AU2018264151B2 - Chitosan-Derivative Compounds and Methods of Controlling Microbial Populations - Google Patents

Abstract

The present invention is directed to chitosan-derivative compounds and structures, methods of making chitosan-derivative compounds and methods for controlling, inhibiting and enhancing microbial populations in a variety of environments. The present invention is also directed to the control, inhibition and enhancement of microbial populations in animals, particularly humans. The microbial populations include bacteria, viruses and other pathogens where control of microbial populations are a necessity. The chitosan-derivative compounds of the present invention include chitosan-arginine compounds, related chitosan-L/D unnatural amino acid compounds, chitosan-acid amine compounds, chitosan-L/D natural amino acid derivative compounds, co-derivatives of the chitosan-derivative compounds, salts of the chitosan derivative compounds, and chitosan-guanidine compounds.

Description

CHITOSAN-DERIVATIVE COMPOUNDS AND METHODS OF CONTROLLING MICROBIAL POPULATIONS

[0001] This application is a divisional of Australian patent application no. 2017200843, filed on

8 February 2017, which is a divisional of Australian patent application no. 2015200865, filed on

February 2015, which is a divisional of Australian patent application no. 2013211460, filed on

July 2013, which is a divisional of Australian patent application no. 2007257436, filed on

24 January 2007, and is related to International patent application no. PCT/US2007/002078, filed

on 24 January 2007 and claims priority from U.S. provisional application no. 60/810,591, filed on

2 June 2006 and from U.S. provisional application no. 60/838,780, filed on 18 August 2006; each

of which is incorporated herein by reference in their entirety.

STATEMENT OF GOVERNMENT INTEREST

[0002] As outlined under 37 CFR 401.14(b), the United States government shall have a

nonexclusive, nontransferable, irrevocable, paid-up license to practice or have practiced for or on

behalf of the United States the subject invention.

BACKGROUND OF THE INVENTION

[00031 The understanding of the role of natural polysaccharides in controlling microbial

populations by selective enhancement or inhibition is emerging. The field of biopolymers has

evolved significantly due to the versatility of their applications and the greater understanding of

their function in many natural symbiotic processes. This development is particularly true for

chitosan, a polysaccharide which is naturally abundant and has been successfully utilized in diverse areas of agriculture, wastewater treatment, food technology, animal food stock, paper/textile manufacture, biotechnology and biomedical devices and products.

OH OH

O O N H2 NH

(1)

[0004] Chitin, a polymer of N-acetyglucosamine, is a cellulose-like biopolymer that is the main

component of crustaceans (e.g. shrimp, crab, lobster) and is also present in the

[Text continues on page 2]

la exoskeleton and the cell wall of fungi, insects and yeast. Chitosan, a principle derivative of chitin, is formed from chitin by deacetylation in the presence of alkali. It is the mostly deacetylated form of the naturally occurring polysaccharide chitin. The process of removing the acetyl group from poly-p(l-+4)-N-acetyl-D-glucosamine to poly-p(--+4)-D-glucosamine causes the formation of primary amines. Chitosan is not a single polymeric molecule, but a class of molecules having different molecular weights and different degrees of deacetylation.

The structure of chitosan and chitin is shown in (1), where p and q are the fractional

relationship of the monomers glucosamine and N-acetyl glucosamine, respectively, and are

between 0 and 1. The sum p + q = 1. For chitin, q -> 1 and the molecule is nearly fully

acetylated. Molecules where q< 0.5 are considered chitosan. A key feature of chitosan is the

positive charge of its amino group (-NH 3+) when pH is below its pKa (- 6.3). When the pH=

pKa, 50% of the amines are positive. The fraction of amines that are positive increases

exponentially as the pH decreases and decreases exponentially as the pH increases, by the

Henderson-Hasselbach equation pH= pK, +log [NH2 ]. At lower pH, native chitosan forms

[NH3]

a polycationic structure that can interact with anionic compounds and macromolecular

structures. While very low molecular weight chitosans with high degree of deacetylation are

soluble at physiological conditions, most chitosans lack positive charge and are only soluble

in acidic conditions. For example, chitosan is highly soluble in aqueous acetic acid.

[00051 Chitosan has numerous biological properties, including antimicrobial activity,

hemostatic activity, acceleration of wound healing, tissue-engineering scaffolds, drug

delivery, and antitumor activity. Additionally, chitosan, when biological burden is removed,

is biodegradable and biocompatible with low toxicity to mammalian cells. It is also important

to note that bacteria generally should not develop chitosan resistance.

[0006] Chitosan's unique biological properties make it medically important. Consequently,

chitosan has been developed for a variety of applications using these properties such as biodegradability, non-toxicity and antibacterial activity against a broad spectrum of microorganisms. However, the use of chitosan is limited because of its insolubility at neutral and physiological pH. The present invention overcomes the limitations of the prior applications and methodologies of making and using chitosan, as discussed below.

[0007 It is well known that biopolymers that are natural polycations have a tendency to be

antimicrobial. Defensins, for example, are small cationic polypeptides with antibacterial

properties that are produced naturally by the human body. See Viljanen et al.; Effect ofsmall

cationic leukocyte peptides (defensins) on the permeability barrier of the outer membrane;

Infect. Immun. September; 56(9): 2324-2329 (1988). Efforts to reproduce the effectiveness

of natural polycations as antibacterials with limited toxicity have been met by limited

success. The complexity of understanding the antibacterial properties results from the

multiplicity of interactions required between the polycation and the bacteria. Some of the

important factors that influence the antibacterial properties include degree of charge,

distribution of charge, molecular weight, degree of hydrophobicity and placement of the

charge relative to the polymer backbone. The magnitude of this latter effect is easily

conceptualized from the experiments comparing poly--lysine vs poly- a -lysine. See Shima

et al.; Antimicrobial action of epsilon-poly-L-lysine. J. Antibiot; 37:1449-1455 (1984). Poly

s -lysine has the positive charges located on the a-amine in close proximity to the linear

polymer backbone. Poly-a-lysine has the positive charges located five bond lengths from the

linear polymer backbone. Poly-s-lysine was more active as an antibacterial than poly-a

lysine against 20 of 22 tested bacterial species. They also determined an optimal molecular

weight. Additionally, they showed the importance of the positive charge by removing the

effective amine by reaction with a variety of carboxylic acids with no positive charge.

Furthermore, they showed that small oligomers of poly- s -lysine interfered with protein synthesis in E. coli more than poly-a-lysine. These results suggest a complicated interplay between charge, molecular weight, and charge placement on antibacterial effectiveness.

[0008] Nonfunctionalized Chitosan And Salts

While several mechanisms have been proposed for chitosan's antimicrobial activity, the

exact mechanism is still unclear. The currently accepted antimicrobial mechanism is based on

the interaction of the positively charged chitosan with the negatively charged residues on

bacterial cell surface. It is believed that this charge interaction alters bacterial surface

morphology and either damages the membrane to induce membrane permeability that causes

leakage of intracellular substances (e.g. electrolytes, proteins, nucleic acids, glucose, and

lactate dehydrogenase), or develops an impermeable layer around the cell and prevents

nutrients from entering the bacteria. Another proposed mechanism suggests that positively

charged chitosan interacts with cellular DNA through chitosan penetration into the cells,

consequently acting as a barrier to RNA and protein synthesis.

[0009] Chitosan or chitosan derivatives that are described with antibacterial activity are

considered to be bacteriocidal or bacteriostatic, often with little distinction between the two

mechanisms. A bacteriocidal material kills the live bacteria or some fraction therein. A

bacteriostatic material hinders the growth of bacteria or some fraction therein but does not

imply whether or not bacteria are killed or their growth inhibited.

[00010] Chitosan's antimicrobial activity is affected by several factors such as source of

chitosan, molecular weight (MW), degree of deacetylation (DD), chitosan concentration, pH,

temperature, solution cations and polyanions, bacterial species and the phase of bacterial

growth. A variety of studies have demonstrated the need for positively charged or other

soluble chitosans in attempts to address the mechanism of activity. See Muzzarelli et al.; N

(carboxymethylidene) chitosan and N-(carboxymethyl) chitosan: novel chelating polyampholytes obtainedfrom chitosan glyoxylate. Carbohyd. Res. 107, 199-214, (1982); and

Moller et al.; Antimicrobial andphysicochemicalproperties of Chitosan-HPMC-basedfilms;

J. Agric. Food. Chem. 52, 6585-6591 (2004).

[0011] Studies on the mechanism of bactericidal activity of chitosan acetate suggest that cell

membranes of Escherichia coli and Staphylococcus aureus are damaged by the chitosan and

become permeable, likely a result of the electrostatic interactions between the positive amines

on the chitosan and the negative phosphoryl groups of the phospholipids comprising the cell

membrane. See Liu, H. et al., Chitosan kills bacteria through cell membrane damage.

Internat. J of Food Microbio 95, 147-155 (2004). Another reference discloses the use of 0

carboxymethylated and N,0-carboxymethylated chitosans as antibacterials due to the

compounds' ability to bind to DNA and the subsequent inhibition of DNA transcription. See

Liu et al.; Antibacterial action of chitosan and carboxymethylated chitosan; J. Appl. Poly.

Sci. 79, 1324-1335 (2001). Another reference discloses a comprehensive survey of the

proposed mechanisms of antimicrobial activity including antifungal, antibacterial and

antiviral properties (highlighting the influence of MW, pH, polynucleotide binding, cell

permeability, binding of essential minerals by chitosan, and its limitations above pH 6.5). See

Ravbeaet al.; Chitosan as antimicrobial agent application and mode of action;

Biomacromolecules 4(6), 1457-1465 (2003). Yet another reference discloses that water

soluble chitins and chitosans without sufficient positive charge or MW are not antibacterial,

whereas insoluble, high MW chitosans are effective if placed in acidic media, again

suggesting that positive charge plays a role in mediating bacteriocide. See Qin et al.; Water

solubility of chitosan and its antimicrobial activity. Carbohydrate Polymers; 63: 367

374(2006).

