino and R3 is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0300
JVWV
Figure AU2019202473A1_D0301
Figure AU2019202473A1_D0302
In one embodiment, R7 is amino and R3 is a lysine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0303
Figure AU2019202473A1_D0304
In one embodiment, R7 is amino and R3 is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0305
Figure AU2019202473A1_D0306
Figure AU2019202473A1_D0307
In one embodiment, at least 1% of R1 substituents are selected from one of the following:
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Figure AU2019202473A1_D0308
Figure AU2019202473A1_D0309
and at least 1 % of R1 substituents are selected from the following:
Figure AU2019202473A1_D0310
Figure AU2019202473A1_D0311
In one embodiment, R2 is amino and R3 is a substituted C]-C6 alkyl.
In one embodiment, R3 is Cj-Ci alkyl substituted with an amino group.
In one embodiment, R3 is Ci alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0312
Figure AU2019202473A1_D0313
Figure AU2019202473A1_D0314
Figure AU2019202473A1_D0315
In one embodiment, R3 is C|-C6 alkyl substituted with a guanidino group.
In one embodiment, R3 is C, alkyl substituted with a guanidino group.
In one embodiment, RJ is C2 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0316
I 14
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In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
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In one embodiment, the nitrogen protecting group is /e/7-butyloxycarbonyl (Boe).
In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
In one embodiment, R2 is amino.
In one embodiment, R2 is hydrogen and R3 is amino.
In one embodiment, R2 is hydrogen and R3 is guanidino.
In one embodiment, R2 is hydrogen and R3 is a substituted Ci-Ce alkyl.
In one embodiment, RJ is Cj-C6 alkyl substituted with an amino group. In one embodiment, R3 is Ci alkyl substituted with an amino group.
In one embodiment, RJ is C? alkyl substituted with an amino group. In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group. In one embodiment, R3 is C.s alkyl substituted with an amino group.
In one embodiment, R
Figure AU2019202473A1_D0317
Figure AU2019202473A1_D0318
is selected from one of the following:
Figure AU2019202473A1_D0319
In one embodiment, R3 is C1-C6 alkyl substituted with a guanidino group.
In one embodiment, RJ is C( alkyl substituted with a guanidino group.
In one embodiment, R3 is C? alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
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Figure AU2019202473A1_D0320
Figure AU2019202473A1_D0321
Figure AU2019202473A1_D0322
Figure AU2019202473A1_D0323
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and 30%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
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In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In another aspect, the invention features a surface skin decontaminant comprising a derivatized chitosan described herein, e.g., used prior to surgery in a subject.
In one embodiment, the subject has a bacterial infection, e.g., bacteria list in Table 4.
In one embodiment, the subject comprises at least one biofilm.
In one embodiment, exopolysaccharides (EPS) of the biolim comprises alginate and/or polysaccharide synthesis locus (Psi) (e.g., in Pseudomonas aeruginosa): acidic polysaccharide (e.g., in Burkholderia cepacia): collanic acid, poly-β-1,6-GlcNAc (PGA) or cellulose (e.g., in Escherichia coli): cellulose (e.g., in Salmonella): N-acetylglucosamine (GlcNAc), D-mannose, 6-deoxy-D-galactose and D-galactose (e.g., in Vibrio cholerae): polysaccharide intercellular adhesion (PIA) (e.g., in Staphylococcus): glucose and mannose rich component (e.g., in Bacillus subtilis): mannose polysaccharide (e.g., in Prevotella intermedia, Capnocytophaga ochracea, or Prevotella nigrescens).
In one embodiment, the biofilm is associated with actin and/or DNA released from bacteria or cells such as neutrophils.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99%, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 1, 2, 5, 10, 50, 100, 200, 500, or 1000 fold, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the biofilm is partially dissolved, e.g, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.999% of the biofilm is dissolved, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the decontaminant further comprises an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic
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In one embodiment, the second agent is administered in a dosage to achieve a synergistic effect.
In one embodiment, the second agent is administered together with the soluble derivatized chitosan (e.g., in the same derivatized chitosan or dosage form).
In one embodiment, the decontaminant further comprises an antibiotic, antiinflammatory, or mucolytic (expectorant) compound to a subject in conjunction with, prior to or subsequent to the administration of the derivatized chitosan.
In one embodiment, the antibiotic, anti-inflammatory, or mucolytic (expectorant) compound is administered in a dosage to achieve a synergistic effect.
In one embodiment, the biofilm is in the wound.
In one embodiment, the subject is infected with planktonic bacteria.
In one embodiment, the derivatized chitosan reduces the viability of planktonic bacteria.
In one embodiment, the derivatized chitosan reduces colonization of the planktonic bacteria.
In one embodiment, the derivatized chitosan is administered topically.
In one embodiment, the decontaminant comprises an effective amount (e.g., therapeutically effective amount) of derivatized chitosan.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 5.0 to about pH 6.0, e.g., in the wound.
In one embodiment, the derivatized chitosan comprises a chitosan of the following formula (I):
Figure AU2019202473A1_D0324
formula (I) wherein:
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2019202473 09 Apr 2019 wvw
Figure AU2019202473A1_D0325
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, C|-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (II).
In one embodiment, between 4-30% of R1 substituents are a group of formula (Π).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
In one embodiment, R2 is amino and R3 is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0326
Figure AU2019202473A1_D0327
In one embodiment, R~ is amino and R3 is a lysine side chain.
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In one embodiment, R1 is selected from one of the following:
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Figure AU2019202473A1_D0328
Figure AU2019202473A1_D0329
In one embodiment, R2 is amino and R3 is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0330
Figure AU2019202473A1_D0331
Figure AU2019202473A1_D0332
In one embodiment, at least 1% of R1 substituents are selected from one of the following:
Figure AU2019202473A1_D0333
Figure AU2019202473A1_D0334
and at least 1 % of R1 substituents are selected from the following:
Figure AU2019202473A1_D0335
In one embodiment, R2 is amino and R3 is a substituted C|-C6 alkyl.
In one embodiment, RJ is C|-C6 alkyl substituted with an amino group.
In one embodiment, R3 is C| alkyl substituted with an amino group.
In one embodiment, R3 is C? alkyl substituted with an amino group.
In one embodiment, R' is C3 alkyl substituted with an amino group.
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In one embodiment, R3 is C.4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, R3 is Ck alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
2019202473 09 Apr 2019
Figure AU2019202473A1_D0336
Figure AU2019202473A1_D0337
Figure AU2019202473A1_D0338
Figure AU2019202473A1_D0339
In one embodiment, R3 is C1-C6 alkyl substituted with a guanidino group.
In one embodiment, R3 is C, alkyl substituted with a guanidino group.
In one embodiment, R3 is C2 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, R3 is C5 alkyl substituted with a guanidino group.
In one embodiment, R’ is Cr, alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0340
In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
In one embodiment, the nitrogen protecting group is /ert-butyloxycarbonvl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
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In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
In one embodiment, R2 is amino.
In one embodiment, R2 is hydrogen and R3 is amino.
In one embodiment, R2 is hydrogen and R3 is guanidino.
In one embodiment, R2 is hydrogen and RJ is a substituted Cj-C6 alkyl.
In one embodiment, R3 is Ci-Ce alkyl substituted with an amino group.
In one embodiment, R3 is C| alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, R3 is Cf, alkyl substituted with an amino group.
Figure AU2019202473A1_D0341
In one embodiment, R3 is C1-C6 alkyl substituted with a guanidino group. In one embodiment, RJ is C| alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, RJ is C\ alkyl substituted with a guanidino group.
In one embodiment, R3 is C(, alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
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Figure AU2019202473A1_D0342
nh2
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and 30%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
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In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the decontaminant has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the decontaminant has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a residual surface skin decontaminant comprising a derivatized chitosan described herein, e.g., used post surgery in a subject.
In one embodiment, the subject has a bacterial infection, e.g., bacteria list in Table 4.
In one embodiment, the subject comprises at least one biofilm.
In one embodiment, exopolysaccharides (EPS) of the biolim comprises alginate and/or polysaccharide synthesis locus (Psi) (e.g., in Pseudomonas aeruginosa)·, acidic polysaccharide (e.g., in Burkholderia cepacia)·, collanic acid, poly-β-1,6-GlcNAc (PGA) or cellulose (e.g., in Escherichia coll)·, cellulose (e.g., in Salmonella)·, N-acetylglucosamine (GlcNAc), D-mannose, 6-deoxy-D-galactose and D-galactose (e.g., in Vibrio cholerae)·, polysaccharide intercellular adhesion (PIA) (e.g., in Staphylococcus)·, glucose and mannose rich component (e.g., in Bacillus subtilis); mannose polysaccharide (e.g., in Prevotella intermedia, Capnocytophaga ochracea, or Prevotella nigrescens).
In one embodiment, the biofilm is associated with actin and/or DNA released from bacteria or cells such as neutrophils.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99%, compared to the biofilm that has not been contacted with the derivatized chitosan.
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In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 1, 2, 5, 10, 50, 100,200, 500, or 1000 fold, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the biofilm is partially dissolved, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.999% of the biofilm is dissolved, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the decontaminant further comprises an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic surfactant). In one embodiment, the second agent comprises another chitosan derivative, e.g., another chitosan derivative described herein.
In one embodiment, the second agent is administered in a dosage to achieve a synergistic effect.
In one embodiment, the second agent is administered together with the soluble derivatized chitosan (e.g., in the same derivatized chitosan or dosage form).
In one embodiment, the decontaminant further comprises an antibiotic, antiinflammatory, or mucolytic (expectorant) compound to a subject in conjunction with, prior to or subsequent to the administration of the derivatized chitosan.
In one embodiment, the antibiotic, anti-inflammatory, or mucolytic (expectorant) compound is administered in a dosage to achieve a synergistic effect.
In one embodiment, the biofilm is in the wound.
In one embodiment, the subject is infected with planktonic bacteria.
In one embodiment, the derivatized chitosan reduces the viability of planktonic bacteria.
In one embodiment, the derivatized chitosan reduces colonization of the planktonic bacteria.
In one embodiment, the derivatized chitosan is administered topically.
In one embodiment, the decontaminant comprises an effective amount (e.g., therapeutically effective amount) of derivatized chitosan.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 5.0 to about pH 6.0, e.g., in the wound.
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In one embodiment, the derivatized chitosan comprises a chitosan of the
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Figure AU2019202473A1_D0343
fonnula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of fonnula (II):
Figure AU2019202473A1_D0344
R3 fonnula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R~ is hydrogen or amino; and
R’ is amino, guanidino, Ci-Ce alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (II).
In one embodiment, between 4-30% of R1 substituents are a group of fonnula (II).
In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of fonnula (II).
In one embodiment, R2 is amino and R3 is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
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Figure AU2019202473A1_D0345
In one embodiment, R* is amino and R is a lysine side chain.
In one embodiment, R1 is selected from one of the following:
jww
Figure AU2019202473A1_D0346
Figure AU2019202473A1_D0347
In one embodiment, R2 is amino and R3 is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0348
In one embodiment, at least 1% of R1 substituents are selected from one of the following:
Figure AU2019202473A1_D0349
Figure AU2019202473A1_D0350
and at least 1% of R1 substituents are selected from the following:
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Figure AU2019202473A1_D0351
In one embodiment, R is amino and R3 is a substituted C]-C6 alkyl.
In one embodiment, RJ is Ci-C6 alkyl substituted with an amino group.
In one embodiment, RJ is Cj alkyl substituted with an amino group.
In one embodiment, RJ is C2 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, RJ is C6 alkyl substituted with an amino group.
In one embodiment, Rl is selected from one of the following:
Figure AU2019202473A1_D0352
Figure AU2019202473A1_D0353
'nh2
Figure AU2019202473A1_D0354
Figure AU2019202473A1_D0355
In one embodiment, R3 is C|-C6alkyl substituted with a guanidino group.
In one embodiment, RJ is C| alkyl substituted with a guanidino group.
In one embodiment, R3 is C2 alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, RJ is C5 alkyl substituted with a guanidino group.
In one embodiment, R3 is Cs alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0356
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In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
In one embodiment, the nitrogen protecting group is iert-butyloxycarbonyl (Boc).
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
In one embodiment, R2 is amino.
In one embodiment, R2 is hydrogen and R3 is amino.
In one embodiment, R2 is hydrogen and RJ is guanidino.
In one embodiment, R2 is hydrogen and RJ is a substituted Ci-Ce alkyl.
In one embodiment, RJ is C|-C6 alkyl substituted with an amino group.
In one embodiment, R3 is Ci alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, R3 is Cb alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, RJ is Ch alkyl substituted with an amino group.
Figure AU2019202473A1_D0357
In one embodiment, RJ is Ci-Cf, alkyl substituted with a guanidino group. In one embodiment, RJ is C1 alkyl substituted with a guanidino group.
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In one embodiment, RJ is C? alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, RJ is C5 alkyl substituted with a guanidino group.
In one embodiment, R3 is Ct alkyl substituted with a guanidino group.
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In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0358
Figure AU2019202473A1_D0359
Figure AU2019202473A1_D0360
Figure AU2019202473A1_D0361
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of fonnula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and 30%.
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In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the decontaminant has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the decontaminant has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
In another aspect, the invention features a method of treating a surface skin in a subject, the method comprising:
administering to the subject an effective amount of a composition comprising a soluble derivatized chitosan, thereby treating the surface skin.
In one embodiment, the skin is treated before a surgery.
In one embodiment, the skin is treated after a surgery.
In one embodiment, the subject has a bacterial infection, e.g., bacteria list in Table 4.
In one embodiment, the subject comprises at least one biofilm.
In one embodiment, exopolysaccharides (EPS) of the biolim comprises alginate and/or polysaccharide synthesis locus (Psi) (e.g., in Pseudomonas aeruginosa); acidic polysaccharide (e.g., in Burkholderia cepacia); collanic acid, poly-P-l,6-GlcNAc (PGA) or cellulose (e.g., in Escherichia coli); cellulose (e.g., in Salmonella); N-acetylglucosamine (GlcNAc), D-mannose, 6-deoxy-D-galactose and D-galactose (e.g., in Vibrio cholerae); polysaccharide intercellular adhesion (PIA) (e.g., in Staphylococcus); glucose and mannose rich component (e.g., in Bacillus
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In one embodiment, the biofilm is associated with actin and/or DNA released from bacteria or cells such as neutrophils.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99%, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the viscosity (e.g., apparent viscosity) of the biofilm is reduced by at least 1,2, 5,10, 50, 100, 200, 500, or 1000 fold, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the biofilm is partially dissolved, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.999% of the biofilm is dissolved, compared to the biofilm that has not been contacted with the derivatized chitosan.
In one embodiment, the method further comprises administering an additional agent, e.g., a pharmaceutical agent, or a non-pharmaceutical agent (e.g., a non-toxic surfactant). In one embodiment, the second agent comprises another chitosan derivative, e.g., another chitosan derivative described herein.
In one embodiment, the second agent is administered in a dosage to achieve a synergistic effect.
In one embodiment, the second agent is administered together with the soluble derivatized chitosan (e.g., in the same derivatized chitosan or dosage form).
In one embodiment, the method further comprises administering an antibiotic, anti-inflammatory, or mucolytic (expectorant) compound to a subject in conjunction with, prior to or subsequent to the administration of the derivatized chitosan.
In one embodiment, the antibiotic, anti-inflammatory, or mucolytic (expectorant) compound is administered in a dosage to achieve a synergistic effect.
In one embodiment, the biofilm is in the wound.
In one embodiment, the subject is infected with planktonic bacteria.
In one embodiment, the derivatized chitosan reduces the viability of planktonic bacteria.
In one embodiment, the derivatized chitosan reduces colonization of the planktonic bacteria.
In one embodiment, the derivatized chitosan is administered topically.
In one embodiment, the effective amount is therapeutically effective amount.
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In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
In one embodiment, the derivatized chitosan is soluble in aqueous solution from about pH 5.0 to about pH 6.0, e.g., in the wound.
In one embodiment, the derivatized chitosan comprises a chitosan of the following formula (I):
HO
HO
-OH formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, Ci-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
In one embodiment, between 25-95% of R1 substituents are hydrogen.