[00121A careful study using temperature and a variety of pH's showed the dramatic effect of

the degree of protonation, ie positive charge, on the antibacterial activity of chitosan against

E. coli. For pH's between 5 and 9, they showed a dramatic and continual increase in cell

death as the pH increased, with nearly no activity at pH 9. See Tsai et al.; Antibacterial

Activity of Shrimp Chitosan against Escherichia coli; J. of Food Protection 62(3); pp. 239

243(1999).

[0013] Several patents disclose applications of nonfunctionalized chitosan for absorbent

materials, drug delivery, biocompatible/bioabsorbable materials, hemostatic agents, filters,

textiles, crosslinked gels, as a carrier for soluble or active antibacterial and antimicrobial

agents, as a chelating and flocculation method for metals in water, as biodegradable and/or

edible films, shellacs and sheets, as well as an odor control agent, hydrophilic absorbent and

biodegradable/biocompatible structural support for co-derived scaffolds. Chitosan is broadly

represented in the patent and academic literature in the insoluble form.

[00141 Chitosan of significant MW is insoluble at physiological pH. However these

references differ from the present invention by disclosing soluble chitosan compounds

formed by intentional breakdown of molecular weight. US Patent 5,730,876 to You et. al

discloses a method for fractionating low molecular weight, soluble chitosans through reaction

with enzymes and acid and subsequent ultrasonic membrane filtration to bring the MW from

over 1OOkDa to 10,OOOkDa or less. Reducing molecular weight is a common way to improve

solubility of chitosan, but it does nothing to increase the positive charge on the

polysaccharide. Patents disclosing the production of water soluble chitosans teach the

desirability of formulations that are soluble at pH 7. In order to make such chitosans that are

not derivatized, the molecular weight must be decreased dramatically, the degree of

deacteylation high (depending on the MW) and may or may not have antibacterial effects,

depending on the composition of the additives. For example, US 4,532,134 to Malette et al.

discloses a water-soluble chitosan that is used to treat wounds as a hemostatic agent. These low MW, low positive charge nonfunctionalized chitosans are not related to the current application.

[0015] Chitosan Mixtures (Compositions)

Many of the prior art references disclose the uses of mixtures of chitosan and atoms,

chemicals (natural or synthetic), polymers or polypeptides which impart new properties to the

entire composition including antibacterial or antimicrobial properties. Mixtures are a

homogenous (same phase) or heterogeneous (different phases) composition of chitosan and

any small molecule, polymeric molecule, or any form of solvent, stabilizing agent, or

polyion. Chemically, physically, and functionally, heterogenous and homogeneous mixtures

are not identical to single molecule comprised of components of the mixture.

[0016] For example, a number of patents discuss mixtures comprised of chitosan and these

other agents, but do not constitute a compound or molecule. For example, US patent

application publication US/2004/0104020 Al to Davidson et al. discloses chitosan

compositions for hair care, odor control, blood management, fabric treatment, plant care,

water purification and drug delivery using a network of nano-sized fibers. This reference is

not directed to antibacterials but is directed to the broad uses of chitosan and the mixtures of

chitosan. This serves as one example of any number of nonfunctionalized chitosan

compositions reflecting its broad usage independent of or including antimicrobial properties.

[0017] The prior art also discloses polycations that are useful in a number of broad

formulations with or without chitosan. Chitosan and arginine or lysine, positively charged

amino acids, or polymeric combinations of poly-arginine or poly-lysine, usually in the L

stereo isomer, of the same often appear simultaneously on these lists as cationic biopolymers,

but are used independently and not dependent on the combined properties for functionality.

Examples of these include the following. US Patent 5,614,204 to Cochrum discloses

"selective vascular embolization" utilizing an occlusion agent that may be alginate, chitosan or poly-L-amino acid. Poly L-amino acids include poly-L-lysine, poly-L-arginine, poly-L glutamic acid, poly-L-histidine, poly-a-D-glutamic acid or a mixture of the above. As a mixture and as an occluding agent, the components of this composition teach away from the applications and activities and compounds of our application. See also US 5,510,102 to

Cochrum. US 4,749,620 to Rha et al discloses microencapsulation including chitosan as a

polymer selected from a group consisting of chitosan and poly-L-lysine. See also US

4,744,933 to Rha et al. The utility and composition of the above references teach away from

the present invention as discussed below. Japanese Patent 10175857 to Sekisui Chem. Co.

Ltd. discloses a healing agent comprising a commercial ointment with a carbohydrate that is a

mixture of positive material in a hydrocarbon base. This reference is directed to the addition

of arginine, glutamate and their derivatives to the ointment, followed by the same commercial

ointment with the addition of chitin, chitosan and their derivatives to ointment. Mutual

benefit is not noted. US 2004/0103821 Al to Shobu, et al., discloses a food and/or

medicament coating of an insoluble shellac comprising a basic amino acid (e.g. arginine)

and/or chitosan. This reference is however directed to an insoluble mixture .

[00181 Interestingly, chitosan and the positively charged amino acids appear in the patent

literature not in combination, but due to their similar constituency as positively charged

polycations in groups where they co-reside. See, for example, US Patent Publication US

2004/0028672 to Bjorck, et al. which discloses methods for identifying agents including L

arginine and chitosan, for treating chronic and acute microbial infections. This patent

discloses polycations that may be screened for antimicrobial activity. Additionally, this

reference is directed to only homopolymers of arginine or chitosan individually.

[0019] Chitosan and antivirals or antibacterials

Because chitosan is often used as a drug carrier, much of the patent literature teaches to

the adhesive and biocompatible properties of chitosan and of chitosan's role as a component

in compositions that contain antiviral or antibacterial active agents. For example, US

6,465,626 October 2002 to Watts et al teaches that chitosan is one of many adhesive

materials that can be used in pharmaceutical applications. The antiviral component is an

agent ICAM-1.

[0020] Similarly, other patents teach to mixtures of low molecular weight chitosans that are

soluble, but in compositions that antimicrobial activity only in the presence of another

molecule, an active antimicrobial agent. For example, US 5,730,876 You et al. teaches

separation and purification of low molecular weight chitosan using multi-step membrane

separation process and only claims antibacterial properties in the presence ofelecampane

(Inula Helenium L.) root extract. The active antimicrobial agent is the elecampane. Further,

the teachings of US 6,521,268 to You et al., disclose a "natural cell carrier" of water-soluble

chitosan and elecampane extract-antibacterial, anti-inflammatory and broad antibacterial

spectrum for food, cosmetics and medicine, and discusses the antibacterial mechanism as

chitosan binding to telechoic acid in all bacterial membranes. It is the carrier, but also

adjuvant to the true antibiotic and inflammatory elecampane. This patent teaches the role of

chitosan in binding a cell membrane which supports the need for a water-soluble chitosan,

but limits the membrane association to a telechoic acid. The patent teaches away from a high

molecular weight antibacterial chitosan derivative.

[00211 Chitosan salts

Chitosan can be prepared in an acidic solution and precipitated with active salts. For

example, US 4,957,908 to Nelson teaches that chitosan salts can be useful to help impart

desirable properties, in this case for antimicrobial activity that does not adsorb quickly into

the skin and can be used for dermatological items such as soaps and shampoos. Zinc and sodium pyrithione are used as antifungals and antibacterials, but absorb into the skin. By making a particular salt of the chitosan, they can make chitosan pyrithione that is as effective as their original material but dissolves more slowly. Similarly, US 5,015,632 to Nelson discloses a salt of chitosan that is to provide slow release of an anionic salt, pyrithione, from films. It is used as an antimicrobial agent in dermatological items, but derives its properties from the pyrithione. This chitosan salt has the same antibacterial efficacy as the sodium salt of the same anion and thus teaches away from the present invention. US 5,300,494 to Brode

II et al. disclose a series of chitosan salts (in particular lactate salts) that act as carrier films

for pharmaceutically active drugs (particularly quaternary ammonia compounds and salts).

As thin films that retain moisture and the ability to retain and slowly deliver a drug, in this

case an antiviral. These serve as examples of any chitosan salts that bear antimicrobial

capacity due to the anionic salt, a salt mixture with an antibacterial salt rather than as the free

compound.

100221 US 6844430 B2 to Pesce et al. disclose the use of aminopolysaccharide salts for the

control of odor in sanitary products, diapers et al. and where a preferred saccharide is

chitosan. The chitosan is prepared with a number of salts including the amino acids such as

arginine and lysine. As salts, they are counterions and not part of the molecule but produce

the desired biocompatibility. This example teaches to the ability to use salts to provide

additional activity in a chitosan formulation, but does not teach the present invention.

[0023] Chitosan Derivatives

Interest has developed over the years in controlling the properties of chitosan by

performing chemical modifications on the polymer backbone. For chemical reactions that

retain the polymeric form of the chitosan, there are only two types of fairly highly reactive

moieties on the monomers: the hydroxyl group and the amine group. The two hydroxyl

groups have slightly different reactivity but can be functionalized by hydroxy active agents at high pH on either the acetylated or deacetylated monomers of the chitosan. The primary amine of the deacetylated monomer of the chitosan is available for reaction at moderate pH above 6 or so where a significant number of the amines are deprotonated. These chemistries provide new chitosan compounds bearing different properties from the original chitosan polymer.

[0024]Hydroxide chemistry

Other patents teach the desirability of functionalization of chitosans to achieve particular

goals. For example, US Patent Publication US 2003/0181416 Al to Comper, discloses

primarily Dextran which when sulfonated is effective in vivo in the treatment or prevention of

viral, bacterial and parasitic infections. This reference teaches that many other

polysaccharides are not antimicrobial or antiviral and teaches the desirability of controlling

the molecular weight of polysaccharides for optimal in vivo and in vitro microbial activity.

This reference also teaches the desirability of controlling the pH to improve deliverability of

the active ingredients. The sulfated polysaccharides include two chitosan derivatives where

the antimicrobial properties are imparted by negatively charged sulfates.

[00251 Carboxyalkylated chitosan derivatives, sulfonyl chitosan derivative, carbohydrate

branched chitosan derivatives, chitosan-iodine complexes and other miscellaneous

derivatives were also developed. See Muzzarelli et al.; N-(carboxymethylidene) chitosan and

N-(carboxymethyl) chitosan: novel chelating polyampholytes obtained from chitosan

glyoxylate. Carbohyd. Res. 107, 199-214 (1982); Chen et al.; Antimicrobial effect and

physical properties of sulfonated chitosan; Advances in Chitin Science, Vol III, 278-282

(1998); Yalpani et al.; Antimicrobial activity ofsome chitosan derivatives, Advances in chitin

and chitosan; 543-548 (1992); US patent 5,538,955 to De Rosa et al (1996); and Muzzarelli

et al.; Fungistatic activity of modified chitosan against Saprolegnia parasitica;

Biomacromolecules, 2, 165-169 (2001). The limited application and range of these derivatives teach to the need for more thoughtful application of chemical knowledge to the control of solubility and antibacterial properties.

[00261 Amine chemistry-quaternization

The primary amine on the glucosamine monomer of chitosan can be the basis for a

number of reactions, the most important in the literature being quaternization. To quaternize

the amine on chitosan, three additional groups must be added, taking the primary amine to a

quaternary amine with a permanent positive charge. A quaternary amine is fairly

electrophilic, but can remain stable in the absence of any available nucleophiles. Many of

these derivatives add functional groups or modify the carbohydrate with non-biological

moieties that render the molecule different than any naturally occurring molecule, and thus

the toxicity of the molecule is unknown.

[0027] Much of chitosan chemistry has centered on the reactive amine that results from the

deacetylation process of chitin. A quatemized chitosan derivative was developed by

introducing quaternary ammonium salts onto the chitosan backbone. See Kim et al.;

Synthesis of chitosan derivatives with quaternary ammonium salt and their antibacterial

activity, Polymer Bulletin. 38, 387-393 (1997) and Zia et al.; Synthesis and antibacterial

activity of quaternary ammonium salt of chitosan; Carbohydrate Res. 333, 1-6 (2001). The

antibacterial activity and water solubility of this derivative was increased with the decrease in

the chain length of alkyl sustituent. Although there are other methods for producing and

analyzing quaternized amines on chitosan, a sampling of methods is presented. See Hamman

et al.; Effect of the type of base and number of reactionsteps on the degree of quaternization

and molecular weight of N-trimethyl chitosan chloride; Drug Dev. And Ind. Pharm 27(5),

373-380 (2001); Avadi et al.; Dimethylmethyl chitosan as an antimicrobialagent: synthesis,

characterizationand antibacterialeffects; Eur. Polymer J. 40, 1355-1361 (2004); Sashiwa et

al.; Chemical modification ofchitin and chitosan 2: preparationand water soluble property of N-acylated or N-alkylated partially deacetylated chitins Carbohydrate Polymers 39, 127

138 (1999); and Sieval et al.; PreparationandNMR characterizationof highly substitutedN

trimethyl chitosan chloride; Carbohydrate Polymers 16, 157-165 (1998). The'quaternary

amine has its positive charge surrounded by bulky methyl or even more sterically

constraining longer hydrocarbons, but clearly teaches to the importance of positive charge in

the antibacterial properties and solubility of chitosan.

[0028] The prior art is rich with the synthesis and applications of chitosans with methodology

to produce the quaternary amine. A series of patents teach the importance of solubility for

materials having necessary activity at physiological pH. For example, a series of patents

describe the creation of and use of the quaternary amines as foaming and stabilizing agents in

cosmetic compositions. For example, US 4,772,689 to Lang et al. discloses the quaternary

chitosan derivatives with hydroxy and propyl substitutions on the amine that are used in

cosmetic compositions for the treatment of hair or skin, characterized by a content of new

quaternary chitosan. Also disclosed are the new quaternary chitosan derivatives per se as well

as processes for their preparation. The chitosan derivatives have a good substantivity,

particularly to hair keratin, and prove to have hair strengthening and hair conditioning

characteristics. US 4,976,952 to Lang et al. discloses a cosmetic agent for the treatment of

the hair and skin that contains macromolecular surface-active, quaternary N-substituted

chitosan derivatives with a variety of degrees of substitution and pendant groups on the

amine. This invention also comprises chitosan derivatives distinguished particularly by their

surface-active properties, for example, their foam-forming and emulsifying properties, and by

their hair-setting and hair-conditioning effect. Similar to these are US 4,921,949; US

4,822,598 and US 4,772,689 all to Lang et al. These describe the action of cation-active

polymers, particularly polymers which have quaternary ammonium groups, as conditioning

compositions in cosmetic compositions, particularly for the treatment of hair. Based upon a reciprocal action between their ammonium groups and the anionic groups of the hair, the cation-active polymers possess a great affinity for keratin fibers. These series of patents teach to the desirable interactions of polycations and polyanions for particular applications and the ability to modify chitosan to achieve and control those interactions.

[0029] Amine chemistry- acid coupling

Amines are typically coupled to carboxylic acids using peptide coupling chemistry. These

chemistries continue to be performed in organic solvents, primarily for the synthesis of

polypeptides and short proteins. Only recently were many of the coupling agents modified to

be active in aqueous solutions See Bioconjugate Techniques Greg. T. Hermanson (Elsevier,

Academic Press: USA) (1996).

[0030] Few descriptions exist of chitosan derivatives N-conjugated with different amino

acids. Jeon et al., describe low molecular weight chitosan polymers (less than 10,000 Da)

with asparagine, glycine, alanine, aspartic acid, cysteine and methionine. However they do

not disclose or describe the inherent solubility issues of their chosen amino acids. Rather

Jeon et al. rely on inherent solubility provided by selecting low molecular weight chitosan,

thereby limiting the applicability of their compounds. Additionally, Jeon et al do not utilize

the positively charged amino acids nor do they disclose or describe any correlation between

solubility and charge. It is important to note that Jeon et al. focus upon aspargine, an amino

acid that is neutral at all pH's. Thus Jeon et al suggest that neutral amines at physiologic pH

contribute to antimicrobial activity. This implication teaches away from the present

invention. Furthermore, the coupling method disclosed by Jeon et al requires the use of

N,N'-dicyclohexylcarbodiimide (DCC)as a coupling agent which is not water soluble. The

reaction is preformed in 4:1 methanol:water mixtures with triethyl amine (TEA) as a base to

bring the pH to 6.8. After reaction, deprotection is performed in trifluoroacetic acid to remove the boc protecting groups thereby further reducing the MW and producing a large distribution that is not addressed for the various chitosan products disclosed by Jeon et al.

Furthermore, the activity disclosed in Jeon et al is not unlike the variability exhibited by low

molecular weight chitosan. Thus, Jeon et al fail to teach control of higher weight chitosans,

as per the present invention. See Jeon et al.; Effect of antimicrobialactivity by Chitosan

oligosaccharide N-conjugated with Asparagine. Microbial. Biotechnol. 11(2): 281-286

(2001).

[00311 It is known that chitosan is an avid coagulant and that chitosan of different molecular

weights are utilized to induce clotting and provide hemostasis. See US 4,394,373 to Malette

et al. It is also important to note that there are scant disclosures of the desirability of

producing arginine bound to chitosan as anticoagulation biomaterials. However, no disclosure

is made of the fact that the methodology as disclosed, produces polymers of their chosen

amino acids. Additionally, no disclosure is made of any manner of limiting the formation or

coupling of poly-amino acids to chitosan as is necessary for the formation of chitosan

arginine. As is well understood by one of ordinary skill, that a mechanism for limiting the

formation or coupling of poly amino acids to chitosan is essential to the formation of

chitosan-argine. Additinally, no disclosure is made as to the relevance of charge density to

antibacterial properties of chitosan-arginine. Thus, in the absence of such a teaching, severe

doubt must be raised as to the probability that the formation of chitosan-arginine has indeed

occurred and in such desirable quantities to perform the described utility. See Liu et al. ; A

chitosan-arginineconjugate as a novel anticoagulationbiomaterial. J. of Materials Science:

Materials in Medicine 15:1199-1203 (2004).

[0032] Chinese Patent 1519035AI provides limited disclosure of the ability to make

chitosan-arginine for the purpose of biomedical polymers for in vivo implants. This reference is directed to the inhibition of hemagglutination by chitosan-arginine based upon their disclosure that arginine derivatized chitosan has a longer hemagglutination time than chitosan alone. However, it is important to note that several patents and purchasable products directly contradict the assertion that chitosan based materials can inhibit hemagglutination.

See US 4,394,373 to Malette et al. and US 6,162,241 to Coury et al. See also, Klokkevold et

al.; Effect of chitosan on lingual hemostasis in rabbits;J. Oral and Maxillofacial Surg. 49(8):

858-863(1991).

100331 The methodology of Chinese Patent 1519035A1 includes peptide coupling that is

well known in the literature, and thereafter subjecting the arginine-chitosan product/pre

product to a magnetic field. No disclosure is made as to the relevance of the magnetic field.