In one embodiment, between 55-90% of R1 substituents are hydrogen.
In one embodiment, between 1-50% of R1 substituents are acetyl.
In one embodiment, between 4-20% of R1 substituents are acetyl.
In one embodiment, between 2-50% of R1 substituents are a group of formula (II).
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In one embodiment, between 4-30% of R1 substituents are a group of formula
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In one embodiment, 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, 4-30% of R1 substituents are a group of formula (II).
In one embodiment, R2 is amino and R3 is an arginine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0362
»A/WW
Figure AU2019202473A1_D0363
Figure AU2019202473A1_D0364
In one embodiment, R7 is amino and R3 is a lysine side chain.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0365
uww
Figure AU2019202473A1_D0366
In one embodiment, R? is amino and R3 is a histidine side chain.
In one embodiment, R1 is selected from one of the following:
</ww
Figure AU2019202473A1_D0367
»/WW
Figure AU2019202473A1_D0368
Figure AU2019202473A1_D0369
In one embodiment, at least 1% of R1 substituents are selected from one of the following:
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Figure AU2019202473A1_D0370
Figure AU2019202473A1_D0371
Figure AU2019202473A1_D0372
In one embodiment, R2 is amino and R3 is a substituted C1-C6 alkyl.
In one embodiment, R3 is Cj-C6 alkyl substituted with an amino group.
In one embodiment, R3 is C’i alkyl substituted with an amino group.
In one embodiment, RJ is C? alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, RJ is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C5 alkyl substituted with an amino group.
In one embodiment, R3 is C7 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0373
In one embodiment, RJ is C1-C6 alkyl substituted with a guanidino group. In one embodiment, R3 is C| alkyl substituted with a guanidino group. In one embodiment, RJ is C? alkyl substituted with a guanidino group. In one embodiment, R3 is C3 alkyl substituted with a guanidino group. In one embodiment, RJ is C4 alkyl substituted with a guanidino group. In one embodiment, RJ is C5 alkyl substituted with a guanidino group. In one embodiment, RJ is C6 alkyl substituted with a guanidino group.
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In one embodiment, R1 is selected from one of the following:
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Figure AU2019202473A1_D0374
In one embodiment, R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
In one embodiment, the derivatized chitosan is made by reacting a chitosan (e.g., a free amino group of one or more of glucosamine monomers of the chitosan) with an amino acid (e.g., a carboxylic acid moiety of the amino acid) wherein the amino group of the amino acid is protected by a protecting group (e.g., Boc). The protecting group can be removed, e.g., by exposure to acid of pH < 3, after the synthesis.
In one embodiment, the nitrogen protecting group is lerl-butyloxycarbonyl (Boc).
In one embodiment, in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group such as Boc.
In one embodiment, R2 is amino.
In one embodiment, R2 is hydrogen and R3 is amino.
In one embodiment, R2 is hydrogen and R3 is guanidino.
In one embodiment, R2 is hydrogen and R3 is a substituted Ci-C6alkyl.
In one embodiment, R3 is C|-C<, alkyl substituted with an amino group.
In one embodiment, RJ is C| alkyl substituted with an amino group.
In one embodiment, R3 is C2 alkyl substituted with an amino group.
In one embodiment, RJ is C3 alkyl substituted with an amino group.
In one embodiment, R3 is C4 alkyl substituted with an amino group.
In one embodiment, R3 is C3 alkyl substituted with an amino group.
In one embodiment, RJ is C6 alkyl substituted with an amino group.
In one embodiment, R1 is selected from one of the following:
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Figure AU2019202473A1_D0375
nh2 η nh2
In one embodiment, R3 is Ci-Ce alkyl substituted with a guanidino group.
In one embodiment, R3 is C, alkyl substituted with a guanidino group.
In one embodiment, R3 is C? alkyl substituted with a guanidino group.
In one embodiment, R3 is C3 alkyl substituted with a guanidino group.
In one embodiment, R3 is C4 alkyl substituted with a guanidino group.
In one embodiment, R3 is C5 alkyl substituted with a guanidino group.
In one embodiment, R3 is C6 alkyl substituted with a guanidino group.
In one embodiment, R1 is selected from one of the following:
Figure AU2019202473A1_D0376
NH2
In one embodiment, at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
In one embodiment, the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
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In one embodiment, the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da.
In one embodiment, the molecular weight of the derivatized chitosan is between 20,000 and 40,000 Da.
In one embodiment, the chitosan is functionalized at between 5% and 50%.
In a preferred embodiment, the chitosan is functionalized at between 20% and 30%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
In one embodiment, the degree of deacetylation (%DDA) of the derivatized chitosan is between 80% and 90%.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
In one embodiment, the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
In one embodiment, the derivatized chitosan is substantially free of other impurities, e.g., salt, e.g., NaCl.
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer wherein one or more of the nitrogencontaining groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In one embodiment, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
A definition of the specific embodiment of the invention claimed herein follows. In a broad format, the invention provides a method of rinsing nasal tissue with an aqueous composition, the method comprising: administering to a subject’s nasal tissue an effective amount of a composition comprising:
a sweetening agent; and a soluble derivatized chitosan; wherein the derivatized chitosan comprises a chitosan of the following formula (I):
OH /OH \ OH
--Λ- -°x I a -o
λ Αχ-θΗ
HO~N^ ----- \ hoLv--' HO-
NH I \ NH/n NH I
R1 R1 R1
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n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
Figure AU2019202473A1_D0377
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino;
R3 is amino, guanidino, Ci-Ce alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II); and wherein administering involves a nasal spray, a nasal mist or a sinus rinse.
The term “comprise” and variants of the term such as “comprises” or “comprising” are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required.
Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia or elsewhere.
Brief Description of the Drawings
Figure 1A shows Gram negative Pseudomonas aeruginosa strain PA01 in ultra-pure water at 200x magnification.
Figure IB shows the clumping of Gram negative Pseudomonas aeruginosa strain PA01 after 1 minute of exposure to chitosan-arginine (24 kDa, 28% functionalized, 83% DDA, 1.54 PDI) at 200x magnification.
Figure 2A shows Gram positive Streptococcus mutans (ATCC 35668) in ultra-pure water at 200x magnification.
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Figure 2B shows the clumping of Gram positive Streptococcus mutans (ATCC 35668) after 1 minute of exposure to chitosan-arginine (24 kDa, 28% functionalized, 83% DDA, 1.54 PD1) at 200x magnification.
Figure 3 shows tire clumping of Gram negative Pseudomonas aeruginosa strain
PA01 with chitosan-acid amine (29 kDa, 39% functionalization) in ultra-pure water at 200x magnification.
Figure 4A shows Gram positive methicillin resistant Staphyloccus aureus strain MW2 (clinical isolate from blood/CSF of community acquired disseminating infection) in ultra-pure water at 200x magnification.
Figure 4B shows Gram positive methicillin resistant Staphyloccus aureus strain MW2 (clinical isolate from blood/CSF of community acquired disseminating infection) after 1 minute of exposure to chitosan-acid amine (29 kDa, 38% functionalized) at 200x magnification.
Figure 5 shows Gram negative Pseudomonas aeruginosa strain PA01, after 24 hours exposure to (A) 100 pg/ml chitosan-lactobionic acid (125 kDa), (B) 100 pg/ml chitosan-arginine (28 kDa, 37% functionalization, 89% DDA, 1.95 PDI), (C) 100 pg/ml chitosan-arginine (40 kDa, 37% functionalization, 89% DDA, 2.454 PDI), and (D) water.
Figure 6 shows quantification of the reduction of Gram negative Pseudomonas aeruginosa strain PA01 in solution after 24 hours exposure to lOOpg/ml chitosanarginine (40 kDa, 37% functionalization, 89% DDA, 2.454 PDI), lOOpg/ml chitosanarginine (28 kDa, 37% functionalization, 89% DDA, 1.95 PDI), and lOOpg/ml chitosan-lactobionic acid (125 kDa) shown visually in Figure 5.
Figure 7A shows clumping of Streptococcus mutans (ATCC 35668) by 2pg/ml of chitosan-arginine (32 kDa, 29% functionalization, 83% DDA, 1.5 PDI) after five minutes incubation at 200x magnification.
Figure 7B shows clumping of Streptococcus mutans (ATCC 35668) by lOpg/ml of chitosan-arginine (32 kDa, 29% functionalization, 83% DDA, 1.5 PDI) after five minutes incubation at 200x magnification.
Figure 7C shows clumping of Streptococcus mutans (ATCC 35668) by 20pg/ml of chitosan-arginine (32 kDa, 29% functionalization, 83% DDA, 1.5 PDI) after five minutes incubation at 200x magnification.
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Figure 8A shows methicillin resistant Staphylococcus aureus strain MW-2 (clinical isolate from blood/CSF of community acquired disseminating infection) in ultra pure water (400x magnification).
Figure 8B shows the ability of chitosan-arginine (24 kDa, 28% functionalized, 83% DDA, 1.5 PDI) to clump methicillin resistant Staphylococcus aureus strain MW-2 (clinical isolate from blood/CSF of community acquired disseminating infection) after 1-minute exposure (400x magnification).
Figure 9A shows optical microscope images taken at 20x of THP-1 human monocyte cell line exposed to 100pg/ml of 40 kDa chitosan-arginine 37% functionalized for 24 hours at high cell density.
Figure 9B shows optical microscope images taken at 20x of THP-1 human monocyte cell line exposed to lOOpg/ml of 40 kDa chitosan-arginine 37% functionalized for 24 hours at lower cell density.
Figure 10 shows reduction in the viscosity of 1% sodium alginate biofilm model treated with 100pg/ml chitosan-arginine (18 kDa, 25% functionalization, 88% DDA, 1.47 PDI) compared to an equal volume of water 6 minutes, 1 hour, and 4 hours after addition. * or ** indicates significant difference p=0.01.
Figure 11 shows reduction in the viscosity of Pseudomonas aeruginosa strain PA01 biofilm/spent media after 1-minute treatment with 100pg/ml chitosan-arginine (18 kDa, 25% functionalization, 88 %DDA, 1.47 PDI).
Figure 12 shows the pourability of cystic fibrosis patient sputum samples treated with 100pg/ml chitosan-arginine (43 kDa, 25% functionalization, 88 %DDA, 2.28 PDI).
Figure 13A shows Gram negative Salmonella enterica serotype Typhimurium (ATCC 700720) in ultra-pure water at 200x magnification.
Figure 13B shows clumping of Gram negative Salmonella enterica serotype Typhimurium (ATCC 700720) after 1 minute exposure to chitosan-lactobionic acid (100 kDa) in ultra-pure water at 200x magnification.
Figure 14A shows Gram positive Clostridium perfringens (ATCC 12919) in ultrapure water at 200x magnification.
Figure 14B shows clumping of Gram positive Clostridium perfringens (ATCC 12919) after 1 minute exposure to chitosan-lactobionic acid (100 kDa) in ultra-pure water at 200x magnification.
Figure 15A shows Gram negative Escherichia coli (ATCC 700728) in ultra-pure water at 200x magnification.
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Figure 15B shows clumping of Gram negative Escherichia coli (ATCC 700728) after 1 minute exposure to chitosan-lactobionic acid (100 l<Da) in ultra-pure water at 200x magnification.
Figure 16A shows Gram positive Streptococcus pyogenes (ATTC 700294D-5) in ultra-pure water at 200x magnification.
Figure 16B shows clumping of Gram positive Streptococcus pyogenes (ATTC 700294D-5) after 1 minute exposure to chitosan-lactobionic acid (100 kDa) in ultrapure water at 200x magnification.
Figure 17A shows Gram negative Shigella flexneri (ATCC 29903) in ultra-pure water at 200x magnification.
Figure 17B shows clumping of Gram negative Shigella flexneri (ATCC 29903) after minute exposure to chitosan-lactobionic acid (100 kDa) in ultra-pure water at 200x magnification.
Figure 18A shows Gram positive Streptococcus mutans (ATCC 35668) in ultra-pure water at 200x magnification.
Figure 18B shows clumping of Gram positive Streptococcus mutans (ATCC 35668) after 1 minute exposure to chitosan-lactobionic acid (100 kDa) in ultra-pure water at 200x magnification.
Figure 19A shows Gram positive Staphylococcus aureus strain MW-2 (clinical isolate from blood/CSF of community acquired disseminating infection) in ultra-pure water at 200x magnification.
Figure 19B shows clumping of Gram positive Staphylococcus aureus strain MW-2 (clinical isolate from blood/CSF of community acquired disseminating infection) after 1 minute exposure to chitosan-lactobionic acid (277 kDa) in ultra-pure water at 200x magnification.
Figure 20 shows clumping of Gram negative Pseudomonas aeruginosa strain PA01 after 1 minute exposure to chitosan-lactobionic acid (100 kDa) in ultra-pure water at 200x magnification.
Figure 21 shows the reduction of Staphylococcus aureus strain MW-2 (clinical isolate from blood/CSF of community acquired disseminating infection) biofilms by chitosan-arginine (24 kDa, 28% functionalized, 18 %DDA, 1.5 PDI).
Figure 22 shows the reduction of Gram negative Pseudomonas aeruginosa strain PA01 biofilms by chitosan-arginine (24 kDa, 28% functionalized, 18 %DDA, 1.5 PDI).
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Figure 23 shows the diffusion of chitosan-arginine from 2% and 20% gelatin formulations in disc diffusion assay. + Auto = autoclaved material.
Figure 24 shows chitosan-arginine (21 kDa, 29% functionalized, 88% DDA, 1.49 PDI) prophylactic activity against MRSA MW-2 (ATCC BAA-1707) compared to other skin decontamination solutions after 1-hour on pigskin. Data is qualitative CFU recovered after 1-hour on the treated skin surface.
Figure 25 shows chitosan-arginine (43 kDa, 25% functionalized, 88% DDA, 2.28 PDI) wound rinse activity against MRSA MW-2 (ATCC BAA-1707) contaminated abraded pigskin. Data is CFU recovered 30 minutes after two 5mL rinses.
Figure 26 shows chitosan-arginine (43 kDa, 25% functionalized, 88% DDA, 2.28 PDI) wound rinse activity against Psudomonas aeruginosa PA01 (ATCC BAA-47) contaminated abraded pigskin. Data is CFU recovered 30 minutes after two 5mL rinses.
Figure 27 shows chitosan-arginine (43 l<Da, 25% functionalized, 88% DDA, 2.28 PDI) dose response against stationary MRSA MW-2 (ATCC BAA-1707) 2 day-old biofilms. Data is CFU recovered after 4-hour treatment.
Figure 28 shows chitosan-arginine (43 kDa, 25% functionalized, 88% DDA, 2.28 PDI) dose response against Klebsiella pneumoniae (ATCC 13883) 2 day-old biofilms grown on pegs. Data is CFU recovered after 5-hour treatment. The * indicates sterilization.
Figure 29 shows chitosan-arginine (43 kDa, 25% functionalized, 88% DDA, 2.28 PDI) dose response against Acinetobacter baumannii (ATCC 19606) 2 day-old biofilms grown on pegs. Data is CFU recovered after 3-hour treatment.
Figure 30 shows chitosan-arginine (43 kDa, 25% functionalized, 88% DDA, 2.28 PDI) dose response against Psudomonas aeruginosa (ATCC BAA-47) 2 day-old biofilms grown on pegs. Data is CFU recovered after 3-hour treatment.
Figure 31 depicts mixed wound biofilms (MRSA MW-2 ATCC BAA-1707, Psudomonas aeruginosa ATCC BAA-47, and Vancomycin-resistant Enterococcus faecalis ATCC 51299) grown in a flow cell overnight then treated with either water or 200 pg/mL chitosan-arginine (43kDa, 25% functionalized, 88% DDA, 2.28 PDI) twice daily for two days and finally rinsed and sonicated for 30 seconds and stained with ciystal violet.
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Figure 32 depicts the amount and consistency of material removed following the final rinse from flow cells treated with either water or chitosan-arginine (43kDa, 25% functionalized, 88% DDA, 2.28 PD1) in Figure 32.