The references cited by the presently discussed publication evidence an absence of interest in

chemically protecting the a-primary amine on the arginine and thus, an inability to control

the chemistry at that amine. The importance of control of the primary active amine results in

the ability to control coupling, as discussed above. Thus, an active primary amine that is

similar in activity to the amine on the chitosan will inevitably react with the coupling agents

to react with the activated carboxylate group of other arginines resulting in poly-arginine,

either attached to the chitosan or copolymerized in solution. A method of controlling activity

on this reaction site is necessary to the production of the chitosan-arginine products as

disclosed, and an absence of such a disclosure must lead one of ordinary skill to the

conclusion, that the present publication has only addressed the desirability of the formation

of such compounds, and not the actual disclosure of such compounds. Additionally, this

reference requires insolubility, a characteristic that teaches away from the present invention,

particularly antibacterial applications.

[00341 A number of patents teach to the addition of amino acids for desirable properties.

US 4,908,404 to Benedict et al. discloses that a polymeric backbone of polypeptides having

primary or secondary amines can be functionalized with combinations of synthetic amino acids for creating strong bioadhesive materials that are compatible with living tissues. While general teachings of the desirability of coupling multiple components on a cationic backbone are disclosed, the reference is markedly distinguished from the present invention as discussed below.

[0035] Further addition of short polypeptides has been attempted on chitosan to stimulate cell

growth or adhesion. See Ho et al..; Preparationand characterizationof RGD-immobilized

chitosan scaffolds; Biomaterials 26: 3197-3206 (2005)]. Ho et al describe attaching the four

amino-acid polypeptide RGDS through a carbodiimide coupling scheme for the purposes of

coupling free amines of the R (arg) peptide as well as the amine on the chitosan. Ho et al also

disclose coupling of short polypeptides to an insoluble chitosan matrix for chitosan scaffolds.

Another reference discloses coupling a longer polypeptide using an activated reagent that

removes the need for protecting the amine on the amino acids. See Masuko, et al.; Chitosan

RGDSGGC conjugate as a scaffold material for musculoskeletal tissue engineerin;.

Biomaterials 26: 5339-5347 (2005). This same coupling agent is also taught in US 7,053,068

to Prinz. Prinz also discloses a method for reacting the amines on chitosan with

iminothiolactones, which impart a positive charge to the chitosan and provide an excellent

coupling group for thiol chemistry. These can be made to gel or crosslinked for controlled

release drug delivery. This reference provides a substantial teaching of the strength of active

amine groups and the need for careful control of the chitosan reactive amine for practical

coupling.

[0036] Textiles

Additional studies include the different responses by bacteria subjected to a range of

molecular weight chitosans prepared on textiles. Though very limited in scope, additional

variables that are considered in a small number of bacterial studies are; degree of

deacetylation, pH, cations and anions present in solution. See Shin et al.; Molecular weight effect on antimicrobial activity of chitosan treated cotton fabrics; J. Apple. Poly Sci., 80,

2495-2501 (2001); and Lim et al.; Review of chitosan and its derivatives as antimicrobial

agents andtheir uses as textile chemicals, J. of Macromolecular Sci. C43(2), 223-269 (2003).

Wound treatments have also been addressed by utilizing a combination of chitosan with

silver sulfadiazine for wound dressings and burns. See Mi et al.; Asymettric chitosan

membranes prepared by dry/wet phase separation: a new type of wound dressing for

controlled antibacterialrelease; J. Membrane Sci.; pp. 212, 237-254 (2003). Additionally

wound dressings have also utilized chitosan in combination with glycerol, chitin and ethylene

oxide. See Marreco et al.; Effects of different sterilization methods on the morphology,

mechanical properties and cytotoxicity of chitosan membranes used as wound dressings,

Wiley periodicals, 268-277(2004).

[00371 Chitosan- Guanidine

The addition of a guanidinium group to a primary amine impart positive charge or polarity

or to act as an intermediate step in chemical reactions has been developed primarily in

organic solvents. Use of a known guanidinylation (guanilating) reagent, formamidine sulfinic

acid, has been demonstrated in absolute methanol for small molecules containing primary

amines. See Katritzky et al.; Recent developments in guanylatingagents. ARKIVOC iv, 49

87 (2005); and Maryanoff et al.; A convenient synthesis of guanidinesfrom thioureas; J.

Org. Chem. 51, 1882-1884 (1986).

[0038] Very little has been reported in the literature for chemical functionalization of the

primary amine on chitosan by direct guanidinylation. Due to chitosan's insolubility in

organic solvents, syntheses are restricted to aqueous solutions. The effectiveness of chitosan

functionalization by typical guanylating agents in water is a challenge, as these are somewhat

different than the conditions under which these reagents were orinigally intended to operate.

Reaction with formamidine sulfinic acid is an atom-economical reaction with no by-products,

and the sulfinate provides the salt to the positive guanidinyl product.

[0039] The chemistry of another guanylating reagent, 1H-pyrazole-l-carboxamidine

hydrochloride, has also been examined for primary amines. See Bernatowics et al.; 1H

pyrazole-1-carboxamidine hydrocholride: an attractive reagent for guanylation of amines

and its applicationto peptide synthesis; J Org. Chem 52, 2497-2502 (1992). This reactant can

be synthesized and characterized in gram quantities in a single synthetic operation.

[0040] The bioactivity of biguanides and chitosan with related biguanidinylations have been

disclosed in the prior art. Japanese Patent 60233102 to Toshio discloses a chitosan derivative

from dicyandiamine or dyanamide to produce a coagulating material and metal ion absorbant

that has biguanide or and or guanidine groups. This reference is distinguished from the

present invention because it fails to teach structure and does not provide disclosure of the

relevance of solubility charge and antimicrobial properties of the compound.

[0041] Microbial Populations

A variety of debilitating diseases and syndromes are the result of poorly regulated

microbial populations. Many symptoms of disease are produced by the concurrent bacterial

infections that encroach upon weakened immune systems and tissues. A dramatic rise in use

of common antibacterials has resulted in the concurrent rise of antibacterial resistant species.

Wounds, lacerations and abrasions as well as burns and ulcers are dermal occurrences that are

easily contaminated by a variety of environmental bacteria. Prosthetic joint sites where

rubbing and abrasion often regularly occur are a common site for chronic infections.

Furthermore, microbial imbalances in the gut are fairly frequent. One important example is

the peptic ulcer, which is primarily caused by the acid-loving Helicobacterpylori, a gram

negative bacterium. Inflammatory bowel syndromes, such as Crohn's disease or ulcerative

colitis, are the result of the body's inability to control bacterial residence in the gut and the leakage of bacteria across the gut membranes. Peritonitis is an inflammation of the peritoneum and can result the dramatic translocation of bacteria across the gut walls and membranes.

100421 Bacteria have been considered free floating organisms, but in the natural world, most

bacteria (-99.5%) aggregate in biofilms and behave differently than their planktonic forms.

See O'Toole et al.; Biofilm formation as microbial development; Annual Review of

Microbiology 54:49 (2000); Watnick et al.; Biofilm city of microbes; Journal of

Bacteriology 182:2675 (2000); Stoodley et al.; Biofilms as Complex Differentiated

Communities; Annual Review of Microbiology; 56:187 (2002). Bacterial biofilm formation

is an industrial problem affecting water purification systems, heat exchangers and biological

sensors. Biofilms serve as a continuous source of planktonic bacteria, which, when released

from biofilms, seed formation of new biofilms in new locations.

[0043] Biofilms are also a major cause of human disease; chronic bladder infections, colitis,

conjunctivitis, and periodontal disease are only a few among many well-established examples

See Davies; Understandingbiofilm resistance to antibacterialagents; Nature Reviews Drug

Discovery 2:114 (2003). Biofilms problematically colonize medical devices such as catheters

(e.g. urinary catheters are among the worst), contact lenses and artificial implants such as

pacemakers, stents, dental and breast implants, and heart-valves among many others. See id.

These biofilms are highly resistant both to clearance by the immune system and to antibiotic

treatments.

100441 Inside the body, biofilms serve as a protected source of continuously shed bacteria and

biofilm fragments. The sloughed-off materials seed into the surrounding tissues and the

circulatory system leading to recurrent acute infection. See Anderson et al.; Intracellular

bacterialbiofilm-like pods in urinary tract infections. Science 301:105(2003). In addition to

releasing the extracellular matrix materials, the biofilm-resident bacteria also show protective behaviors (e.g. expression of multidrug efflux pumps) which may play an important role in evolution of multi-drug resistant nosocomial infection. For example, Pseudomonas aeruginosa, a common nosocomial pathogen and an adept biofilm-former, is multi-drug resistant at an alarming rate of 95% in the planktonic form.

[00451 Biofilm bacteria display profoundly decreased sensitivity to biocides and antibiotics,

becoming 10-1000 fold more resistant than the same type of bacteria grown in planktonic

culture. See Luppens et al.; Development of a standard test to assess the resistance of

Staphylococcus aureus biofilm cells to disinfectants; Applied & Environmental Microbiology

68:4194 (2002). Controlling bacterial populations in biofilms is clearly a challenge.

10046] The most common causes of diarrheal diseases are E. coli, Campylobacterfejuniand

Shigella. There are no vaccines approved by the FDA to prevent infection. Intense

psychological and physical stressors often lead to respiratory infections such as bacterial

pneumonia and streptococcal infections as well as increased susceptibility to viral influenza.

Escherichia coli are responsible for a variety of diarrheal and intestinal diseases. Diarrhea

treated with ciprofloxacin substantially increases the antimicrobial resistance rates for

multiple antibiotics, although they appear to return to pretreatment levels within a month. See

Shannon et al.; Antimicrobial Agents and Chemotherapy; pp. 2571-2572 49:6 (2005).

Outbreaks of E. coli poisoning due to improper food storage are not infrequent and can lead

to high mortality rates due to the specific shiga-like toxins produced by strains such as the

0157:H7. Other enteropathic bacteria with devastating effects include Campylobacter

species, Salmonella species, Shigella species, and Vibrio species, which can produce

gastroenteritis with severe diarrhea, nausea and vomiting.

[0047] A number of bacteria are associated with battlefield wounds but translate directly into

the civilian population. Acinetobacter baumannii, a bacterium found in soil and water, has

resulted in wound, respiratory, and bloodstream infections. The bacterium poses a danger due to its ability to survive on surfaces for up to 20 days and its apparent resistance to most known antibacterials. Acinetobacter is one of the most common gram-negative bacteria to colonize the skin of hospital personnel, potentially increasing the likelihood of nosocomial infection amongst other patients. A. baumanniican easily lose susceptibility to the antibiotics available. Only three drugs have been known to have exhibited efficacy against A.

Baumannii. Imipenem carries a risk of seizure, amikacin, does not work for bone infections

and has not been effective against some strains of the bacteria, and colistin, an antibiotic with

severe toxic effects on the kidneys. See Aronson et al.; In Harm's Way: Infections in

Deployed American Military Forces; Clinical Inf. Disease Volume 43: 1045 - 1051(2006).

[00481 Major complications of burn injuries include fluid loss and wound sepsis due to

bacterial infections; a common cause is the Pseudomonas aeruginosaorganism, which can be

difficult to treat due to its resistance to antibiotics. Oral antibiotics, with the exceptions of the

fluoroquinolones, are generally ineffective against most serious skin and soft tissue infections

by P. aeruginosa. See Dale et al.; Therapeutic Efficacy of "Nubiotics"againstBurn Wound

Infection by Pseudomonas aeruginosa;Antimicrobial Agents and Chemotherapy; pp.2918

2923, 48:8 (2004). Burrowing bacteria such as the Proteus are particularly difficult to treat in

deep wounds and bums.

[0049] A variety of other pathogens have emerged as multi drug resistant. Klebsiella

pneumoniae can cause nosocomial wound infections and is resistant to ampicillin. Many

strains have acquired resistance to carbenicillin, quinolones, and increasingly to ceftazidime.

The bacteria remain largely susceptible to aminoglycosides and cephalosporins. Cutaneous

infection from Leishmania major generally results in chronic, painless skin lesions.

Leishmania tropica and Leishmania infantum-donovani may be associated with

visceralization and more chronic, reactivating illness. While treatment controls the clinical

disease, it does not destroy the organism. Methicillin resistant Staphylococcus aureus

(MRSA) is resistant to methicillin and other more common antibiotics such as oxacillin,

penicillin and amoxicillin. MRSA infections occur most frequently among persons in

hospitals and healthcare facilities who have weakened immune systems. From 1995 to 2004,

the percentage of resistant bacteria in ICU patients has increased from <40 to 60% [National

Nosocomial Infections Surveillance System, CDC].

[0050] In addition, viruses are an important set of microbes that infect almost any type of

body tissue, including the brain. Often the use of antibiotics complicates viral infections.

Most treatments for viral infections are preventative in the form of vaccines, as the vast

majority of human viral infections are controlled by the immune system. However, materials

are needed to prevent or treat serious viral infections or reduce viral infectivity.

[0051] The present invention overcomes the limitations of the prior art as discussed above

and present chitosan derivative compounds that are discussed hereinbelow.

SUMMARY OF THE INVENTION

[00521 It is, therefore, an objective of the present invention to control microbial populations

in a variety of environments.

[0053] It is also another objective of the present invention to inhibit and/or enhance

microbial subpopulations and pathogens in a wide variety of environments.

[0054] It is yet another objective of the present invention to inhibit of growth of exogenous

bacteria, mycoplasma and biofilms.

[0055] It is yet another objective of the present invention to promote wound healing due to

second and third degree bums, chronic ulcers, bed sores, etc.

[00561 It is yet another objective of the present invention to provide chitosan derivative

compounds that are capable of controlling, treating and preventing the growth of microbial

populations.

[0057] It is yet another objective of the present invention to provide chitosan-arginine

compounds, and chitosan-guanidine compounds, that are soluble at physiologic pH and

utilized to control, treat and prevent the growth of microbial populations.

[00581 It is yet another objective of the present invention to provide chitosan-unnatural

amino acid compounds, chitosan-acid amine compounds, chitosan-natural amino acid

compounds, co-derivatives of chitosan derivative compounds, salts of chitosan derivative

compounds and salts of co-derivatives of chitosan derivative compounds that are soluble at

physiologic pH and utilized to control, treat and prevent the growth of microbial populations.

[00591 It is yet another objective of the present invention to provide a method of

synthesizing chitosan-arginine compounds that are soluble at physiologic pH for controlling,

treating and preventing the growth of microbial populations.

[00601 It is also another objective of the present invention to provide a method of

synthesizing chitosan-unnatural amino acid compounds, chitosan-acid amine compounds,

chitosan-natural amino acid compounds, co-derivatives of chitosan derivative compounds,

salts of chitosan derivative compounds and salts of co-derivatives of chitosan derivative

compounds that are soluble at physiologic pH for controlling, treating and preventing the

growth of microbial populations.

[0061] These and other objectives are discussed hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1(a) shows the effect of chitosan-arginine (7% functionalized) on E. coli at 24

hours.

[0063] Figure1(b) shows the effect of chitosan-arginine (7% functionalized) on E. coli at

48 hours.

10064] Figure 1(c) shows the effect of chitosan-arginine (7% functionalized) on B. subtilis at

24 hours.

[00651 Figure 1(d) shows the effect of chitosan-arginine(7% functionalized) on B. subtilis at

48 hours.

[00661 Figure 1(e) shows the effect of chitosan-arginine (7% functionalized) on S.

epidermidis 35984 at 24 hours.

[0067] Figure 1(f) shows the effect of chitosan-arginine (7% functionalized) on S.

epidermidis35984 at 48 hours.

[0068] Figure 1(g) shows the effect of chitosan-arginine (7% functionalized) on P.

fluorescens 700830 at 24 hours.

[0069] Figure 1(h) shows the effect of chitosan-arginine (7% functionalized) on P.

fluorescens 700830 at 48 hours.

[0070] Figure 1(i) shows the summary inhibitory effect of chitosan-arginine (7%

functionalized) on E. coli, B. sublilis, S. epidermidis 35984 and P. fluorescens 700830 at 48

hours incubation

[00711 Figure 2 (a) shows the effect of chitosan-arginine (7% functionalized) on Klebsiella

pneumoniae 85W 1880 at 24 hours.

[0072] Figure 2 (b) shows the effect of chitosan-arginine (7% functionalized) on Shigella

flexneri 85W 2332 at 24 hours.

[00731 Figure 2 (c) shows the effect of chitosan-arginine (7% functionalized) on Salmonella enteritidis 8W2310 at 24 hours.

[0074] Figure 3(a) shows the bacteriocidal effect of chitosan-arginine (6.3% functionalized)

for Streptococcus pyogenes (Strep A) in cell culture media for 107 cells; Figure 3(b) shows

the bacteriocidal effect of chitosan-arginine (30 % functionalized) for Streptococcus

pyogenes (Strep A) in cell culture media for 107 cells; Figure 3(c) shows the bacteriocidal

effect of chitosan-arginine (6.3% functionalized) for Streptococcuspyogenes (Strep A) in cell

culture media for 2 x 105 cells; and Figure 3(d) shows the bacteriocidal effect of chitosan

arginine (30% functionalized) for Streptococcuspyogenes (Strep A) in cell culture media for

2 x 105 cells.

[0075] Figure 4 shows chitosan-arginine's ability to inhibit the formation of biofilms ofS

epidermidis.

[0076] Figure 5(a) shows chitosan-arginine's bacteriocidal activity in natural water

preformed biofilms.

[0077] Figure 5(b) shows chitosan-arginine's dose response of bacteriocidal activity

preformed biofilms of S. aureus.

[0078] Figure 6(a) shows the charge density and broad spectrum antibacterial effect of

chitosan-arginine (6.3% and 30% fictionalized) for A. baumannii, E. coli, P. fluorescens, S.

pyogenes (StaphA), B. subtilis, S. epidermidis,and S. aureus at 24 hrs., with a 1:50 dilution.

[0079] Figure 6(b) shows the charge density and broad spectrum antibacterial effect of

chitosan-arginine (6.3% and 30% functionalized) for A. baumannii, E. coli, P. fluorescens, S.

pyogenes (Staph A), B. subtilis, S. epidermidis, and S. aureus at 48 hrs., with a 1:50 dilution.

[0080] Figure 6(c) shows the charge density and broad spectrum antibacterial effect of

chitosan-arginine (6.3% and 30% functionalized) for A. baumannii, E. coli, P. fluorescens, S.

pyogenes (Staph A), B. subtiis, S epidermidis, and S. aureus at 24 hrs., with a 1:1000

dilution.

[0081] Figure 6(d) shows the charge density and broad spectrum antibacterial effect of

chitosan-arginine (6.3% and 30% functionalized) for A. baumannii, E. coli, P. fluorescens, S.

pyogenes (Staph A), B. subtilis, S epidermidis, and S aureus at 48 hrs., with a 1:1000

dilution.

[00821 Figure 7(a) shows the bacteriostatic effects of chtiosan-arginine in cell culture media

for S. epidermidis 35984.

[0083] Figure 7(b) shows the bacteriostatic effects of chtiosan-arginine in cell culture media

for S. aureus 29213.

[00841 Figure 7(c) shows the bacteriostatic effects of chtiosan-arginine in cell culture media

for P.fluorescens 700830.

[00851 Figure 8(a) shows the limited cytotoxic effects of chitosan-arginine ~ 30% on HeLa

cells.