Figure 33 depicts the immediate dispersal of Acinetobacter baumannii (ATCC 19606) 2-day old stationary biofilms treated with 100 pg/ml chitosan-arginine (43kDa, 25% functionalized, 88% DDA, 2.28 PDI). The biofilms were rinsed, stained with crystal violet and treated with either water or chitosan-arginine for 5 minutes and rinsed.
Detailed Description
Described herein are methods and compositions that contain a soluble chitosan or chitosan derivative. The compositions can be used, for example, for reducing preformed biofilms, preventing formation of biofilms, treating or preventing complications of cystic fibrosis, or treating or preventing gastrointestinal infections. In some embodiments, a composition described herein can result in the clumping of bacteria, for example, to a soluble chitosan or chitosan derivative. The clumped bacteria can be discarded, for example, expirated by a subject, or can be ingested.
In some embodiments, the soluble chitosans or derivatized chitosans exhibit one or more of the following characteristics: for example, long shelf lives, ability to be stored as a dry powder, or ability to dissolve in water, saline, or other neutral solution (e.g., in the human body) and to be dispersed as needed (e.g., as a solid, semisolid, or liquid composition). Exemplary compounds include, but are not limited to soluble chitosan compounds, chitosan-arginine compounds, chitosan-guanidine compounds, chitosan-unnatural amino acid compounds, chitosan-acid-amine compounds, chitosan-natural amino acid compounds, and co-derivatives of the just described compounds and the salts thereof. These compounds and their antimicrobial activity are disclosed in US Patent applications 11/657,382 and 11/985,057, which is herein incorporated by reference. Exemplary compounds also include neutral chitosan compounds (e.g., monosaccharide-containing chitosan compounds, chitosanlactobionic acid compounds, and chitosan-glycolic acid compounds), and coderivatives of these compounds and the salts thereof.
Biofilm
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Methods and compositions described herein can be used to disrupt (e.g., reduce the viscosity of, or dissolve) a preformed biofilm in a subject. As used herein, the term “dissolve” or “dissolving” means breaking up cohesion in a preformed biofilm such that some or all can be rinsed, flushed or washed away.
A biofilm is a structured community of microorganisms encapsulated within a self-developed polymeric matrix and adherent to a living or inert surface. Biofilms are also often characterized by surface attachment, structural heterogeneity, genetic diversity, complex community interactions, and an extracellular matrix of polymeric substances.
Formation of a biofilm begins with the attachment of free-floating microorganisms to a surface. Colonization begins with adherence to the surface initially through weak, reversible van der Waals forces. If the colonizing bacteria are not immediately separated from the surface, they can anchor themselves more permanently using cell adhesion structures such as pili. The first colonizing bacteria facilitate the arrival of other cells by providing more diverse adhesion sites and beginning to build the matrix that holds the biofilm together. Once colonization has begun, the biofilm grows through a combination of cell division and recruitment. The final stage of biofilm formation is known as development, and is the stage in which the biofilm is established and may only change in shape and size. This development of biofilm environment and communication pathway allows for the cells to become more antibiotic resistant.
Biofilms can contain many different types of microorganisms, e.g. bacteria, archaea, protozoa, fungi and algae; each group performing specialized metabolic functions. Microorganisms can also form monospecies films.
The biofilm is held together and protected by a matrix of excreted polymeric compounds called Extracellular polymeric substance (EPS). This matrix protects the cells within it and facilitates communication among them through biochemical signals.
Bacteria living in a biofilm have different properties from free-floating bacteria of the same species, as the dense and protected environment of the film allows them to cooperate and interact in various ways. One benefit of this environment to the bacteria is increased resistance to detergents and antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community.
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Exemplary bacteria associated with biofilm include Gram-positive (e.g., Staphylococcus aureus (e.g., strain MW-2), Streptococcus mutans, Clostridium perfringens, Streptococcus pyogenes (GAS), Clostridium difficile and Streptococcus sanguis) and Gram-negative bacteria (E. coli (e.g., strain 0:157 H:7), Shigella flexneri, Salmonella typhimurium, Acinetobacter baumannii, Pseudomonas aeruginosa and Legionella bacteria (e.g., L. pneumophila)).
Exemplary oral, gastro and pulmonary bacteria are listed in Tables 1-3, respectively.
Table 1. Exemplary oral bacteria strains
Strain (oral) Description
Streptococcus mutans Causes cavities
Streptococcus sanguinis Causes cavities
Lactobacillus acidophilus Causes cavities
Actinomyces viscosus Causes actinomysis/oral infection
Table 2. Exemplary gastro bacteria strains
Strain (gastro) Description
Escherichia coli Shiga-like toxin producer, such as 0157:H7
Shigella flexneri Shiga toxin producer
Salmonella typhimurium Causes gastroenteritis, food poisoning
Clostridium difficile Causes food poisoning, forms spores
Enterococcus faecalis Vancomycin resistant, gastrointestinal
Helicobacter pylori Gastrointestinal ulcers
Bacillus subtilis Spore former
Listeria monocytogenes Intracellular pathogen
Campylobacter jejuni Causes food poisoning, non-spore former
Staphylococcus aureus Gastroenteritis
Klebsiella pneumoniae Causes pneumonia, many drug resistant strains
Table 3. Exemplary pulmonary bacteria strains
Strain (Pulmonary) Characteristics
Staphylococcus aureus Broadly infective, wounds,body fluids, tissue, pulmonary, highly multi-durg resistant strains including MRSA and mupirocin resistant MRSA
Pseudomonas aeruginosa Causes pneumonia, primary pathogen in patients with cystic fibrosis, many MDR strains, forms thick biofilms
Burkholdaria cepacia genomvar cenocepatia Virulent pathogen in lungs of patients with cystic fibrosis
Acinetobacter baumannii Slow-growing, colonization, causes penumonia
Streptococcus pneumoniae Aquatic bacterium, colonizes breathing and feeding tubes
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Stenotrophomonas maltophilia Similar to pseudomonas, pulmonary infections
Burkholdaria cepacia genomvar dots a Virulent pathogen in lungs of patients with cystic fibrosis
Klebsiella pneumoniae Causes pneumonia and wound infections, many MDR strains
Burkholdaria cepacia complex Virulent pathogen in lungs of patients with cystic fibrosis
Table 4. Exemplary wound strains
Strain (wound) Description
Staphylococcus aureus Senisitive and MDR strains, can produce toxin, found on skin, in wounds, in tissue
Escherichia coli Many resistant strains, found in wounds
Acinetobacter baumannii Wound and tissue infections, slow growing, many MDR strains (including calcoaceticus complex)
Klebsiella pneumoniae Nosocomical and independent wound infections, many MDR strains
Vancomycin resistant enterococcus Higly resistant, thrives in wounds
Pseudomonas aeruginosa Causes wound infections, colonizes medical devices, many MDR strains
In some embodiments, bacteria associated with biofnn can include antibiotic resistant bacteria such as Methicillin resistant Staphylococcus aureus, Mupirocin resisistant Staphylococcus aureus , Mupirocin and Methicillin resistant Staphylococcus aureus , Fluoroquinolone resistant Staphylococcus aureus, Vancomycin intermediate resistant Staphylococcus aureus, Linezolid resistant Staphylococcus aureus, Penicillin resistant Streptococcus pneumoniae, Macrolide resistant Streptococcus pneumoniae, Fluoroquinolone resistant Streptococcus pneumoniae. Vancomycin resistant Enterococcus faecalis, Linezolid resistant Enterococcus faecalis, Fluoroquinolone resistant Enterococcus faecalis, Vancomycin resistant Enterococcus faecium, Linezolid resistant Enterococcus faecium. Fluoroquinolone resistant Enterococcus faecium, Ampicillin resistant Enterococcus faecium, Macrolide resistant Haemophilus influenzae, β-lactam resistant Haemophilus influenzae, Fluoroquinolone resistant Haemophilus influenzae, β-lactam resistant Moraxella catarrhalis, Methicillin resistant Staphylococcus epidermidis, Methicillin resistant Staphylococcus epidermidis, Vancomycin resistant Staphylococcus epidermidis, Fluoroquinolone resistant Staphylococcus epidermidis, Macrolide resistant Mycoplasma pneumoniae, Isoniazid resistant Mycobacterium tuberculosis, Rifampin resistant Mycobacterium tuberculosis, Methicillin resistant coagulase negative Staphylococci, Fluoroquinolone resistant coagulase negative Staphylococci,
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Glycopeptide intermediate resistant Staphylococcus aureus, Vancomycin resistant Staphylococcus aureus, Hetero vancomycin intermediate resistant Staphylococcus aureus, Hetero vancomycin resistant Staphylococcus aureus, Macrolide-LincosamideStreptogramin resistant Staphylococcus, β-lactam resistant Enterococcus faecalis, βlactam resistant Enterococcus faecium, Ketolide resistant Streptococcus pneumoniae, Ketolide resistant Streptococcus pyogenes, Macrolide resistant Streptococcus pyogenes, Vancomycin resistant Staphylococcus epidermidis, multidrug resistant Clostridium difficile, multidrug resistant Acinetibacter baumannii, multidrug resistant Kelbsiella pneumoniae, or multidrug resistant Escherichia coli.
As used herein resistant microorganism or bacterium means, an organism which has become resistant to an antibacterial agent. Also, resistant microorganism or bacterium means its effective MIC has exceeded the effective dosage according to Clinical Laboratory Standards Institute (CLSI) resistance breaktpoints, predefined national or internationally accepted limits, at or above which administration of an effective dose of antibiotic produces undesirable side effects. In some embodiments, the minimum inhibitory concentration of an antibacterial agent for a resistant bacterium will be at least, 2, 5, 10, or 100 fold greater than that seen with a sensitive bacterium for a selected antibacterial agent.
In an embodiment, bacteria associated with biofim can include, e.g., Salmonella choleraesuis. Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter aerogenes, Pseudomonas aeruginosa, MRSA, E. coli, vancomycin resistant Enterococcus faecalis, Acinetobacter baumannii, MDR Acinetobacter baumannii, or MDR Klebsiella pneumoniae.
Biofilms can be associated with a variety of diseases or conditions, e.g., urinary tract infections, catheter infections, middle-ear infections, throat infection, formation of dental plaque, gingivitis, dental caries, halitosis, gastrointestinal tract infections, respiratory tract infections (e.g., airway infections, lung infections, pneumonia and chronic sinusitis), complications of contact lenses, eye infections, conjunctivitis, endocarditis, complications (e.g., infections) of cystic fibrosis, complications (e.g., infections) in immunocompromised patient, impairing cutaneous wound healing, chronic wounds, infections due to bums, reducing topical antibacterial efficacy in infected skin wounds, or infections of pennanent indwelling devices such as joint prostheses, intrauterine devices or heart valves.
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Exemplary bacteria associated with biofilm also include bacteria causing urinary tract infections, catheter infections, middle-ear infections, throat infection, formation of dental plaque, gingivitis, dental caries, halitosis, gastrointestinal tract infections, respiratory tract infections (e.g., airway infections, lung infections, pneumonia and chronic sinusitis), complications of contact lenses, endocarditis, complications (e.g., infections) of cystic fibrosis, complications (e.g., infections) in immunocompromised patient, impairing cutaneous wound healing, infections due to bums, reducing topical antibacterial efficacy in infected skin wounds, or infections of permanent indwelling devices such as joint prostheses, intrauterine devices or heart valves.
Exemplary diseases and conditions associated with biofilm can also include diseases characterized by the presence of one or more of the bacteria that cause resistant bacterial infections as described herein.
Bacterial clumping
Bacterial population, e.g., in a body cavity or epithelial/mucosal surfaces in a subject, can be reduced (e.g., to a level closer to the normal microbial level) by clumping using compounds and compositions described herein. Described herein are also methods of treatment for the colonization of e.g., the ear, nose, throat, sinus, respiratory system, skin, wounds, or gastrointestinal tract by harmful bacteria. This clumping can, in some embodiments, act as a “barrier,” for example, when a composition described herein is used to contact a bacterial population so as to result in clumping of the bacteria onto the soluble chitosan derivative, and the resulting composition is discarded by the subject (e.g., spit out, for example, as an oral rinse).
The method of clumping (e.g., barrier clumping) includes the step of contacting compositions or compounds described herein (e.g., soluble chitosans or derivatized chitosans) with bacteria, e.g., in a body cavity or epithelial/mucosal surfaces. The soluble chitosan or chitosan derivatives described herein can interact with more than one bacterium simultaneously, linking them via contact with a part of the polymer chain. Thus, the contact causes the bacteria to aggregate with one another. These bacterial clumps are limited in their ability to bind to other surfaces, thereby creating a barrier to colonization by the bacteria. This barrier is also a result of the decreased bacterial surface area available for colonization, the bacterial trapping within the aggregate as well as limitation of the exposure of bacterial surface
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The positively charged characteristic of chitosan derivatives, e.g., chitosanarginine, can allow the composition described herein to be effective in clumping and aggregating bacteria. The positively charged polymer can interact with the negatively charged cell wall of the bacteria electrostatically. This, in turn, can allow the long polymer chains of chitosan derivatives, e.g., chitosan-arginine, to interact with the bacterial cell surface and bridge between bacteria cells. This interaction can allow for clumping and aggregation of the bacteria cells and prevents them from adhering to mucosal or tissue surfaces. This mechanical action can allow for easy removal of the bacteria from the surface of a body orifice, wound, gastrointestinal tract or pulmonary surface.
The neutral soluble chitosan derivatives, e.g. chitosan lactobionic acid, can allow the composition described herein to be effective in clumping and aggregating bacteria. The long polymer chains of chitosan derivatives, e.g., chitosan-arginine, interact with the bacterial cell surface and bridge between bacteria cells. This interaction can allow for clumping and aggregation of the bacteria cells and prevents them from adhering to mucosal or tissue surfaces. This mechanical action can allow for easy removal of the bacteria from the surface of a body orifice, wound, gastrointestinal tract or pulmonary surface.
Treatment
The compositions and compounds described herein (e.g., a soluble chitosan or a derivatized chitosan) can be administered to planktonic cells in culture or in biofilms, e.g. in vitro or ex vivo, or to a subject, e.g., in vivo, to treat, prevent, and/or diagnose a variety of disorders, including those described herein below.
As used herein, the term “treat” or “treatment” is defined as the application or administration of a composition or compound (e.g., a compound described herein (e.g., a soluble derivatized chitosan) to a subject, e.g., a patient, or application or administration of the composition or compound to an isolated tissue or cell, e.g., cell line, from a subject, e.g., a patient, who has a disorder (e.g., a disorder as described herein), a symptom of a disorder, or a predisposition toward a disorder, with the
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As used herein, the term “prevent” or “prevention” is defined as the application or administration of a composition or compound (e.g., a compound described herein (e.g., a soluble derivatized chitosan)) to a subject, e.g., a subject who is at risk for a disorder (e.g., a disorder described herein), or has a disposition toward a disorder, or application or administration of the compound to an isolated tissue or cell, e.g., cell line, from a subject, e.g., a subject who is at risk for a disorder (e.g., a disorder as described herein), or has a predisposition toward a disorder, with the purpose to avoid or preclude the disorder, or affect the predisposition toward the disorder (e.g., to prevent at least one symptom of the disorder or to delay onset of at least one symptom of the disorder).
As used herein, an amount of a composition or compound effective to treat a disorder, or a “therapeutically effective amount” refers to an amount of the composition or compound which is effective, upon single or multiple dose administration to a subject, in treating a cell, or in curing, alleviating, relieving or improving a subject with a disorder beyond that expected in the absence of such treatment.
As used herein, an amount of a composition or compound effective to prevent a disorder, or “a prophylactically effective amount” of the composition or compound refers to an amount effective, upon single- or multiple-dose administration to the subject, in preventing or delaying the occurrence of the onset or recurrence of a disorder or a symptom of the disorder.
As used herein, the term “subject” is intended to include human and nonhuman animals. Exemplary human subjects include a human patient having a disorder, e.g., a disorder described herein or a normal subject. The term “non-human animals” of the invention includes all vertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, domesticated and/or agriculturally useful animals, e.g., sheep, dog, cat, cow, pig, etc.
As used herein, administered in combination or a combined administration of two agents means that two or more agents (e.g., compounds described herein) are administered to a subject at the same time or within an interval such that there is
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In a preferred embodiment, the agents are administered essentially simultaneously, for example two unit dosages administered at the same time, or a combined unit dosage of the two agents. In another preferred embodiment, the agents are delivered in separate unit dosages. The agents can be administered in any order, or as one or more preparations that includes two or more agents. In a preferred embodiment, at least one administration of one of the agents, e.g., the first agent, is made within minutes, one, two, three, or four hours, or even within one or two days of the other agent, e.g., the second agent. In some cases, combinations can achieve synergistic results, e.g., greater than additive results, e.g., at least 1.25, 1.5, 2, 4, 10, 20, 40, or 100 times greater than additive.
Subject
The subject can be a human or an animal. Suitable animal subjects include: but are not limited to, pet, wild, zoo, laboratory, and farm animals. Suitable animal subjects include primates, mammals, rodents, and birds. Examples of said animals include, but not limited to, guinea pigs, hamsters, gerbils, rat, mice, rabbits, dogs, cats, horses, pigs, sheep, cows, goats, deer, rhesus monkeys, monkeys, tamarinds, apes, baboons, gorillas, chimpanzees, orangutans, gibbons, fowl, e.g., pheasant, quail (or other gamebirds), a waterfowl, ostriches, chickens, turkeys, ducks, and geese or free flying bird.
In some embodiments, the subject has urinary tract infections, catheter infections, middle-ear infections, throat infections, eye infections, dental plaque,
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In some embodiments, the subject has diseases or conditions characterized by the presence of one or more of the bacteria that cause resistant bacterial infection as described herein.
Cystic Fibrosis
The compositions described herein can be used to treat or prevent complications of cystic fibrosis in a subject. For example, liquid or solid particulate compositions comprising soluble chitosans or derivatized chitosans described herein can be used to treat or prevent complications of cystic fibrosis, e.g., lung infections or respiratory tract congestion, in a subject. Treatment or prevention includes administration of soluble chitosans or derivatized chitosans alone or in combination with drugs or treatments described below.
Cystic Fibrosis (also known as CF, mucovoidosis, or mucoviscidosis) is a hereditary disease affecting the exocrine (mucous) glands of the lungs, liver, pancreas, and intestines, causing progressive disability due to multisystem failure. CF is caused by a mutation in the gene cystic fibrosis transmembrane conductance regulator (CFTR). The product of this gene is a chloride ion channel important in creating sweat, digestive juices and mucus. CF is considered an autosomal recessive disease.
Symptomatic diseases and complications associated with CF include, e.g., lung and sinus diseases; gastrointestinal, liver and pancreatic diseases; endocrine diseases; and infertility. For example, lung disease results from clogging the airways due to mucosa buildup and resulting inflammation. Some of these symptoms occur when bacteria that normally inhabit the thick mucus grow out of control and cause pneumonia. In later stages of CF, changes in the architecture of the lung further
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Complications of CF, e.g., lung diseases, can be treated or prevented using soluble chitosans or derivatized chitosans described herein, in combination with one or more of agents or therapeutics. Exemplary agents to treat complications of CF, e.g., lung diseases include antibiotics such as xylitol, vancomycin, tobramycin, meropenem, ciprofloxacin, or piperacillin, administered e.g., intravenously. Inhaled therapy with antibiotics such as tobramycin, colistin or aztreonam can also be given to improve lung function by impeding the growth of colonized bacteria. Oral antibiotics such as ciprofloxacin or azithromycin can be given to help prevent infection or to control ongoing infection. Other methods to treat lung disease include, e.g., chest physiotherapy (CPT), Biphasic Cuirass Ventilation, or aerosolized medications (e.g., DNase (e.g., domase (Pulmozyme®)), hypertonic saline, Ν-acetylcysteine, albuterol, or ipratropium). In some embodiments, the administrations of a combination of agents and therapeutics are spaced sufficiently close together such that a synergistic effect is achieved.
In one embodiment, a soluble chitosan or derivatized chitosan (e.g., a soluble chitosan or derivatized chitosan described herein) is used in combination with a DNase (e.g., Pulmozyme®) to treat a complication of cystic fibrosis, e.g., a lung disease. For example, the DNase (e.g., Pulmozyme®) can be administered at a dosage of less than about 2.5 mg twice a day, 2.0 mg twice a day, 1.5 mg twice a day, 1.0 mg twice a day, 0.5 mg twice a day, 2.5 mg once daily, 2.0 mg once daily, 1.5 mg once daily, 1.0 mg once daily, or 0.5 mg once daily.
Respiratory tract infections
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The compositions described herein can be used to treat or prevent respiratory tract infections in a subject. For example, liquid or solid particulate compositions comprising soluble chitosans or derivatized chitosans described herein can be used to treat or prevent respiratory tract infections, e.g., respiratory tract bacterial infections, in a subject. Treatment or prevention includes administration of soluble chitosans or derivatized chitosans alone or incombination with drugs or treatments described below.
Respiratory tract infections can be caused by e.g., bacteria, viruses, parasites or fungi. Exemplary respiratory tract bacterial infections include upper respiratory tract infections such as sinusitis, pharygitis, epiglotittis, laryngitis, tracheitis, and rhinitis; and lower respiratory tract infections such as bronchitis and pneumonia.
Symptoms of respiratory tract infections include, e.g., pain, inflammation, fever, fatigue, lack of breath, nausea, diarrhea, cough, and death.
Respiratory tract infections can be treated or prevented using soluble chitosans or derivatized chitosans described herein, in combination with one or more of agents or therapeutics. Exemplary agents and therapeutics to treat respiratory tract infections includes systemic antibiotics, inhaled antibiotics, anti-inflammatory agents and steroids, mucolytic agents, and supplemental oxygen. In some embodiments, the administrations of a combination of agents and therapeutics are spaced sufficiently close together such that a synergistic effect is achieved.
Gastrointestinal tract infections
The compositions described herein can be used to treat or prevent gastrointestinal tract infections in a subject. For example, liquid or solid particulate compositions comprising soluble chitosans or derivatized chitosans described herein can be used to treat or prevent gastrointestinal tract infections, e.g., gastrointestinal tract bacterial infections, in a subject. Treatment or prevention includes administration of soluble chitosans or derivatized chitosans alone or incombination with drugs or treatments described below.
Gastrointestinal tract infections can be caused by e.g., bacteria (e.g., enteric bacteria), viruses, parasites or fungi. Exemplary gastrointestinal tract bacterial infections include noninflammatory gastroenteritis caused by e.g., Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Clostridium difficile or Clostridium botulinum; inflammatory gastroenteritis caused by e.g., Vibrio cholerae,
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Enterotoxigenic (ETEC) Escherichia coli, Enteropathogenic (EPEC) Escherichia coli, Enteroaggregative (EAggEC) Escherichia coli, Clostridium dificile, Vibrio parahemolyticus, or Bacillus anthracis; or invasive gastroenteritis caused by e.g., Shigella sp., Salmonella sp., Campylobacter jejuni, Enteroinvasive (EIEC) Escherichia coli, Enterohemorrhagic (EHEC) Escherichia coli, Vibrion vulnificus, Yersinia sp., Francisella tularensis, or Helicobacter pylori.
Symptoms of gastrointestinal tract infections include, e.g., diarrhea, vomiting, abdominal pain, cramps, fecal leukocytes, fever, dysentery, and/or blood in stool.
Gastrointestinal tract infections can be treated or prevented using soluble chitosans or derivatized chitosans described herein, in combination with one or more of agents or therapeutics. Exemplary agents and therapeutics to treat gastrointestinal tract infections includes rehydration, dietary therapy, probiotics, zinc, pharmacologic therapy (e.g., antibiotics (e.g., fluoroquinolone, metronidazole or vancomycin), antidiarrheal agents (e.g., loperamide or bismuth subsalicylate (BSS)), or antiemetic drugs (e.g., ondansetron or metoclopramide)). In some embodiments, the administrations of a combination of agents and therapeutics are spaced sufficiently close together such that a synergistic effect is achieved.
Antibacterials
The compositions and compounds described herein (e.g., soluble chitosans or derivatized chitosans) can be used in combination of one or more of antibiotics, to treat one or more diseases and conditions described herein. General classes of antibiotics include, e.g., aminoglycosides, bacitracin, beta-lactam antibiotics, cephalosporins, chloramphenicol, glycopeptides, macrolides, lincosamides, penicillins, quinolones, rifampin, glycopeptide, tetracyclines, trimethoprim and sulfonamides. In some embodiments, the administrations of a combination of agents and therapeutics are spaced sufficiently close together such that a synergistic effect is achieved.
Exemplary antibiotics within the classes recited above are provided as follows. Exemplary aminoglycosides include Streptomycin, Neomycin, Framycetin, Patpmycin, Ribostamycin, Kanamycin, Amikacin, Dibekacin, Tobramycin, Hygromycin B, Spectinomycin, Gentamicin, Netilmicin, Sisomicin, Isepamicin, Verdamicin, Amikin, Garamycin, Kantrex, Netromycin, Nebcin, and Humatin. Exemplary carbacephems include Loracarbef (Lorabid). Exemplary carbapenems include Ertapenem, Invanz, Doripenem, Finibax, Imipenem/Cilastatin,
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Primaxin, Meropenem, and Merrem. Exemplary cephalosporins include Cefadroxil, Durisef, Cefazolin, Ancef, Cefalotin, Cefalothin, Keflin, Cefalexin, Keflex, Cefaclor, Ceclor, Cefamandole, Mandole, Cefoxitin, Mefoxin, Cefprozill, Cefzil, Cefuroxime, Ceftin, Zinnat, Cefixime, Suprax, Cefdinir, Omnicef, Cefditoren, Spectracef, Cefoperazone, Cefobid, Cefotaxime, Claforan, Cefpodoxime, Fortaz, Ceftibuten, Cedax, Ceftizoxime, Ceftriaxone, Rocephin, Cefepime, Maxipime, and Ceftrobriprole. Exemplary glycopeptides include Dalbavancin, Oritavancin, Teicoplanin, Vancomycin, and Vancocin. Exemplary macrolides include Azithromycin, Sithromax, Sumamed, Zitrocin, Clarithromycin, Biaxin, Dirithromycin, Erythromycin, Erythocin, Erythroped, Roxithromycin, Troleandomycin, Telithromycin, Ketek, and Spectinomycin. Exemplary monobactams include Aztreonam. Exemplary penicillins include Amoxicillin, Novamox, Aoxil, Ampicillin, Alocillin, Carbenicillin, Coxacillin, Diloxacillin, Flucloxacillin Floxapen, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin, and Ticarcillin. Exemplary polypeptides include Bacitracin, Colistin, and Polymyxin B. Exemplary quiniolones include Ciproflaxin, Cipro, Ciproxin, Ciprobay, Enoxacin, Gatifloxacin, Tequin, Levofloxacin, Levaquin, Lomefloxacin, Moxifloxacin, Avelox, Norfloxacin, Noroxin, Ofloxacin, Ocuflox, Trovafloxacin, and Trovan. Exemplary sulfonamides include Mefenide, Prontosil (archaic), Sulfacetamide, Sulfamethizole, Sulfanilamide (archaic), Sulfasalazine, Sulfisoxazole, Trimethoprim, Trimethoprim-Sulfamethoxazole (cotrimoxazole), and Bactrim. Exemplary tetracyclines include Demeclocyline, Doxycycline, Vibramycin, Minocycline, Minocin, Oxytetracycline, Terracin, Tetracycline, and Sumycin. Other exemplary antibiotics include Salvarsan, Chloamphenicol, Chloromycetin, Clindamycin, Cleocin, Linomycin, Ethambutol, Fosfomycin, Fusidic Acid, Fucidin, Furazolidone, Isoniazid, Linezolid, Zyvox, Metronidazole, Flagyl, Mupirocin, Bactroban, Nitrofurantion, Macrodantin, Macrobid, Platensimycin, Pyrazinamide, Quinupristin/Dalfopristin (Syncerid), Rifampin (Rifampicin), and Tinidazole. Exemplary antibiotics also include xylitol.
Anti-inflammatory
The compositions and compounds described herein (e.g., soluble chitosans and derivatized chitosans) can be used in combination with one or more anti-inflammatory drugs, e.g., steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs (NSAIDs), to treat one or more diseases or conditions described herein. In
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Exemplary steroidal anti-inflammatory drugs include glucocorticoids (corticosteroids), e.g., Hydrocortisone (Cortisol), Cortisone acetate, Prednisone, Prednisolone, Methylprednisolone, Dexamethasone, Betamethasone, Triamcinolone, Beclometasone, Fludrocortisone acetate, Deoxycorticosterone acetate (DOCA), and Aldosterone. Exemplary non-steroidal anti-inflammatory drugs include Aspirin, Choline and magnesium salicylates, Choline salicylate, Celecoxib, Diclofenac potassium, Diclofenac sodium, Diclofenac sodium with Isoprostol, Diflunisal, Etodolac, Fenoprofen calcium, Flurbiprofen, Ibuprofen, Indomethacin, Ketoprofen, Magnesium salicylate, Meclofenamate sodium, Mefenamic acid, Meloxicam, Nabumetone, Naproxen, Naproxen sodium, Oxaprozin, Piroxicam, Rofecoxib, Salsalate, Sodium salicylate, Sulindac, Tolmetin sodium, and Valdecoxib. Exemplary non-steroidal anti-inflammatory agents (e.g., peptides) include regulatory cytokines, such as interleukins, e.g., IL-1, IL-4, IL-6, IL-10, IL-11, and IL-13.
Mucolytic agent (expectorant)
The compositions and compounds described herein (e.g., soluble chitosans and derivatized chitosans) can be used in combination with one or more mucolytic agents, to treat one or more diseases and conditions described herein. A mucolytic agent or expectorant is an agent which dissolves thick mucus and is used to help relieve respiratory difficulties. It does so by hydrolyzing glycosaminoglycans, tending to break down/lower the viscosity of mucin-containing body secretions/components. The viscosity of mucous secretions in the lungs is dependent upon the concentrations of mucoprotein, the presence of disulfide bonds between these macromolecules and DNA.
An expectorant can reduce the thickness or viscosity of bronchial secretions and help bring up mucus and other material from the lungs, bronchi, and trachea. An example of as expectorant is guaifenesin which promotes drainage of mucus from the lungs by thinning the mucus and also lubricates the irritated respiratory tract. Other exemplary mucolytic agents or expectorants include Althea root, Antimony pentasulfide, Creosote, Guaiacolsulfonate, Guaifenesin, Ipecacuanha (Syrup of ipecac), Levoverbenone, Potassium iodide, Senega, Tyloxapol, Acetylcysteine,
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Ambroxol, Bromhexine, Carbocisteine, Domiodol, Domase alfa, Eprazinone,
Erdosteine, Letosteine, Mesna, Neltenexine, Sobrerol, Stepronin, and Tiopronm.
Soluble chitosans and chitosan derivatives
The compositions described herein include a soluble chitosan or a functionalized chitosan derivative.
Chitosan is an insoluble polymer derived from chitin, which is a polymer of N-acetylglucosamine that is the main component of the exoskeletons of crustaceans (e.g. shrimp, crab, lobster). Chitosan is formed from chitin by deacetylation, and as such is not a single polymeric molecule, but a class of molecules having various molecular weights and varius degrees of deacetylation. The percent deacetylation in commercial chitosans is typically between 50-100%. The chitosan derivatives described herein are generated by functionalizing the resulting free amino groups with positively charged or neutral moieties, as described herein. The degrees of deacetylation and functionalization impart a specific charge density to the functionalized chitosan derivative. The resulting charge density affects solubility, and the strength of interaction with bacterial cell walls and membranes. The molecular weight is also an important factor in the tenacity of bacterial wall interaction and thus bactericidal activity. Thus, in accordance with the present invention, the degree of deacetylation, the functionalization and the molecular weight must be optimized for optimal efficacy. The derivatized chitosans described herein have a number of properties which are advantageous including solubility at physiologic pH and antimicrobial activity when in solution or dry at any pH less than about 9.
A soluble chitosan as described herein, refers to a water soluble chitosan that is not derivatized on the hydroxyl or amine moieties. A soluble chitosan is comprised of glucosamine and acetylglucosamine monomers. Generally a water soluble chitosan has a molecular weight of less than or equal to about 10 kDa and a degree of deactylation equal or greater than 80%. Water soluble is defined as being fully dissolvable in water at pH 7.