[00861 Figure 8(b) shows the limited cytotoxic effects of chitosan-arginine 6% on HeLa cells

[00871 Figure 9(a) shows an SEM image of amino-functionalized glass

[00881 Figure 9(b) shows an SEM image of chitosan arginine bound to amino

functionalizedglass.

[0089] Figure 9(c) shows an SEM image of amino-functionalized glass exposed to

Pseudomonasfluorescens.

[00901 Figure 9(d) shows an SEM image of chitosan-arginine coated glass exposed to

Pseudomonasfuorescens.

[0091] Figure 10 shows chitosan-arginine's effect on viral infectivity of herpes virus,

EMCV, influenza virus and vaccinia virus.

[0092] Figure 11(a) shows the morphology of MDCK cells exposed to nonfunctionalized

chitosan and influenza virus (HI, N [control]).

[0093] Figure 11(b) shows the morphology of MDCK cells exposed to chitosan-arginine

and influenza virus (HI, NI).

[0094] Figure 11(c) shows the morphology of MDCK cells infected by influenza virus (HI,

NI).

10095] Figure 12(a) shows amino-functionalized glass exposed to influenza virus (HI, NI).

[0096] Figure 12(b) shows amino-functionalized glass bound to chitosan-arginine (6%

functionalized).

[0097] Figure 12(c) shows amino-functionalized glass-bound to chitosan-arginine and

exposed to influenza virus (H1, N1).

[0098] Figure 13 shows the ability of chitosan-arginine to improve survivability by 100%

in-vivo mouse 3d degree burns infected with Proteus mirabilis.

[0099] Figure 14 shows MW data of chitosan prior to functionalization and post-arginine

functionalization with 19 hours deprotection in 95% TFA.

[0100] Figure 15 shows dynamic light scattering measurements of chitosan and chitosan

arginine at different deprotection times.

[0101] Figure 16 NMR spectrum of chitosan at 70°C with water peak suppression.

[0102] Figure 17(a) NMR spectrum of chitosan-arginine at 70°C with water peak

suppression.

[0103] Figure 17(b) NMR spectrum of chitosan-arginine at room temperature at a limited

range between 2.2 and 1.3 ppm.

[0104] Figure 18 (a) shows chitosan-guanidine's bacteriocidal effect on E. coli.

[0105] Figure 18 (b) shows chitosan-guanidine's bacteriocidal effect on S. pyogenes (Strep

A).

[0106] Figure 18 (c) shows chitosan-guanidine's bacteriocidal effect on B. subtilis.

[01071 Figure 18 (d) shows chitosan-guanidine's bacteriocidal effect on A. baumannii.

[0108] Figure 18 (e) shows chitosan-guanidine's bacteriocidal effect on S. epidermidis.

[01091 Figure 18 () shows chitosan-guanidine's bacteriocidal effect on S. aureus; and

Figure 18 (g) shows chitosan-guanidine's bacteriocidal effect on P. fluorescens.

[0110] Figure 18 (h) shows the summary of chitosan-guanidine's bacteriocidal effect on E.

coli, S. pyogenes (Strep A), B. subtilis, A. baumannii, S. epidermidis, S. aureus, and P.

fluorescens.

[0111] Figure 19 (a) shows chitosan-guanidine's rapid antibacterial effect on a broad

spectrum of bacteria: S. aureus, S. pyogenes (Strep A), A. baumannii, S. epidermidis, P.

fluorescens and E. coli.

[01121 Figure 19 (b) shows the bacteria count of S. aureus after1 hour exposure to chitosan

guanidine.

[01131 Figure 19 (c) shows the bacteria count of S. pyogenes (StrepA) after1 hour exposure

to chitosan-guanidine.

[0114] Figure 19 (d) shows the bacteria count of A. baumannii after 1 hour exposure to

chitosan-guanidine.

[01151 Figure 19 (e) shows the bacteria count of S. epdermidis after 1 hour exposure to

chitosan-guanidine.

[0116] Figure 19 (f) shows the bacteria count of P. fluorescens after 1 hour exposure to

chitosan-guanidine.

[01171 Figure 19 (g) shows the bacteria count of E. coli after 1 hour exposure to chitosan

guanidine.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0118] The present invention is directed to the control of microbial populations in a variety of

environments. Inhibition and/or enhancement of particular subpopulations of microbes and

pathogens is desirable in a wide variety of environments. For the purposes of the present

invention, microbial populations include, but are not limited to, bacteria, mycobacteria,

mycoplasma, viruses, protozoas and prions. For example, control of bacterial populations,

including the general microbiota balance, is important in gut health. Additionally, the

impermeability of the peritoneum to selective bacteria, control of inflammatory bowel

syndrome and control of enteropathic diseases are also highly desired. Growth of cells in cell

culture media requires the inhibition of growth of exogenous bacteria, fungi and mycoplasma.

Wound healing, such as in chronic ulcers, bed sores, lacerations and other sources of

potential infections, requires inhibition of bacterial growth as well as enhancing cellular

signals that promote wound healing. Oral care involves selective control of bacterial

populations as well. Control of bacterial populations in burn induced wounds, particularly in

2 nd and 3rd degree burn wounds, requires a soluble antibacterial that penetrates into

corrugated and necrotic tissue crevices where bacteria reside. Bacterial populations as well as

biofilm growth, particularly in the lung must also be minimized. Growth enhancement of

livestock, such as poultry, ruminants, etc, are also controlled by selective use of

antibacterials as food additives. As is understood to one skilled in the art, the applications of

the present invention can be extended to any areas where control and/or inhibition of

microbial populations is desirable.

[0119] The present invention is also directed to the prevention of bacterial and/or pathogenic

infection or contamination, as well as the prevention of biofilm formation. Additionally, this

invention is particularly important in precluding contamination of pre-sterilized medical materials such as, but not limited to, textiles, IV bags and tubing, catheters, masks, medical materials such as medical grade latex, PVC, silicone, rubber, or the like.

[0120] The present invention accomplishes the control of microbial populations, the

prevention of bacterial or and/or pathogenic infection or contamination, and prevention of

biofilm formation by providing chitosan derivative compounds including but not limited to

chitosan-arginine compounds, chitosan-guanidine coumpounds, chitosan-unnatural amino

acid compounds, chitosan-acid-amine compounds, chitosan-natural amino acid compounds,

co-derivatives of the just described compounds and the salts thereof. These compounds are

capable of being formed into soluble powders, films, coatings, gels, laminate, nanoparticles,

impregnated onto biohazard masks or the basis of the mask, woven and non-woven fibers,

nanofibers, encapsulated for pharmaceutical applications, time-released pharmaceutical

composition, and mixtures of different chitosan derivative compounds. Additionally, the

chitosan derivative compounds of the present invention have ultra-low endotoxin levels. The

chitosan-derivative compounds of the present invention are efficacious over a broad pH

range, ie, 1-10. However in accordance with the present invention these chitosan-derivative

compounds provide markedly improved antimicrobial properties and solubility at physiologic

pH. Physiologic pH is defined as the pH range between 6.8 and 7.4.

[01211 Chitosan is the deacetylated form of chitin. Although chitosan itself has some

antibacterial activity, this activity is limited by its solubility and lack of significant positive

charge at physiological pH. It is known that at low pH, chitosan forms a polycationic

structure that can interact with anionic compounds and macromolecular structures. Since the

degree of protonation increases with increased acidic conditions, solubility in aqueous

solutions also increases. This increased solubility and protonation is directly proportional to

increased antibacterial activity. Conversely, the degree of aqueous solubility decreases with

decreased acidic conditions, as does antibacterial activity.

[0122] The chitosan-derivative compounds, of the present invention, overcome the issues of

solubility and charge as well as molecular weight, charge density, charge distribution and

placement of charge in order to provide improved and selective antibacterial activity. Like the

fibrous material of all polysaccharides including chitosan, these chitosan derivative

compounds retain the properties of a natural biomaterial that is not metabolized, not

absorbed in the body and does not cross the blood brain barrier. Because of these properties,

these chitosan derivative compounds are a novel and powerful class of antibiotics.

[01231 Control of molecular weight, charge density, charge distribution and placement of

charge from the chitosan backbone allows for selective microbicidal activity. The backbone

of the chitosan polysaccharide, as shown in (1) above, is a linear chain of glucose-substituted

monomers (also referred to as "glucose monomers", below) connected by a glycosidic bond.

Each of the glucose monomers has either an amine or an acetyl amine, indicated by the

fractions p and q, respectively, where p + q = 1 and where p >10.5 . Functionalization of the

chitosan backbone, occurs at either the primary amine or either of the two hydroxyl groups on

the glucose ring. To optimize functionalization opportunities, the primary amine of the

chitosan backbone is the reactive site. To optimize control of microbial populations, the

present invention is directed to correlating the sensitivity of different microbial species and

strains to the molecular weight of the chitosan-derivative compounds.

[0124] In accordance with the present invention, the sensitivity of different microbial species

and strains is also dependent on the charge density of the chitosan derivative compounds,

which is in turn directly related to the degree of functionalization. Functionalization is

defined as the coupling of a molecule containing a carboxylic acid to the primary amine.

Functionalization is also defined as the direct guanidinylation of the primary amine. The

functionalized chitosan-derivative compounds of the present invention are developed to vary in molecular weights and charge densities depending upon their use. The degree of functionalization imparts a particular charge density to the chitosan derivative compounds.

For a particular molecular weight, the higher the functionalization with positive charge, the

higher the charge density on the polymer chain. However, it is important to note that

compounds having very high degrees of funcationalization may be toxic to mammalian cells.

Increased charge density produces higher solubility and higher efficiency of microbiostatic or

microbiocidal activity. Increased charge density also increases the interactions with

mammalian cell membranes. Thus, this invention is also directed to balancing

functionalization with toxicity in mammals and humans, so as to provide optimal efficacy and

minimal toxicity in all environments and utilities.