The chitosan derivatives described herein are generated by functionalizing the resulting free amino groups with positively charged or neutral moieties, as described herein.
Chitosans with any degree of deacetylation (DDA) greater than 50% are used in the present invention, with functionalization between 2% and 50% of the available
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2019202473 09 Apr 2019 amines. The degree of deacetylation determines the relative content of free amino groups to total monomers in the chitosan polymer. Methods that can be used for determination of the degree of deacetylation of chitosan include, e.g, ninhydrin test, linear potentiometric titration, near-infrared spectroscopy, nuclear magnetic resonance spectroscopy, hydrogen bromide titrimetry, infrared spectroscopy, and first derivative UV-spectrophotometry. Preferably, the degree of deacetylation of a soluble chitosan or a derivatized chitosan described herein is determined by quantitave infrared spectroscopy. Percent functionalization is determined as the % of derivatized amines relative to the total number of available amino moieties prior to reaction on the chitosan polymer. Preferably, the percent functionalization of a derivatized chitosan desccribed herein is determined by H-NMR or quantitative elemental analysis. The degrees of deacetylation and functionalization impart a specific charge density to the functionalized chitosan derivative. The resulting charge density affects solubility, and strength of interaction with bacterial cell walls and membranes. The molecular weight is important in controlling the size of the bacterial clumps. Thus, in accordance with the present invention, these properties must be optimized for optimal efficacy. Exemplary chitosan derivatives are described in Baker et al; 11/657,382 filed on January 24, 2007, which is incorporated herein by reference.
The chitosan derivatives described herein have a range of polydispersity index (PDI) between about 1.0 to about 2.5. As used herein, the polydispersity index (PDI), is a measure of the distribution of molecular weights in a given polymer sample. The PDI calculated is the weight averaged molecular weight divided by the number averaged molecular weight. This calculation indicates the distribution of individual molecular weights in a batch of polymers. The PDI has a value always greater than 1, but as the polymer chains approach uniform chain length, the PDI approaches unity (1). The PDI of a polymer derived from a natural source depends on the natural source (e.g. chitin or chitosan from crab vs. shrimp vs. fungi) and can be affected by a variety of reaction, production, processing, handling, storage and purifying conditions. Methods to determine the polydispersity include, e.g., gel permeation chromatography (also known as size exclusion chromatography); light scattering measurements; and direct calculation from MALDI or from electrospray mass spectrometry. Preferably, the PDI of a soluble chitosan or a derivatized chitosan described herein is determined by HPLC and multi angle light scattering methods.
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The chitosan derivatives described herein have a variety of selected molecular weights that are soluble at neutral and physiological pH, and include for the purposes of this invention molecular weights ranging from 5-1,000 kDa. Embodiments described herein are feature medium range molecular weight of derivatized chitosans (25 kDa, e.g., from about 15 to about 300 kDa) which can have clumping, diffusible and biofilm disruption properties.
The functionalized chitosan derivatives described herein include the following:
(A) Chitosan-arginine compounds;
(B) Chitosan-natural amino acid derivative compounds;
(C) Chitosan-unnatural amino acid compounds;
(D) Chitosan-acid amine compounds;
(E) Chitosan-guanidine compounds; and (F) Neutral chitosan derivative compounds.
(A) Chitosan-arginine compounds
In some embodiments, the present invention is directed to chitosan-arginine compounds, where the arginine is bound through a peptide (amide) bond via its carbonyl to the primary amine on the glucosamines of chitosan:
OH / OH \ OH
Λ I .^0
HO \ YD \ \ OH
ΗΟ-Χ-''''V VA ho-V-'
NH \ ] NH/n NH 1
R1 R1 R1
wherein each R1 is independently selected from hydrogen, acetyl, and a group of the following formula:
Figure AU2019202473A1_D0378
or a racemic mixture thereof, wherein at least 25% of Rl substituents are H, at least 1 % are acetyl, and at least 2% are a group of the formula shown above.
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PCT/US2010/047758 (B) Chitosan-natural amino acid derivative compounds
In some embodiments, the present invention is directed to chitosan-natural amino acid derivative compounds, wherein the natural amino acid may be histidine or lysine. The amino is bound through a peptide (amide) bond via its carbonyl to the primary amine on the glucosamines of chitosan:
2019202473 09 Apr 2019
Figure AU2019202473A1_D0379
wherein each R1 is independently selected from hydrogen, acetyl, and a group of the following formula:
Figure AU2019202473A1_D0380
or a racemic mixture thereof, wherein at least 25% of R1 substituents are H, at least 1% are acetyl, and at least 2% are a group of the formula shown above; OR a group of the following formula:
Figure AU2019202473A1_D0381
or a racemic mixture thereof, wherein at least 25% of R1 substituents are H, at least 1 % are acetyl, and at least 2% are a group of the formula shown above.
(C) Chitosan-unnatural amino acid compounds
In some embodiments, the present invention is directed to chitosan-unnatural amino acid compounds, where the unnatural amino acid is bound through a peptide (amide) bond via its carbonyl to the primary amine on the glucosamines of chitosan:
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OH / OH \ OH
E A\ -ο I Ax ^0
HO^\ \ ^-N^OH
HO-A-- Λ'Μ' ho-V-' HO-A--'
NH X I NH/n NH [
R1 R1 R1
wherein each R1 is independently selected from hydrogen, acetyl, and a group of the following formula:
WWW
Figure AU2019202473A1_D0382
wherein RJ is an unnatural amino acid side chain, and wherein at least 25% of R1 substituents are H, at least 1% are acetyl, and at least 2% are a group of the formula shown above.
Unnatural amino acids are those with side chains not normally found in biological systems, such as ornithine (2,5-diaminopentanoic acid). Any unnatural amino acid may be used in accordance with the invention. In some embodiments, the unnatural amino acids coupled to chitosan have the following formulae:
Figure AU2019202473A1_D0383
(D) Chitosan-acid amine and guanidine compounds
In some embodiments, the present invention is directed to chitosan-acid amine compounds, or their guanidylated counterparts. The acid amine is bound through a peptide (amide) bond via its carbonyl to the primary amine on the glucosamines of chitosan:
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Figure AU2019202473A1_D0384
wherein each R1 is independently selected from hydrogen, acetyl, and a group of the following formula:
Figure AU2019202473A1_D0385
R3 wherein RJ is selected from amino, guanidino, and C|-Ce alkyl substituted with an amino or a guanidino group, wherein at least 25% of R1 substituents are H, at least 1 % are acetyl, and at least 2% are a group of the formula shown above.
In some embodiments, R1 is selected from one of the following:
Figure AU2019202473A1_D0386
Figure AU2019202473A1_D0387
Figure AU2019202473A1_D0388
Figure AU2019202473A1_D0389
Figure AU2019202473A1_D0390
Figure AU2019202473A1_D0391
(Έ) Chitosan-guanidine compounds
In some embodiments, the present invention is directed to chitosan-guanidine compounds.
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Figure AU2019202473A1_D0392
wherein each R1 is independently selected from hydrogen, acetyl, and or together with the nitrogen to which it is attached, forms a guanidine moiety; wherein at least 25% of R1 substituents are H, at least 1% are acetyl, and at least 2% are a group of the formula shown above.
(F) Neutral chitosan derivative compounds
In some embodiments, the present invention is directed to neutral chitosan derivative compounds. Exemplary neutral chitosan derivative compounds include those where one or more amine nitrogens of the chitosan has been covalently attached to a neutral moiety such as a sugar:
Figure AU2019202473A1_D0393
wherein each R1 is independently selected from hydrogen, acetyl, and a sugar (e.g., a naturally occurring or modified sugar) or an α-hydroxy acid. Sugars can be monosaccharides, disaccharides or polysaccharides such as glucose, mannose, lactose, maltose, cellubiose, sucrose, amylose, glycogen, cellulose, gluconate, or pyruvate. Sugars can be covalently attached via a apacer or via the carboxylic acid, ketone or aldehyde group of the terminal sugar. Examples of oc-hydroxy acids include glycolic acid, lactic acid, and citric acid. In some preferred embodiments, the neutral chitosan derivative is chitosan-lactobionic acid compound or chitosan-glycolic acid compound. Exemplary salts and coderivatives include those known in the art, for example, those described in US 20070281904, the contents of which is incorporated by reference in its entirety.
Compositions
Described herein are also compositions comprising a soluble chitosan or a functionalized chitosan derivative, e.g., a soluble or derivatized chitosan described
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2019202473 09 Apr 2019 herein. In some embodiments, the composition is a liquid, solid, or semisolid composition. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is a reaction mixture.
In some embodiments, the composition further comprises one or more additional compound or agent. In some embodiments, the second compound or agent is another chitosan derivative, e.g., a soluble or derivaized chitosan described herein.
In some embodiments, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid, e.g., polyarginine (e.g., diargine, triargine, etc).
In some embodiments, the composition has less than about 20%, 15%, 10%, 5%, 2%, or 1%, or is substantially free, of a chitosan polymer having a molecular weight of less than 15,000 Da, 10,000 Da, or 5,000 Da.
The composition described herein can be used to disrupt a preformed biofilm or prevent the formation of a biofilm in a subject. The composition described herein can also be used to treat or prevent a disease or a symptom of a disease described herein.
Formulations and routes of administration
The compounds described herein can be formulated in a variety of manners, including for oral or topical delivery (e.g., administered orally (e.g., oral rinse, throat gargle), by inhalation spray (e.g., nasal spray, nasal mists, or sinus spray), nebulizer, topically, rectally, nasally, buccally. In some embodiments, inhalation sprays (e.g., nasal spray, nasal mists, or sinus spray), are used for the nasal delivery of a compound descried herein, to locally treat or prevent an infection or disorder described herein, e.g., a nosocomial infection or MRS A infection. Inclusion in feed, water or an inhaled formulation is particularly desirable for use with animals. In some embodiments, a compound is formulated so as to allow the soluble chitosan or soluble chitosan derivative thereof to diffuse into a subject (e.g., into the wound, body cavities, or skin of a subject) upon administration to the subject or to be ingested, inhaled or swabbed while incorporated into a time release formulation.
The compound described herein (e.g., a soluble chitosan or a derivatized chitosan) can be administered before or after the onset of the disorder described herein. The methods herein contemplate administration of an effective amount of
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2019202473 09 Apr 2019 compound or compound composition to achieve the desired or stated effect. Typically, the compositions of this invention will be administered from about 1 to about 6 times per day. Alternatively, the compounds can be administered as a continuous timerelease. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical solution preparation will contain from about 1 pg/mL to 1000 pg/mL, about 5 pg/mL to 500 pg/mL, about 10 pg/mL to about 250 pg/mL, about 20 pg/mL to about 100 pg/mL, or about 40 pg/mL to about 60 pg/mL. A typical solid diffusible preparation will contain from about 1% to about 20%, about 2% to about 15%, or about 5% to about 10% by weight. A typical solid dissolvable preparation will contain from about 2% to about 95% by weight.
Lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the type and nature of the bacteria, the patient’s disposition to the disease, condition or symptoms, and the judgment of the treating physician.
In an embodiment, the compounds described herein (e.g., a soluble chitosan or a derivatized chitosan) can be formulated, e.g., as a solution, gel, or dressing, e.g., for treating a wound. In an embodiment, the dosage (e.g., solution dosage) is from about 10 pg/mL to about 1000 pg/mL, about 100 pg/mL to about 750 pg/mL, or about 250 pg/mL to about 500 pg/mL, applied e.g., sufficiently to rinse a wound area. In an embodiment, the dosage (e.g., solution dosage) is about 10 to about lOOOpg/mL, about 100 pg/mL to about 750 pg/mL, or about 250 pg/mL to about 500 pg/mL, applied to coat the wound at least 1, 2, 3, or 4 times daily. In an embodiment, the solid diffusible composition (dressing) is from about 1% to about 10%, about 3% to about 8%, or about 5% to about 6%, by weight applied to cover the wound at least 1, 2, 3, or 4 times daily. In an embodiment, the composition is applied to a thickness of at least about 1/128, 1/64, 1/32, or 1/16 inch.
In an embodiment, the compounds described herein (e.g., a soluble chitosan or a derivatized chitosan) can be formulated, e.g., as a solution, encapsulated time release, gel, or enema, e.g., for treating gastrointestinal disorder or condition. In an
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In an embodiment, the compounds described herein (e.g., a soluble chitosan or a derivatized chitosan) can be formulated as a nebulized solution or powder, or lavage, e.g., for treating a pulmonary disorder or condition. In a preferred embodiment, the dosage is from about 500 pg to about 50000 pg, about 1000 pg to about 25000 pg, about 2000 pg to about 10000 pg, or about 4000 pg to about 6000 pg, every 2, 4, 6, 8, 10, 12, or 24 hours. In an embodiment, the composition is administered at least 1, 2, 3, or 4 times daily.
In an embodiment, the compounds described herein (e.g., a soluble chitosan or a derivatized chitosan) can be formulated, e.g., as a spray, ointment, gel or inhalant, e.g., for treating a disorder or condition or ear, nose or throat. In a preferred embodiment, the dosage is from about 10 to 1000 pg/mL, about 100 pg/mL to about 750 pg/mL, or about 250 pg/mL to about 500 pg/mL in solution, or from about 0.1% to about 10%, about 0.5% to about 5%, or about 1% to about 2% by weight in an ointment or gel. In an embodiment, the composition is administered at least I, 2, 3, or 4 times daily.
In an embodiment, the compounds described herein (e.g., a soluble chitosan or a derivatized chitosan) can be formulated as an ointment, solution, gel, or dressing, e.g., for skin decontamination or treating a skin condition. The dosage (e.g., solution dosage) is from about 10 pg/mL to about 1000 pg/mL, about 100 pg/mL to about 750 pg/mL, or about 250 pg/mL to about 500 pg/mL, applied e.g., sufficiently to rinse a skin area. In an embodiment, the dosage (e.g., solution dosage) is from about 10 to about lOOOpg/mL, about 100 pg/mL to about 750 pg/mL, or about 250 pg/mL to about 500 pg/mL applied to coat the skin at least 1, 2, 3, or 4 times daily. In an embodiment, the solid diffusible composition (dressing) is from about 1 % to about 10%, about 3% to about 8%, or about 5% to about 6% by weight applied to coat the
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Upon improvement of a patient’s condition, a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
Pharmaceutical compositions of this invention comprise a compound of the formulae described herein or a pharmaceutically acceptable salt thereof; an additional compound including for example, a steroid or an analgesic; and any pharmaceutically acceptable carrier, adjuvant or vehicle. Alternate compositions of this invention comprise a compound described herein or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier, adjuvant or vehicle. The compositions delineated herein include the compounds described herein, as well as additional therapeutic compounds if present, in amounts effective for achieving a modulation of disease or disease symptoms.
The compositions are generally made by methods including the steps of combining a compound described herein with one or more carriers and, optionally, one or more additional therapeutic compounds delineated herein.
The term “pharmaceutically acceptable carrier or adjuvant” refers to a carrier or adjuvant that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.
The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried com starch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase which can be combined
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2019202473 09 Apr 2019 with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
The compounds of this invention may be administered by aerosol, nebulizer, or inhalation. In some embodiments, the composition is in the form of a dry powder, a suspension, or a solution. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Exemplary methods and devices for aerosol or inhalation include those described in US Patent No. 6,962,151, which is incorporated herein by reference in its entirety.
Compositions formulated for inhaled delivery generally include particles having a mean diameter of from about 0.1 pm to about 50 pm (e.g., from about 0.1 pm to about 10 pm, or from about 0.2 pm to about 5 pm. In some embodiments, the composition includes a dispersion of suitably-sized dry particles, for example, precipitants or crystals) or a dispersion of a solution (e.g., droplets) of a suitable size.
The pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block
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In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form for delivery in particular regions of the body, such as the colon.
The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solublizing or dispersing agents known in the art.
When the compositions of this invention comprise a combination of compounds described herein, both the compounds are generally present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen. Additionally, combinations of a plurality of compounds described herein are also envisioned. The compounds may be administered separately, as part of a multiple dose regimen, from the compounds of this invention. The compounds may be administered in a manner and dose where they act synergistically as describe e.g., in US Patent Application 61/113904, which is incorporated herein by reference in its entirety. Alternatively, those compounds may be part of a single dosage form, mixed together with the compounds of this invention in a single composition.
In some embodiments, the pharmaceutical compositions described herein can be administered topically, e.g., via liquid, semi-sold, gel, or time release, by irrigation, or by incorporation into a dressing.
Kits
A compound described herein (e.g., a soluble chitosan or a derivatized chitosan) can be provided in a kit. The kit includes (a) a composition that includes a compound described herein, and, optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material
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The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to use of the compound described herein to treat a disorder described herein.
In one embodiment, the informational material can include instructions to administer the compound described herein in a suitable manner to perform the methods described herein, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material can include instructions to administer the compound described herein to a suitable subject, e.g., a human, e.g., a human having or at risk for a disorder described herein. For example, the material can include instructions to administer the compound described herein to such a subject.
The informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet. However, the informational material can also be provided in other formats, such as computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is contact infonnation, e.g., a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about a compound described herein and/or its use in the methods described herein. Of course, the informational material can also be provided in any combination of formats.
In addition to a compound described herein, the composition of the kit can include other ingredients, such as a solvent or buffer, a stabilizer, a preservative, and/or a second compound for treating a condition or disorder described herein. Alternatively, the other ingredients can be included in the kit, but in different compositions or containers than the compound described herein. In such embodiments, the kit can include instructions for admixing the compound described
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The compound described herein can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that the compound described herein be substantially pure and/or sterile. When the compound described herein is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. When the compound described herein is provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.
The kit can include one or more containers for the composition containing the compound described herein. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of a compound described herein. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of a compound described herein. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.
The kit optionally includes a device suitable for administration of the composition, e.g., a syringe, inhalant, pipette, forceps, measured spoon, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device. In a preferred embodiment, the device is an implantable delivery device.
Examples
Example 1. Chitosan-arginine clumps both Gram positive and Gram negative bacteria under planktonic conditions.
Planktonic conditions are defined for bacteria that are in solution, as opposed to conditions where bacteria are already attached to a surface. Clumping of the bacteria by chitosan-arginine occurs very rapidly in solution as shown in the SYTOX
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2019202473 09 Apr 2019 green/propidium iodide stained fluorescent images at 200x magnification. Figure 1A shows a solution of Gram negative Pseudomonas aeruginosa strain PA01 (ATCC BAA-47) in ultra-pure water. Note that the bacteria in the images are very small as they are independent cells. Figure IB shows clumped Pseudomonas aeruginosa after 1 minute of exposure to 100pg/ml of chitosan-arginine (24kDa, 28% functionalized, 83 %DDA, 1.54 PDI). Note that this Gram negative bacteria clumps dramatically into very large agglomerates in a very short time.
Chitosan derivatives also clump Gram positive bacteria. Figure 2A shows a solution of Gram positive Streptococcus mutans (strain) in ultra-pure water. Note that by their nature, Streptococci grow in chains or pairs. As seen in Figure 2A, the bacteria exist as very small clumps. However, the dramatic clumping of S. mutans after 1 minute of exposure to 100pg/ml of chitosan-arginine (24 kDa, 28% functionalized, 83 %DDA, 1.54 PDI) is seen in Figure 2B. The chitosan-arginine provides a mechanism for very tight clumping of the bacteria.
Example 2. Chitosan-acid amines clump both Gram positive and Gram negative bacteria under planktonic conditions.
Chitosan derivatives known as chitosan-acid amines, derived from 6-aminohexanoic acid were observed to clump both Gram positive and Gram negative bacteria under planktonic conditions. Clumping occurs very rapidly, in solution, as shown in the SYTOX green/propidium iodide stained fluorescent images at 200x magnification. Figure 3 shows the clumping of the Pseudomonas aeruginosa strain PAO1 (ATCC BAA-47) after 1 minute of exposure to lOOpg/ml of chitosan-acid amine (29 kDa, 39% functionalized with 6-amino-hexanoic acid) compare to the untreated control (See Figure 1A). Figure 4A shows Gram positive methicillin resistant Staphylococcus aureus strain MW-2 in ultra-pure water. Note that Staphylococcus can make very small clusters by their natural growth cycle. Figure 4B shows the same Staphylococcus aureus strain MW-2 being clumped after 1 minute of exposure to 100pg/ml of chitosan-acid amine (29 kDa, 39% functionalized with 6-aminohexanoic acid). Note that the morphology of the clumping is different than with Pseudomonas aeruginosa, but that clumping nonetheless occurs. The morphology of the clumping is dependent on the strain of the bacteria, the molecular weight and chain length of the derivative and the nature of the chitosan derivative moiety.
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Example 3. Higher molecular weight chitosan derivatives aggregate bacteria into larger clumps than lower molecular weight chitosan derivatives.
While chitosan derivatives can clump a broad spectrum of bacteria, the molecular weight of the derivative is an important parameter because different molecular weights provide different clumping morphologies, regardless of the nature of the chitosan derivative. As shown in Figures 5 and 6, higher molecular weight chitosan derivatives aggregate bacteria into larger clumps than lower molecular weight chitosan derivatives. Specifically in Figure 5, Gram negative Pseudomonas aeruginosa strain PA01 shows visible evidence of clumping after 24 hours treatment with 100 ng/ml chitosan-lactobionic acid the neutral chitosan derivative (125 kDa) (Tube A), 100pg/ml chitosan-arginine (28 kDa, 37% functionalization, 83 %DDA, 1.95 PDI) (Tube B), 100pg/ml chitosan-arginine (40 kDa, 37% functionalization, 83 %DDA, 2.454 PDI) (Tube C), and compared to water alone (Tube D). The difference between the 28 kDa chitosan derivative (Tube B) and the 125 kDa chitosan derivative (Tube A) shows that greater clumping occurs following treatment with the larger molecule. This data is quantified in Figure 6. The derivatives as described above, were added to the Gram negative Pseudomonas aeruginosa strain PA01 suspension (total volume 5 mL). Following 24 hours treatment, three samples of each treatment in a volume of 100 pl was obtained from the middle (to avoid misrepresentation of the observation because of natural bacterial settling due to gravity) of each bacterial suspension. The OD 595 was measured. A decrease in the OD 595 with respect to the PA01 in water alone indicated the bacteria were aggregating and precipitating out of the suspension, corresponding to visible observations in Figure 5.
Example 4. Reduction of the earner state of Staphylococcus aureus and/or Acinetobacter baumannii in the nose (anterior nares).
It has been shown that the reduction of the carrier state of bacteria in the anterior nares is sufficient to reduce nosocomial infections. A chitosan derivative nasal spray in hypotonic, neutral solution is sufficient to clump over 108 bacteria/ml. Chitosan derivatives clump Acinetobacter baumannii and multiple strains of MRSA as shown above. Figures 7A-7C shows that Streptococcus mutans is clumped by various doses of chitosan-arginine after five minutes incubation at 200x magnification. Figures 7A through 7C show clumping of Streptococcus mutans by 2pg/ml, 10 pg/ml, and 20 pg/ml of chitosan-arginine, respectively, at 200x
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2019202473 09 Apr 2019 magnification. It is important to note that the lowest of these doses is sufficient to cause significant aggregation of the bacteria. Furthermore, clumping is also observed in known resistant bacteria such as MRSA as shown in Figures 8A and 8B. Figure 8A shows Staphylococcus aureus strain MW-2, a blood/CSF isolate that is methicillin resistant after a 1 -minute exposure to water. It is important to note that Staphylococci tend to exist naturally in very small clusters. However, after the addition of chitosanarginine as shown in Figure 8B, clumping dramatically increases.
Table 5. Exemplary bacteria clumped by positive and neutral chitosan derivatives
Gram positive Gram negative
Staphylococcus aureus MW-2 Escherichia coli 0:157 H:7
Streptococcus mutans Shigella flexneri
Clostridium perfringens Salmonella typhimurium
Streptococcus pyogenes (GAS) Acinetobacter baumannii
Pseudomonas aeruginosa
Example 5. Mammalian cells are not clumped by these chitosan derivatives.
A number of human cells have been treated with up to 1 mg/ml of chitosanarginine with no change in viability nor any indication of clumping. Figures 9A and 9B shows optical microscope images taken at 20x of THP-1 human monocyte cell line exposed to 100pg/ml of 40kDa chitosan-arginine 37% functionalized for 24 hours at high (A) and lower (B) cell density (106 cells/ml and 105 cells/ml, respectively). The viability and structure of mammalian cells is not affected by the presence of the chitosan derivatives up to doses that are highly effective at clumping bacteria. These positively charged, soluble polymer chitosan derivatives are very broadly effective at clumping bacteria, with somewhat higher efficiency of clumping Gram positive relative to Gram-negative bacteria. As will be understood by one of ordinary skill, selection of an appropriate MW and functionalization depends on the strain and species of the bacteria.
Example 6. The ability of chitosan derivatives to clump and reduce viable Clostridium difficile
The ability of chitosan derivatives to treat enteric infection by reducing the effective bacterial load via clumping and reducing viable Clostridium difficile is tested. Clostridium difficile is closely related to Clostridium perfringens, having
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2019202473 09 Apr 2019 similar etiology in the gut. Clostridium difficile is mostly found in humans, and Clostridium perfringens is mostly found in poultry. Efficacious doses of chitosan derivatives are determined in vitro for significant reduction of Clostridium difficile viability alone and in synergistic combinations with current antibiotic therapies. Additional information about the potential role for chitosan-lactobionic acid can complement these studies and provide a neutral control aiding in elucidating the role of the positive charge versus the chitosan backbone itself in bacterial interaction. A treatment formulation to be tested in animal models can be developed. Oral therapies of chitosan-arginine can be tested in well-established in vivo models of Clostridium difficile infections to ascertain the feasibility and effectiveness of this antimicrobial polysaccharide. Potential applications, although focused here on Clostridium difficile infections, extend broadly to enteric infections.
Example 7. Viscosity studies
1% Sodium alginate biofilm model treated with chitosan-arginine
Homogeneous 1% sodium alginate solutions (35mL each) were prepared in water. Viscosity was immediately (0.1 hours) measured on a Brookfield digital viscometer (Model DV-E) using spindle 62 at speed 30 rpm, after adding either 100 pg/mL chitosan-arginine (in 2 mL, 25% functionalized, 18kDa, 88 %DDA, PDI 1.47) or an equivalent amount of water (control). Viscosity was also measured 1 and 4 hours following treatment.
As shown in Figure 10, after 1 and 4 hours, chitosan-arginine showed a significant (p=0.01) reduction in the viscosity of the sodium alginate solution compared to water treatment alone.
Pseudomonas aeruginosa biofilm/spent media viscosity study
Pseudomonas aeruginosa biofilms were grown in polystyrene plates for 2.5 days at 37 °C. The biofilm supematant/spent media is very viscous due to alginate production and DNA sequestration. We tested the ability of chitosan-arginine to reduce the viscosity of P. aeruginosa biofilm supematants/spent media following a 1minute treatment with 100 pg/mL chitosan-arginine. First, a dramatic visual reduction in viscosity was observed upon addition of the chitosan-arginine to the gelatinous material. Subsequently, a Brookfield spinning viscometer was used to measure the viscosity of biofilm supernatants that were removed from the polystyrene plates and
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2019202473 09 Apr 2019 placed in 1.5 ml tubes. Four samples each were tested before chitosan-arginine treatment and 1-minute after 100 pg/mL chitosan-arginine (28% functionalized, 24kDa, 83 %DDA, PDI 1.54) treatment. Viscosity readings in centipoises (cP) were obtained with spindle number 64 at a speed of 50 rpm. As shown in Figure 11, the average viscosity before chitosan-arginine treatment was 950 ± 280 cP. Following the 1-minute chitosan-arginine treatment, this measurement was reduced to 270 ± 80 cP and the material appeared visually less opaque than untreated biofilm supernatant.
CFpatient sputum study: Pourability
Two sputum samples were obtained from cystic fibrosis patients during routine clinical practice and collected immediately from the hospital. The each sample was briefly vortexed and a volume of 200 pL was placed in two microfuge tubes. The controls were treated with 10 pL of water only. The others were treated with 100 pg/mLof chitsan-arginine (25% functionalized, 43kDa, 88 %DDA, PDI 2.28) in 10 pL of water. After a brief mixing step the tubes were incubated at 37 °C for 1 hour to simulate conditions in the patient. The tubes were removed and tested for pourability.
Both control sputum samples were unable to pour, however the chitosanarginine treated sputum was pourable as shown in Figure 12. This qualitatively demonstrates the ability of chitosan-arginine to reduce the viscosity of viscous sputum associated with cystic fibrosis.
Example 8. Tobramycin synergy experiment
Chronic antibacterial treatment of Pseudomonas aeruginosa infections in the lungs of cystic fibrosis patients is thought to facilitate antibacterial resistance (Speert et al., 1990; Govan & Deretic, 1996). Recently, sub-inhibitory aminoglycoside concentrations have been associated with induction of biofilm formation by P. aeruginosa (Hoffman et al., 2005).
A checkerboard assay was used to screen 64 unique combinations of antimicrobial concentrations in triplicate. For each study, approximately 102 cells per mL were treated with chitosan-arginine (64-0.5 pg/mL) and/or tobramycin (4-0.031 pg/mL). Bacteria were incubated at 37 °C for 20 hours. The fluorescent pigment pyocyanin produced by Pseudomonas aeruginosa can be correlated with bacterial growth. The Minimum Inhibitory Concentration (MIC) values obtained with this
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2019202473 09 Apr 2019 protocol (fluorescence was measured 485nm excitation, 535nm emission) are the same as those obtained using optical density (Hoffman et. cil., 2005).
Three independent checkerboard assays were completed in triplicate using different lots of chitosan-arginine and tobramycin with the MIC of each antimicrobial alone reported in Table 6 (column 1). The amounts of chitosan-arginine (pg/ml) coadministered are in bold. The tobramycin MIC reported in response to coadministered C/A is noted above the bolded values. It is clear that the addition of 0.5 ug/ml chitosan-arginine caused the MIC of tobramycin to be reduced 4 to 8-fold. The MIC of tobramycin is reduced 8 to 32-fold upon the addition of 4 pg/rnl of chitosanarginine (Table 6).
Table 6. Summary of the MIC data and FIC calculations to determine synergy
MIC Antimicrobial Alone (pg/ml) Tobramycin MIC (pg/ml) FIC
0.125 0.063 0.031
C/A1 (16) Tobramycin (1) C/A Treatment (pg/’ml) 0.5 2 4 <0.28 (S)4
C/A2 (32) Tobramycin (0.5) 0.5 2 4 <0.27 (S)
C/A3 (32) Tobramycin (0.5) 0.5 4 8 <0.30 (S)
127%, 32kDa;2 31%, 54kDa;3 25%, 40kD; 4 (S)=Synergistic
All of the treatment concentrations shown in Table 6 were shown to exhibit synergistic relationships as defined by the Fractional Inhibitory Concentration (FIC). The FIC is an interaction coefficient indicating whether the combined inhibitory/bacteristatic effect of drugs is synergistic, additive or antagonistic (FIC = A + B) where: A = (MIC of X with Y) / (MIC of drag X alone) and, B = (MIC of Y with X) / (MIC of drag Y alone). If the FIC <0.5 the relationship is synergistic, additive if the FIC - 1, or antagonistic if the FIC > 4.
These analyses determined that co-administration of tobramycin and chitosanarginine is synergistic. Specifically, chitosan-arginine lowers the MIC of tobramycin and works synergistically to eliminate Pseudomonas aeruginosa.