+NH 3 (2)

+ NH ,NH - NH H2N=< 2 NH2N2H NH2 NH2 +NH2 (3)

[0125] The sensitivity of different microbial species and strains is also dependent upon the

charge distribution of the compounds. The charge distribution is either a single point charge

as shown in (2) or a resonance structure as shown (3). A point charge must be located at a

particular site for optimal interaction whereas a charge distribution interacts simultaneously

with multiple negative charges or polar groups.

[0126] The sensitivity of different microbial species and strains is dependent upon the

position of the charge relative to the chitosan backbone, (1), of the chitosan-derivative compounds, discussed below. Additionally, the position of the charge is also varied by choice of the functionalizing group, as described below. Finally, the sensitivity of different microbial species and strains is also dependent on compound dosage relative the concentration of microbial species.

[01271 The present invention provides protonated and positively charged chitosan-derivative

compounds that are soluble and/or active at physiologic pH while imparting significant

antimicrobial activity. Depending on its application, the present invention incorporates these

positively charged chitosan compounds as bacteriocial or bacteriostatic agents. These

compounds will have minimal residual endotoxin and protein concentrations.

[0128] This invention is also directed to the control of microbial populations through the

antimicrobial activity of chitosan-derivative compounds, an activity that is controlled by

charge density imparted by the degree of functionalization of the chitosan-derivative

compounds and by overall molecular weight. The antibacterial activity is broad spectrum and

is concentration dependent. In accordance with the present invention the molecular weight of

the chitosan derivative compounds is between 25,000 Da and 1,000,000 Da.

[0129] Particular classes and/or species of microbes can be targeted by manipulation of

composition and formulation of the chitosan-derivative compounds. These include but are

not limited to gram-positive bacteria, gram negative bacteria, mycobacteria, mycoplasma, and

viruses, protozoas and prions. Specific targeted bacteria include but are not limited to those

listed in Table 1 .

[01301 Table 1

Gram negative

Escherichiacoli gastroenteritis, urinary tract infections

Pseudomonasaeruginosa predominant infection in patients with cystic

fibrosis

Pseudomonasfluorescens plant infections and a model for P. aeruginosa (CF

and burn ward infections A cinetobacterbaumannii battle wounds

Klebsiellapneumonia pneumonia in community settings (tends to affect

people with underlying diseases, such as

alcoholism, diabetes and chronic lung disease Shigellaflexneri gastroenteritis and a model for dysentery

Salmonella typhi gastroenteritis

Proteusmirabilis wound infection, septicemia and pneumonias, mostly in hospitalized patients

Gram positive

Bacillus subtilis model for anthrax and Bacillus cereus

Staphylococcus aureus boils, hospital infections, toxic shock

Staphylococcus epidermidis hospital infections, especially catheters

Streptococcusmutans key bacteria in biofilm of teeth, required for most tooth decay

Streptococcuspyogenes(Strep A) a cause of 'strep throat", impetigo and necrotizing

fasciitis

[0131] Viruses are small particles containing either DNA or RNA encapsulated by protein

coat, occasionally containing lipids. In their infective form outside a cell, the particle is a

virion that may be infective. The present invention is also directed to the prevention of viral

infection. In a preferred embodiment, the chitosan-derivative compounds of the present invention are utilized as surface binding agents. Surface binding applications include, but are not limited to masks, gloves, clothing, textiles, wipes, etc. Surface applications also include, but are not limited to chitosan-derivative compounds as binding agents in solution. Table II, below, lists viruses that were studied, and the applications of the chitosan-derivative compounds against the viruses. As is understood by one of ordinary skill, Table II is exemplary of tested viruses and does not encompass the entire group of viral pathogens against whom the compounds of the present invention are effective.

[01321 Table II

Viruses Tested Description

influenza A (H1, N1) major pathogen of humans and a model for H5Nl (bird flu)

vaccinia virus model for small pox, monkey pox and Molluscom Contagium (a pathogen of

(Copenhagen strain) HIV patients)

herpes simplex 1 (sp7 pathogenic, neuroinvasive strain obtain from a fatal neonatal infection.

strain)

encephalomyocarditis picornavirus that is a model for polio, foot and mouth disease (a major threat

virus to cattle, sheep, horses) and hepatitis A

[0133] The present invention is directed to the following chitosan-derivative compounds:

(A) Chitosan-Arginine compounds;

(B) Related chitosan-L/D unnatural amino acid compounds;

(C) Chitosan- acid amine compounds;

(D) Chitosan-L/D natural amino acid derivative compounds;

(E) Co-derivatives of the chitosan-derivative compounds;

(F) Salts of the chitosan derivative compounds; and

(G) Chitosan-guanidine compounds.

[01341 In accordance with a preferred embodiment, selectivity of microbiocidal activity is

controlled by degree of chitosan-derivative functionalization and by use of particular

molecular weights. Targeted delivery of chitosan-arginine and the chitosan-derivative

compounds to particular locations in the body is controlled by manipulation of composition

and formulation of these chitosan-derivative compounds. It is within the scope of the present

invention to control solubility via selective natural and unnatural amino acid derivatization,

via acid-amine derivatization, and by mixtures of derivatives and antibacterial properties at

various pH.

[0135] (A) Chitosan-arginine compounds:

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. Chitosans with any degree of deacetylation greater than 50% are

used with arginine functionalization between 2% and 50%. The degree of functionalization

imparts a specific charge density to the chitosan-arginine compound. For a particular

molecular weight, the higher the functionalization with positive charge, the higher the charge

density on the polymer chain. However, it is important to note that the issue of increased

functionality is tempered by potential increases in toxicity with mammalian cells at very high

degrees of functionalization. Increased charge density produces higher solubility and higher

efficiency of microbiostatic or microbiocidal activity. Increased charge density also increases the interactions with mammalian cell membranes. In accordance with this present invention, it is important to balance functionalization with toxicity to provide optimal efficacy and minimal toxicity in all uses or utilities.

10136] Broad control over bacterial populations is achieved by utilizing a large range of

molecular weights of chitosan-arginine compounds. In accordance with the present

invention, the chitosan-arginine compounds have molecular weights (MW) between 25,000

and 1,000,000 Da. Lower MW's chitosan-arginine compounds are effective and soluble with

lower per cent functionalization than larger MW chitosan-arginine compounds. Depending

upon application, the chitosan-arginine compounds having greater MW distribution and

narrowly focused polydispersity are within the scope of the present invention.

OH OH OH

p HO NH2 NH N 0 3 (4)

where X is:

0 NH2

NH HN=( NH2 (5a)

NH2

NH HN=( NH 2 (5b)

[0137] In accordance with the present invention, chitosan-arginine compounds, where X is as

shown in (5a) as L-arginine and in (5b) as D-arginine, exhibits a random distribution of

arginine on the primary amines with a numerical ratio of functionalized to nonfunctionalized

amines as indicated in (4) as p and q. The guanidine group on the arginine can be protonated

as shown in (3). In this case, s = 1. The position of each of the m monomers with the arginine

functionalized primary amine is distributed along the backbone where m is 0.02 - 0.50,

related to the degree of functionalization of the primary amines. The subscripts p and q

represent the degree of deacetylation such that q is between 0.50 and 0.01 and the position of

each of the p or q monomers is distributed along the backbone randomly. As is understood by

one of ordinary skill in the art, the representation of monomers in this format indicates a

number distribution and not a physical placement as a block copolymer. In a preferred

embodiment q is less than 0.20. The sum p + q + m = 1. Note that not all of the monomers

on the chitosan backbone are deacetylated. Given a particular degree of deacetylation the

number of free amines is calculated. Upon reaction with arginine, the degree of

functionalization is determined relative to the number of free amines and is presented as a

percent, m/(1-q) • 100%. The upper limit of functionalization is determined by both sterics

and electrostatics. The tert-butyloxycarbonyl (boc)-arginine is similar in size to a glucose

monomer, and has some fairly extensive rotational degrees of freedom. However, the positive

charge with its extensive resonance over three atoms as shown in (3) provides an electrostatic repulsion that is difficult toovercome, even in very high salt concentrations. Consequently, an upper limit of approximately 0.50 fractional functionalization is reasonably achieved.

10138] Non- boc- protected arginine, polyarginine functionalization is as described in (4)

above where s, a polymerization factor between 1 and 10 as described by comb-like polymers

with up to 50% of the total MW fulfilled by arginine, and a random distribution of

polyarginine lengths and positions. One of ordinary skill in the art will recognize that the

functionalized chitosans will have an average degree of functionalization and polymerization.

The polymerization facter, s, thus need not be an integer as s is averaged over m reactive

sites.

[0139] A preferred embodiment of the present chitosan-arginine compound shown in (6),

below is the L-stereoisomer of arginine coupled to chitosan. Here, m is 0.02 - 0.50, related

to the degree of functionalization of the primary amines. The subscripts p and q represent the

degree of deacetylation such that q is between 0.50 and 0.01 and the position of each of the p

or q monomers is distributed along the backbone randomly. As is understood by one of

ordinary skill in the art, the representation of monomers in this format indicates a number

distribution and not a physical placement as a block copolymer. In a preferred embodiment q

is less than 0.20. The sum p + q + m = 1. Note that not all of the monomers on the chitosan

backbone are deacetylated. Given a particular degree of deacetylation the number of free

amines is calculated. Upon reaction with arginine, the degree of functionalization is

determined relative to the number of free amines and is presented as a percent, m/(1-q) •

100%. The upper limit of functionalization is determined by both sterics and electrostatics.

The tert-butyloxycarbonyl (boc)-arginine is similar in size to a glucose monomer, and has

some fairly extensive rotational degrees of freedom. However, the positive charge with its

extensive resonance over three atoms as shown in (3) provides an electrostatic repulsion that is difficult to overcome, even in very high salt concentrations. Consequently, an upper limit of approximately 0.50 fractional functionalization is reasonably achieved.