Example 9. Neutral soluble chitosan derivative clumping
The ability of chitosan-lactobionic acid (LOT 007; about lOOkDa), a neutral charge chitosan derivative, to clump various bacterial species was evaluated using SYTOX green/propidium iodide staining after a 1-minute incubation with either treatment. As shown in Figures 13-20 (magnification 200x), chitosan-lactobionic acid is able to clump these bacteria within a minute of exposure, similar to chitosan178
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2019202473 09 Apr 2019 arginine. Bacteria tested were Salmonella enterica serotype Typhimurium (Figure 13), Clostridium perfringens (Figure 14), Escherichia coli strain O157:H7 (Figure 15), Streptococcus pyrogenes also know as Group A Strep (Figure 16), Shigella flexneri (Figure 17), Streptococcus mutans (Figure 18), methicillin resistant Staphylococcus aureus strain MW-2 (Figure 19), and Pseudomonas aeruginosa strain PA01 (Figure 20, see Figure 1A for untreated control). Figures 13-19A depict the bacteria before treatment (control) compared to Figures 13-19B after 1-minute treatment with 100pg/ml of chitosan-lactobionic acid. This observation demonstrates that innate properties of the chitosan backbone, in addition to or instead of the positive charge provided by positivie derivatives, is a critical component of the clumping mechanism. This observation also demonstrates that neutral derivatives can be effectively used for clumping in addition to positively charge derivatives.
Example 10. Wound biofilms
Bacteria associated with wound bio films were also tested for sensitivity to chitosan-arginine. Methicillin resistant Staphylococcus aureus strain MW-2 biofilms were grown up in THB stationary at 37 °C for 2.5 days. The biofilms were rinsed and treated with 0, 10, 50, or 100 pg/ml of chitosan-arginine (24 kDa, 28% functionalization, 83 %DDA, 1.5 PDI), in triplicate for 4 hours. Then the biofilms were scraped from the wells, diluted and plated to obtain CFU. Figure 21 shows a 3log reduction with 10 pg/ml after 4-hours and 4-log reduction at 50 and 100 pg/ml.
Chitosan-arginine efficacy against wound pathogen Pseudomonas aeruginosa strain PA01 biofilms was also tested. Biofilms were grown for 3 days in 12 well plates in yeast extract and treated with 25, 50, or 100 pg /ml chitosan-arginine for 6hours. The virtual CFU were enumerated following the technique of Brewster (Brewster, 2003). Pseudomonas aeruginosa strain PA01 biofilms are significantly reduced after 6-hour incubation with chitosan-arginine as seen in Figure 22. A marked dose response was also observed as the concentration of chitosan-arginine was increased from 25-100 pg/ml. The highest dose (100pg/ml) reduced the virtual CFU by 4 logs with a decrease in efficacy as seen in the figure at lower doses. It is notable that almost half of the data points for the replicates of the 100pg/ml chitosanarginine treatment did not show any growth at all.
Example 11. Disc diffusion assay
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To test the ability of chitosan-arginine to diffuse from 2% or 20% gelatin formulation, antibiotic test wafers were impregnated with 50 mM silver nitrate (positive control), gelatin alone (negative control), or chitosan-arginine (31%, 9kDa). Following 18h incubation at 37°C, diffusion was evident by the observation of a zone of inhibition that occurred around the discs (either autoclave or not) that contained the 9 kDa chitosan-arginine delivered in 2 or 20% gelatin, as shown in Figure 23. In this instance, autoclaving did not have an effect on activity.
This example suggests that the ability of chitosan-arginine to be delivered in a viscous or gel-like substance facilitates the slow release of chitosan-arginine into the local area of treatment. Maintaining a higher concentration of chitosan-arginine over an extended period of time can extend the spectrum of antibacterial activity and reduces the need for re-application.
Example 12. The prophylactic activity of chitosan-arginine against MRSA on pigskin
Pigskin was obtained from Sinclair Research Center, Inc. (Colombia, MO) shaved, cleaned, and divided into 1 -inch square sections. Each skin used was placed in sterile Petri dishe and UV sterilized on both sides for 1 -hour each to prepare for testing. In triplicate each skin was treated with 3% hydrogen peroxide, 70% ethanol, Chloroprep (commercial product consisting of 2% chlorohexidine in 70% ethanol), and 60 pg/cm2 chitosan-arginine in 70% ethanol, or no treatment and allowed to dry for 1-hour. Approximately 103 CFU of MRSA MW-2 was placed onto the pigskins and left at room temperature to adhere for 1-hour. Three pieces were excluded from treatment for controls to indicate sterilization of the skin occurred. The skin surface was pressed onto a BHI agar plate to obtain a qualitative indication of the amount of bacteria remaining on the skin surface. As shown in Figure 24, chitosan-arginine and Chloroprep maintained good prophylactic activity while hydrogen peroxide and 70% ethanol had no prophylactic activity.
Example 13. The wound rinse activity of chitosan-arginine against bacteria contaminated abraded pigskin.
Pigskin was obtained from Sinclair Research Center, Inc. (Colombia, MO) shaved, cleaned, and divided into 1-inch square sections. Each skin used was placed in sterile Petri dishe and UV sterilized on both sides for 1-hour each to prepare for testing. Then pigskins were abraded repeatedly with a hypodermic needle and
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2019202473 09 Apr 2019 approximately 105 CFU of MRS A (Figure 25) pr P. aeruginosa (Figure 26) was placed in triplicate onto the pigskins and left at room temperature to adhere for 30 minutes. Controls consisted of abraded skin only and abraded skin with bacterial contamination left untreated. The skins were irrigated with syringes twice with 5 mF of chitosan-arginine (43kDa, 25% functionalized, 88% DDA, 2.28 PDI) and left at room temperature for 30 minutes. Following treatment the pigskin were placed in 10 ml of PBS, sonicated and the remaining CFU were enumerated by plate counts. Chitosan-arginine was shown to rinse away more bacteria than the standard of care (saline).
Example 14, Chitosan-arginine dose response against stationary MRSA biofilms
The MRSA MW-2 biofilms were grown in 12-well untreated tissue culture plates containing BHI media for approximately 3 days. The biofilms were rinsed with water three times and treated with increasing doses of chitosan-arginine for 4-hours. Following treatment the biofilms were rinsed three times and the chitosan-arginine treated biofilms were resuspended, sonicated, diluted and plated to obtain CFU remaining. As shown in Figure 27, a 4-log reduction was achieved with 50 pg/ml treatment.
Example 15. Chitosan-arginine dose response against Klebsiella pneumoniae biofilms
Chitosan-arginine was analyzed with respect to reduction of mature K. pneumoniae biofilms with previously established methods (Harrison, J.J. et al. (2005) High-throughput metal susceptibility testing of microbial biofilms Environ Microbiol 7, 981-994). The biofilms were grown according to MBEC™ for High-throughput Screening (Innovotech, Edmonton, AB Canada) methods on a peg lid placed in trough containing EB media for 36 hours. The pegs were rinsed and placed into a 96-well plate with serial dilutions of the chitosan derivative or controls and exposed for 5hours at room temperature. The biofilms were rinsed, and the pegs removed and placed into microfuge tubes in 200μ1 of water. The tubes were sonicated to remove the peg biofilm. Aliquots of recovered biofilms were diluted and plated onto EB agar to quantify growth. Testing was done in duplicate and representative assays are depicted. The K. pneumoniae biofilms showed that the bacterial CFU were significantly reduced by chitosan-arginine. As shown in Figure 28, a 3-log reduction was observed with 125 pg/ml treatment.
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Example 16. Chitosan-arginine dose response against Acinetobacter baumannii biofilms
Chitosan-arginine was analyzed with respect to reduction of mature A. baumannii biofilms with previously established methods (Harrison, J.J. et al. (2005) High-throughput metal susceptibility testing of microbial biofilms Environ Microbiol 7, 981—994). The biofilms were grown according to MBEC™ for High-throughput Screening (Innovotech, Edmonton, AB Canada) methods on a peg lid placed in trough containing LB media for 36 hours. The pegs were rinsed and placed into a 96-well plate with serial dilutions of the chitosan derivative or controls and exposed for 3hours at room temperature. The biofilms were rinsed, and the pegs removed and placed into microfuge tubes in 200μΐ of water. The tubes were sonicated to remove the peg biofilm. Aliquots of recovered biofilms were diluted and plated onto LB agar to quantify growth. Testing was done in duplicate and representative assays are depicted. The A. baumannii biofilms showed that the bacterial CFU were significantly reduced by chitosan-arginine. As shown in Figure 29, a 4-log reduction was observed with 250 pg/ml treatment.
Example 17. Chitosan-arginine dose response against P.sz+fcww/u.s' aeruginosa biofilms
Chitosan-arginine was analyzed with respect to reduction of mature P. aeruginosa biofilms with previously established methods (Harrison, J.J. et al. (2005) High-throughput metal susceptibility testing of microbial biofilms Environ Microbiol 7, 981-994). The biofilms were grown according to MBEC™ for High-throughput Screening (Innovotech, Edmonton, AB Canada) methods on a peg lid placed in trough containing LB media for 36 hours. The pegs were rinsed and placed into a 96-well plate with serial dilutions of the chitosan derivative or controls and exposed for 3hours at room temperature. The biofilms were rinsed, and the pegs removed and placed into microfuge tubes in 200μ1 of water. The tubes were sonicated to remove the peg biofilm. Aliquots of recovered biofilms were diluted and plated onto LB agar to quantify growth. Testing was done in duplicate and representative assays are depicted. The A. baumannii biofilms showed that the bacterial CFU were significantly reduced by chitosan-arginine. As shown in Figure 30, a 3-log reduction in CFU was observed with 125 pg/ml chitosan-arginine treatment.
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Example 18. The ability of chitosan-arginine to reduce the cohesion of mixed biofilms The interactions of chitosan derivatives with biofilms were evaluated in more detail in order to determine the effect of mixed wound infections. In these experiments mixed bacterial populations consisting of MRSA MW-2, P. aeruginosa PA01, and Vancomycin-resistant E. faecalis were use to initiate biofilm growth in a flow cell to examine biofilm cohesion and in an artificial model. This experiment examined the ability of chitosan-arginine to reduce the cohesion of mixed biofilms. Each convertible flow cell slide chamber (Stovall Life Science Inc., CFCAS0003) was assembled into the convertible flow cell apparatus (Stovall Life Science Inc., CFCAS0001) including a bubble trap (Stovall Life Science Inc., ACCFL0002). The bacteria were grown overnight in LB media at 37°C under anaerobic conditions, centrifuged and resuspended approximately 108 cfii/ml of each in LB media. Each flow cell was primed with approximately 10 ml of the bacterial suspension. An initial attachment phase was carried out for 1-hour with a flow rate of 1.5 ml/min facilitated by an IsmaTec Low Flow, High Accuracy Multichannel Peristaltic Pump (Stovall Life Science Inc., ACCFL0013). Following the attachment phase the flow cells were rinsed and LB media was pumped in at a flow rate of 0.24 ml/min for 8-hours. The flow cells were rinsed for 2 minutes at approximately 29 ml/min with either water or chitosan-arginine at 200 pg/ml then media pumping was resumed overnight. Rinses were repeated at 22 and 26 hours post attachment. For the final rinse the flow cells were disconnected and place in a Petri dish full of water for 5 minutes. Excess water was wiped or drained from the slide careful not to disrupt the biofilm, then dried in a humid chamber 37 °C for 10 minutes. Cohesion was examined by submerging each slide in a beaker of water then sonicating for 30 seconds at amplitude 18 pm at the liquid surface. The slides were removed and excess water was wiped or drained from the slide careful not to disrupt the biofilm, then dried in a humid chamber 37 °C for 10 minutes. The slides were stained with crystal violet for 2-minutes, rinsed and qualitative assessment of biofilm remaining following mechanical disruption to simulate debridement was completed. As shown in Figure 31, Mixed biofilms treated with chitosan-arginine were less cohesive and were more easily dispersed than untreated biofilms. Further, as shown in Figure 32, the material removed from the chitosan-arginine treated flow cell during the final rinse was more aggregated and dense and in a larger amount than the untreated mixed biofilm.
183
WO 2011/028967
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Example 19. The effect of chitosan-arginine on Acinetobacter baumannii biofilms
The Acinetobacter baumannii biofilms were grown in 12-well untreated tissue culture plates containing LB media for approximately 3 days. The biofilms were rinsed with water three times and stained with crystal violet for 2 minutes. The biofilms were then rinsed with water three times then treated with 100 pg/ml of chitosan-arginine or water for 5-minutes. Following treatment the biofilms were rinsed three times. As shown in Figure 33, chitosan-arginine treated biofilms were removed from the surface while the water only treated biofilm was unaffected.

Claims (111)

1. A method of disrupting a preformed biofdm in a subject, the method comprising:
administering to the subject an effective amount of a composition comprising a soluble derivatized chitosan wherein the soluble derivatized chitosan when administered contacts the preformed biofilm, thereby disrupting the preformed biofilm;
wherein the derivatized chitosan comprises a chitosan of the following formula (I);
formula (I) wherein:
n is an integer between 20 and <000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of fonnula (II):
A-VuV
R3 fonnula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, C|-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
2. The method of claim 1, wherein the viscosity of the biofilm is reduced by at least 50%, compared to the biofilm that has not been contacted with the composition.
184a
2019202473 09 Apr 2019
3. The method of claim 1, wherein the viscosity of the biofilm is reduced by at least two-f'old, compared to the biofilm that has not been contacted with the composition.
4. The method of claim 1, wherein the biofilm is partially dissolved compared to the biofilm that has not been contacted with the composition.
5. The method of claim 1, further comprising administering an additional agent.
6. The method of claim 5, wherein the second agent is administered in a dosage to achieve a synergistic effect.
7. The method of claim 5, wherein the second agent is administered together with the soluble derivatized chitosan.
8. The method of claim 1, further comprising administering an antibiotic, antiinflammatory, or mucolytic compound to a subject in conjunction with, prior to, or subsequent to the administration of the composition.
9. The method of claim 8, wherein the antibiotic, anti-inflammatory, or mucolytic compound is administered in a dosage to achieve a synergistic effect.
10. The method of claim 1, wherein the biofilm is in the respiratory system.
11. The method of claim 1, wherein the subject has a complication of cystic fibrosis, uppor or lowerrespiratory tract infection, pneumonia, bronchitis, dental plaque, oral disease, ear infection, throat infection, a complication in immunocompromised patient, gastrointestinal infection, enteritis, ora symptom thereof.
12. The method of claim 1, wherein the subject is infected with bacteria listed in Table 1,2, 3, or 4.
186
184b
2019202473 09 Apr 2019
13. The method of claim 1, wherein the composition is administered topically, orally, or enterally.
14. The method of claim 1, wherein the composition is administered by inhalation.
15. The method of claim 1, wherein the effective amount is therapeutically effective amount.
16. The method of claim 1, wherein the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
17. The method of claim 1, wherein the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
18. The method of claim 1, wherein the derivatized chitosan is soluble in aqueous solution from about pH 5.0 to about pH 6.0.
19. The method of claim 1, wherein the derivatized chitosan is soluble in aqueous solution from about pH 2.0 to about pH 4.0.
20. The method of claim 1, wherein the derivatized chitosan is soluble in aqueous solution from about pH 8.0 to about pH 8.5.
21. The method of claim 1, wherein between 25-95% of R1 substituents are hydrogen.
22. The method of claim 1, wherein between 55-90% of R1 substituents are hydrogen.
23. The method of claim 1, wherein between 1 -50% of R1 substituents are acetyl.
24. The method of claim 1, wherein between 4-20% of R1 substituents are acetyl.
IS7
184c
25. The method of claim 1, wherein between 2-50% of R1 substituents are a group of formula (II).
2019202473 09 Apr 2019
26. The method of claim 1, wherein between 4-30% of R1 substituents are a group of formula (II).
27. The method of claim 1, wherein 55-90% of R1 substituents are hydrogen, 420% of R1 substituents are acetyl, 4-30% of R1 substituents arc a group of formula (II).
28.
29.
30.
31.
32.
33.
The method of claim 1, wherein R is amino and R3 is an arginine side chain.
The method of claim 1, wherein R1 is selected from one of the following:
2 and
The method of claim 1,
The method of claim 1, wherein R2 is amino and RJ is a lysine side chain.
wherein R1 is selected from one of the following:
The method of claim 1, wherein R2 is amino and RJ is a histidine side chain.
The method of claim 1, wherein R1 is selected from one of the following:
I <88
184d
34. The method of claim 1, wherein at least 1% of R1 substituents are selected from one of the following:
2019202473 09 Apr 2019 vww
AND at least 1% of R1 substituents are selected from the following:
35.
The method of claim 1, wherein R? is amino and R3 is a substituted C i -C6 alkyl.
36.
The method of claim 1, wherein R3 is Ci-Οό alkyl substituted with an amino group.
37.
The method of claim 36, wherein R3 is C i alkyl substituted with an amino group.
38.
The method of claim 36, wherein R’ is C2 alkyl substituted with an amino group.
184e
39. The method of claim 36, wherein R3 is C3 alkyl substituted with an amino
2019202473 09 Apr 2019 group.
40.
group.
41. group.
42. group.
43.
The method of claim 36, wherein R3 is C4 alkyl substituted with an amino
The method of claim 36, wherein R3 is C5 alkyl substituted with an amino
The method of claim 36, wherein R3 is C6 alkyl substituted with an amino
The method of claim 1, wherein R1 is selected from one of the following:
44. group.
45. group.
46. group.
47.
group.
48.
group.
The method of claim 1, wherein R3 is C1-C6 alkyl substituted with a guanidino
The method of claim 44, wherein R3 is C, alkyl substituted with a guanidino
The method of claim 44, wherein R3 is C? alkyl substituted with a guanidino
The method of claim 44, wherein RJ is C3 alkyl substituted with a guanidino
The method of claim 44, wherein RJ is C4 alkyl substituted with a guanidino
184f
2019202473 09 Apr 2019
49. The method of claim 44, wherein R3 is C5 alkyl substituted with a guanidino group.
50. The method of claim 44, wherein R3 is C6 alkyl substituted with a guanidino group.
51. The method of claim 1, wherein R1 is selected from one of the following:
WWV
52. The method of any one of claims 1 and 21-51, wherein R2 is amino that is substituted with a nitrogen protecting group prior to substitution on chitosan and removed subsequent to substitution on chitosan.
53. The method of claim 1, wherein the derivatized chitosan is made by reacting a chitosan with an amino acid wherein the amino group of the amino acid is protected by a protecting group.
54. The method of claim 53, wherein in the synthetic process a nitrogen protecting group is used, which can provide an intermediate polymer having a nitrogen protecting group.
55. The method of claims 52 or 54, wherein the nitrogen protecting group is tertbutyloxycarbonyl (Boc).
56. The method of any one of claims 1 and 21-52, wherein R2 is amino.
7 3
57. The method of claim 1, wherein R~ is hydrogen and R is amino.
191
184g
2019202473 09 Apr 2019
7 3
58. The method of claim 1, wherein R is hydrogen and R is guanidino.
59. The method of claim 1, wherein R2 is hydrogen and R3 is a substituted C,-C6 alkyl.
60. The method of claim 1, wherein R3 is C|-C6 alkyl substituted with an amino group.
61. The method of claim 60, wherein R3 is Ci alkyl substituted with an amino group.
62. The method of claim 60, wherein R3 is C2 alkyl substituted with an amino group.
63. The method of claim 60, wherein R3 is C3 alkyl substituted with an amino gr»up.
64. The method of claim 60, wherein R3 is C4 alkyl substituted with an amino group.
65. The method of claim 60, wherein R3 is C3 alkyl substituted with an amino group.
66. The method of claim 60, wherein R3 is C(, alkyl substituted with an amino group.
67.
The method of claim 1, wherein R1 is selected from one of the following:
184h
2019202473 09 Apr 2019
68. The method of claim 1, wherein R3 is C[-C6 alkyl substituted with a guanidino group.
69. The method of claim 68, wherein R3 is Cj alkyl substituted with a guanidino group.
70. The method of claim 68, wherein R3 is C? alkyl substituted with a guanidino group.
71. The method of claim 68, wherein R3 is C3 alkyl substituted with a guanidino group.
72. The method of claim 68, wherein R3 is C4 alkyl substituted with a guanidino group.
73. The method of claim 68, wherein R3 is C, alkyl substituted with a guanidino group.
74. The method of claim 68, wherein R3 is C6 alkyl substituted with a guanidino group.
75. The method of claim 1, wherein R1 is selected from one of the following:
193
184Ϊ
2019202473 09 Apr 2019
76. The method of claim 1, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents independently selected from any of the formulae specifically shown above.
77. The method of claim 1, wherein the chitosan of formula (I) may be further derivatized on the free hydroxyl moieties.
78. The method of claim 1, wherein the molecular weight of the derivatized chitosan is between 5,000 and 1,000,000 Da.
79. The method of claim 1, wherein the molecular weight of the derivatized chitosan is between 5,000 and 350,000 Da.
80. The method of claim 1, wherein the molecular weight of the derivatized chitosan is between 10,000 and 150,000 Da.
81. The method of claim 1, wherein the molecular weight of the derivatized chitosan is between 15,000 and 100,000 Da.
82. The method of claim 1, wherein the molecular weight of the derivatized chitosan is between 15,000 and 50,000 Da, e.g., between 20,000 and 40,000 Da.
83. The method of claim 1, wherein the chitosan is functionalized at between 5% and 50%.
84. The method of claim 1, wherein the chitosan is functionalized at between 20% and 30%.
85. The method of claim 1, wherein the degree of deacetylation (%DDA) of the derivatized chitosan is between 75% and 95%.
86. The method of claim 1, wherein the degree of deacetylation (%DD A) of the derivatized chitosan is between 80% and 90%.
194
184j
2019202473 09 Apr 2019
87. The method of claim 1, wherein the polydispersity index (PDI) of the derivatized chitosan is between 1.0 and 2.5.
88. The method of claim 1, wherein the polydispersity index (PDI) of the derivatized chitosan is between 1.5 and 2.0.
89. The method of claim 1, wherein the functionalized chitosan is substantially free of other impurities.
90. The method of claim 1, wherenin the composition is substantially free of a chitosan polymer wherein one or more of the nitrogen-containing groups of the glucosamine monomer is substituted with a polymerized amino acid.
91. The method of claim 1, wherein the composition is substantially free of a chitosan polymer having a molecular weight of less than 15,000 Da.
92. A method of preventing the formation of a biofilm in the respiratory system of a subject, the method comprising:
administering to the respiratory system of the subject an effective amount of a composition comprising a soluble derivatized chitosan, wherein the derivatized chitosan comprises a chitosan of the following formula (I):
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected f»r each occurrence from hydrogen, acetyl, and a group of formula (II):
195
184k
2019202473 09 Apr 2019 or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, C]-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II), thereby preventing or treating the formation of a biofilm in the respiratory system of the subject.
93. A method of preventing the formation of a biofilm in the mouth, nose, ear, eye, skin, or digestive tract of a subject, the method comprising:
administering to the mouth, nose, ear or digestive tract of the subject an effective amount of a composition comprising a soluble derivatized chitosan, wherein the derivatized chitosan comprises a chitosan of the following formula (I):
OH / OH \ OH _X XI . ^o HO \ HOXv^V \ \ -X HO~-X^ Ύ-χ HO-X^ NH \ I NH/n NH I R1 formula (I) wherein: n is an integer between 20 and R1 6000; and R1
each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, Cj-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain,
196
1841
2019202473 09 Apr 2019 wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II), thereby preventing the formation of a biofilm in the mouth, nose, ear, skin, or digestive tract of the subject.
94. A method of treating or preventing a complication of cystic fibrosis in a subject, the method comprising:
administering to the subject an effective amount of a composition comprising a soluble chitosan or derivatized chitosan, wherein the derivatized chitosan comprises a chitosan of the following formula (I):
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occunence from hydrogen, acetyl, and a group of formula (II):
JWUV
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, C1-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II), thereby treating or preventing a complication of cystic fibrosis.
95. A method of treating or preventing a gastrointestinal tract infection in a subject, the method comprising:
197
184m
2019202473 09 Apr 2019 administering to the subject an effective amount of a composition comprising a soluble derivatized chitosan, wherein the derivatized chitosan comprises a chitosan of the following formula (I):
fonnula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of fonnula (II):
R3 fonnula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
RJ is amino, guanidino, Ci-Cf, alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II), thereby treating or preventing the gastrointestinal tract infection.
96. A liquid composition comprising a soluble derivatized chitosan, wherein the derivatized chitosan comprises a chitosan of the following fonnula (I):
formula (I) wherein:
n is an integer between 20 and 6000; and
198
184n
2019202473 09 Apr 2019 each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
RJ is amino, guanidino, C|-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of Rl substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
97. A solid particulate composition comprising a soluble derivatized chitosan, wherein the derivatized chitosan comprises a chitosan of the following formula (I):
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
WWW
R3 formula (11) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, Cj-Cf, alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain,
199
184o
2019202473 09 Apr 2019 wherein at least 25% of R1 substituents are H, at least 1 % of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
98. A semisolid composition comprising a soluble derivatized chitosan, wherein the derivatized chitosan comprises a chitosan of the following formula (1):
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
uww
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R2’ is amino, guanidino, C\-C(, alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
99. A dressing or an ointment comprising soluble derivatized chitosan, wherein the derivatized chitosan comprises a chitosan of the following formula (I):
OH / OH \ OH
0 ^-0 ηοΆ Lo'^V' hoA Γ hoA hoA^ NH | NH/n NH [ R1 R1 R1
formula (I) wherein:
200
184p
2019202473 09 Apr 2019 n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
R3 foimula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R“ is hydrogen or amino; and
R3 is amino, guanidino, Ci-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II), wherein the dressing or ointment is formulated such that the soluble derivatized chitosan is diffusible into a subject upon administration to the subject.
100. A kit comprising:
a soluble derivatized chitosan, wherein the derivatized chitosan comprises a chitosan of the following formula (I):
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
•ZWW
R3 formula (II)
184q
2019202473 09 Apr 2019 or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R‘ is hydrogen or amino; and
R3 is amino, guanidino, Cj-Q alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II); and instructions to disrupt a performed biofilm in a subject.
101. A kit comprising:
a soluble derivatized chitosan, wherein the derivatized chitosan comprises a formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, C|-C', alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II); and instructions to prevent the formation of a biofilm in the respiratory system of a subject.
202
184r
2019202473 09 Apr 2019
102. A kit comprising:
a soluble derivatized chitosan, wherein the derivatized chitosan comprises a chitosan of the following formula (I):
HO HO
OH formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (11):
O'formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, C,-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II); and instructions to prevent or treat a complication of cystic fibrosis in a subject.
103. A kit comprising:
a soluble derivatized chitosan, wherein the derivatized chitosan comprises a chitosan of the following formula (I):
HO
HO fonnula (I)
184s
2019202473 09 Apr 2019 wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
fonnula (II) orR!, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, 'J wherein R is hydrogen or amino; and
R3 is amino, guanidino, Ci-Cg alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II); and instructions to prevent or treat a gastrointestinal tract infection in a subject.
104. A medical device constructed to fit into the respiratory system of a subject, wherein the surface of the medical device is coated with a soluble derivatzed chitosan, wherein the derivatized chitosan comprises a chitosan of the following formula (I):
HO HO
OH
NH
I „
NH i,
NH
R1 fonnula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
o·
R3 fonnula (II)
204
184t
2019202473 09 Apr 2019 or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, C]-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
105. A nebulizer comprising a solution or powder of soluble derivatized chitosan, wherein the derivatized chitosan comprises a chitosan of the following formula (I):
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R is hydrogen or amino: and
R3 is amino, guanidino, Ci-C6 alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
106. A surface skin decontaminant comprising a derivatized chitosan, wherein the derivatized chitosan comprises a chitosan of the following formula (1):
184u
OH
OH
OH
2019202473 09 Apr 2019 wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of foimula (II):
JVWVV
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, Ci-Cg alkyl substituted with an amino or guanidino moiety, or a natural or unnatural ammo acid side chain, wherein at least 25% of R1 substituents are II, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
107. A residual surface skin decontaminant comprising a soluble derivatized chitosan, wherein the derivatized chitosan comprises a chitosan of the following formula (I):
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II) :
184v
2019202473 09 Apr 2019
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R is hydrogen or amino; and
R3 is amino, guanidino, Ci-C^ alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
108. A method of treating a surface skin in a subject, the method comprising: administering to the subject an effective amount of a composition comprising a soluble derivatized chitosan, wherein the derivatized chitosan comprises a chitosan formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
R3 is amino, guanidino, Cj-Cf, alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain,
207
184w wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II), thereby treating the surface skin.
2019202473 09 Apr 2019
109. A method of treating or preventing a wound infection in a subject, the method comprising administering to the wound of the subject an effective amount of a composition comprising a soluble derivatized chitosan, wherein die derivatized chitosan comprises a chitosan of the following fonnula (I):
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
R3 fonnula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
RJ is amino, guanidino, C|-Ce alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of fonnula (II), thereby treating or preventing the wound infection.
110. The method of claim 109, wherein the wound damages or penetrates the skin of a subject.
184x
111. A medical device constructed to contact a wound of a subject, wherein the wound damages or penetrates the skin of the subject, wherein the surface of the medical device is coated with a soluble derivatzed chitosan, wherein the derivatized chitosan comprises a chitosan of the following formula (I):
2019202473 09 Apr 2019
-OH formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino; and
RJ is amino, guanidino, Cj-C(> alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R! substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II).
209 184y
2019202473 09 Apr 2019
1. A method of rinsing nasal tissue with an aqueous composition, the method comprising: administering to a subject’s nasal tissue an effective amount of a composition comprising:
a sweetening agent; and a soluble derivatized chitosan; wherein the derivatized chitosan comprises a chitosan of the following formula (I):
formula (I) wherein:
n is an integer between 20 and 6000; and each R1 is independently selected for each occurrence from hydrogen, acetyl, and a group of formula (II):
R3 formula (II) or R1, when taken together with the nitrogen to which it is attached, forms a guanidine moiety, wherein R2 is hydrogen or amino;
R3 is amino, guanidino, Ci-Ce alkyl substituted with an amino or guanidino moiety, or a natural or unnatural amino acid side chain, wherein at least 25% of R1 substituents are H, at least 1% of R1 substituents are acetyl, and at least 2% of R1 substituents are a group of formula (II); and wherein administering involves a nasal spray, a nasal mist or a sinus rinse.
2. The method of claim 1, wherein the derivatized chitosan is soluble in aqueous solution from about pH 3 to about pH 9.
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3. The method of claim 1, wherein the derivatized chitosan is soluble in aqueous solution from about pH 6.8 to about pH 7.4.
4. The method of claim 1, wherein the molecular weight of the derivatized chitosan is between 10,000 and 100,000 Da.
5. The method of claim 1, wherein the polydispersity index of the derivatized chitosan is from 1.0 to 2.5.
6. The method of claim 1, wherein 25-95% of R1 substituents are hydrogen.
7. The method of claim 1, wherein 55-90% of R1 substituents are hydrogen.
8. The method of claim 1, wherein 1-50% of R1 substituents are acetyl.
9. The method of claim 1, wherein 4-20% of R1 substituents are acetyl.
10. The method of claim 1, wherein 2-50% of R1 substituents are a group of formula (II).
11. The method of claim 1, wherein 4-30% of R1 substituents are a group of formula (II).
12. The method of claim 1, wherein 55-90% of R1 substituents are hydrogen, 4-20% of R1 substituents are acetyl, and 4-30% of R1 substituents are a group of formula (II).
13. The method of claim 1, wherein R2 is amino and R3 is an arginine side chain.
14. The method of claim 1, wherein R1 is selected from one of the following:
186
15. The method of claim 1, wherein R2 is amino and R3 is a lysine side chain.
2019202473 09 Apr 2019
16.
WW
WW
The method of claim 1, wherein R1 is selected from one of the following:
WW
17.
The method of claim 1, wherein R2 is amino and R3 is a histidine side chain.
18.
The method of claim 1, wherein R1 is selected from one of the following:
19.
The method of claim 1, wherein the method of rinsing treats or prevents a wound infection.
20. The method of claim 19, wherein the wound damages or penetrates the nasal tissue of a subject.
AU2019202473A 2009-09-02 2019-04-09 Methods And Compositions For Disrupting Biofilm Utilizing Chitosan-Derivative Compounds Abandoned AU2019202473A1 (en)

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