OH OH OH

NH 2 NH NH

NH 2

NH HN=( NH 2 (6)

[01401 In a preferred embodiment, soluble chitosan-arginine is used as an antibacterial in

burn and wound treatment. A solution of chitosan arginine (dissolved in any biocompatible

aqueous solution, salve or cream) is delivered to the burn or wound site. Chitosan-arginine is

used as a preventative treatment or a treatment to reduce infection. Many wounds become

chronically infected such as dermal ulcers including diabetic ulcers and decubitous ulcers

(bed sores) or appear to be caused by infection such as the gastric ulcer byH pylori. Other

ulcers of the gut and digestive system include peptic, gastric, stomach, aphtous, duodenal, or

esophageal ulcers.

[0141] Burn morphologies, particularly in severe cases of 3 rd degree bums, provide an

exceptional medium for bacteria to grow. Necrotic and dying tissue provides nooks and

crannies in which bacteria thrive and gauzes and bandages do not access. The soluble

chitosan-arginine provides an increased antibacterial efficacy

[0142] Examples 1 though 13, below show in-vitro activity of chitosan-arginine (L

stereochemistry) compounds with a preferred molecular weight distribution between 25kDa

and 350KDa. As shown below, these compounds are soluble at physiologic pH and have

broad antimicrobial activity against both gram-negative and gram-positive bacteria. Also as

shown below, the chitosan-arginine compounds are also active against both planktonic

bacteria and bacteria in biofilms. In addition to having bactericidal activity against bacteria

in preformed biofilms, the chitosan-arginine compounds also inhibits biofilm formation.

Furthermore, chitosan-arginine with broad MW distribution is used to control bacterial

growth in tissue culture media at a concentration that is non-toxic to mammalian cells. The

activity of chitosan-arginine is dependent upon the charge density and shows different

efficacy with different bacteria. The examples also show in-vitro activity of chitosan

arginine to bind virions and reduce infectivity.

Finally, the chitosan-arginine compounds show an increase in survivability by 100% over

standard burn care protocol:

[0143] Example 1-Broad bacteriocidal properties of chitosan arginine: Planktonic

Growth

The bacteriocidal activities of chitosan-arginine was compared for both gram-positive

and gram-negative bacteria. 6% functionalized chitosan-arginine that is highly positively

charged without the addition of an acid, and having broad molecular weight distribution

between 25kDa and 350KDa, was tested at physiological pH. Both pathogenic and non

pathogenic bacteria 'model' bacteria were tested either in either planktonic growth

conditions. Five gram-negative bacteria, Escherichia coli, Pseudomonas fluorescens,

Klebsiella pneumoniae, Shigellaflexneri and Salmonella enteritidis; and two gram-positive

bacteria, Bacillus subtilis, Staphylococcus epidermidis were tested. The non-pathogenic bacteria were chosen because they are models for their pathogenic counterparts: Escherichia coli K12 is a model for enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enterohemorrhagic 0157:H7 E. coli (EHEC), enteropathogenic E. coli (EPEC), and enteroaggregative E. coli (EAggEC). Pseudomonasfluorescensis a model for P. aeruginosa and Burkholderia. Bacillus subtilis is a model forB. anthracisand B. cereus. Staphylococcus epidermidis is a model for S. aureus and S. saprophiicus. The effect of chitosan-arginine on the bacterial growth in planktonic cultures was determined by measuring the optical densities

(600 rn) of the liquid growth medium at various times post inoculation. All were grown

overnight in trypticase soy broth (TSB). The original cell density is uniformly ~ 109 cells at

which point the media is replaced by media containing 0.1% chitosan-arginine 7%

functionalized. As seen in figures 1(a) through 1(h) , 7% functionalized chitosan-arginine

was very effective at bacteriocide of B. subtilis, S. epidermidis, and P. fluorescens at 24 and

48 hours at physiological pH and displayed a weaker inhibitory activity against E. coli. The

two bars in the data indicate the initial OD at Omin that contains both live and dead cells and

the OD at 30 min which indicates the remaining live bacteria. This data demonstrates a

dramatic inhibition of the planktonic proliferation of B. subtilis, S. epidermidis, and P.

fluorescens, with viability decreasing by >99% at the concentration « 0.1% . Samples for

viable cell counts were taken from the solutions above. 100pL of each solution were plated

on agar and plates were incubated for -26hrs at 37°C. Live cells per mL of original solution

were calculated, giving rise to the summary corroborating data in figure 1(i). Thus, with the

exception of E. coli which is inhibited -80%, chitosan-arginine effectively kills >99% of

these bacteria at very low doses and possesses a broad and varied spectrum of antibacterial

properties.

[01441 Example 2-Minimum Inhibitory Dose of Pathogen Models

The effect of chitosan-arginine on the bacterial growth in planktonic cultures of the gram

negative pathogens Klebsiella pneumoniae, Shigellaflexneri and Salmonella enteritidis was

determined by measuring the optical densities (600 nm) of the liquid growth medium at

various times post inoculation. The bacteria Klebsiella pneumoniae, Shigella flexneri and

Salmonella enteritidis were grown overnight in Nutrient Broth, all at 37°C with shaking. The

original cell density was uniformly - 109 cells, and the 0 dose of chitosan-arginine was

performed using nanopure water as the control. The effect on viability as a function of dose

of chitosan arginine was determined via standard plate counts. 7% functionalized chitosan

arginine at physiological pH dramatically inhibited the planktonic proliferation of K

pneumoniae, S. flexneri, and S. enteritidisin 24 hrs as demonstrated in figures 2(a), 2(b) and

2(c) respectively, with viability decreasing by >99% at the concentration between 10 and

500ppm. The minimum inhibitory concentrations (MICs) were observed for K pneumoniae,

S. flexneri, and S. enteritidis to be ~ 100 ppm, ~ 10 ppm, and - 20 ppm respectively. Based

on these findings, the effectiveness of chitosan-arginine against other bacterial types can be

determined based upon optimal minimal inhibitory concentration ( MIC) of chitosan-arginine

for each bacteria.

[01451 Example 3-Effect of Charge Density on Bacteriocide

The mechanism of antibacterial action of chitosan-arginine is dependant on the addition of

positive charges and increased solubility. As demonstrated in figure 3, the amount of positive

charge on the chitosan-argine affects its bacteriocidal dose. The effect of two derivatives of

chitosan-arginine, functionalized at 7% and 30% with a broad MW distribution between

kDa and 300kDa were demonstrated for Stapphylococcus pyogenes (Staph A) in cell

culture media for two different innoculation doses of Staph A. Staph A was inoculated into

cell culture media, RPMI, for an overnight growth at 37°C with shaking, where the growth saturates as the nutrients get depleted, resulting in a consistent density that is about 109(

OD 6oo « 0.7) cells per ml for all strains tested. This fresh overnight growth was diluted to

1:100 or 1:2000 into RPMI and initial optical density (OD) measurements at 600 nm were

taken to establish the baseline OD for a given concentration of cells. To individual wells,

doses of chitosan-arginine, as described above, were added at 100, 50, 25, 12.5 and 6.25 ppm

for the 7% chitosan-arginine and 50, 25, 12.5, 6.25 and 3.125 ppm for the 30% chitosan

arginine. The surviving bacteria were grown for the subsequent 24 hours in the presence of

the chitosan-arginine at 37°C in the presence of CO2 with shaking. The final ODs

(corroborated by plate counts) were plotted, as shown in figure 3. As demonstrated in figures

3(a) and 3(c) the 7% functionalized chitosan-arginine inhibited the 1:100 fully at 100ppm

and the 1:2000 dilution fully at 25ppm. At concentrations lower than these optimal relative

amounts to cell density, the 7% chitosan-arginine was not 100% efficient. However, as

demonstrated in figures 3(b) and 3(d), the 30% functionalized chitosan-arginine was

effective at inhibiting bacterial growth with nearly 100% efficacy for all doses examined for

both Strep A concentrations. The efficiency of bacteriocide was controlled by the charge

density as determined by functionaizaion.

[0146] Example 4- Biofilm Inhibition

The efficacy of chitosan-arginine on biofilm formation was demonstrated using the

following assay: bacteria (S. epidermdis) were inoculated into LB and grown overnight to

saturation; this resulted in a final density of about 109 ( OD 6 0 0 « 0.7) cells per ml for all

strains tested. This fresh overnight growth was then diluted 1:1000 into LB to the final

bacterial concentration of 109 ( OD6 0 0 « 0.1). Equal aliquots of thus diluted bacteria were

placed either into 30 mm tissue culture plates and the functionalized chitosan added to the

desired final concentration ( 0.1% or 1000ppm in this figure). The plates were incubated for

24 hours at 37 deg. C without shaking. Thereafter, the media with the non-adherent,

planktonic bacteria, was poured off and the biofilm which adheres to the bottom of the dish,

stained with 1% crystal violet and photographed. The extent of biofilm formation was

quantified by staining with 1% crystal violet. As Seen in figure 4, the film grew wells in the

absence of chitosan arginine and chitosan-arginine significantly inhibited the formation of

biofilm.

[01471 Example 5-Bacteriocidal Activity in Biofilms

The efficacy of chitosan-arginine on controling bacteria in preformed biofilms was

demonstrated using liquid growth media inoculated with S. epidermitis in the absence of

chitosan-arginine. The biofilm was allowed to form. The biofilms were grown as described

above, in LB and in the absence of any AC, using either naturally occurring bacteria in

produced water or a selection of biofilm-forming pathogens. After the 24 hour growth period,

the planktonic bacteria were removed as above and the biofilms exposed to 5000 ppm of 30%

arginine-chitosan for one hour. The chitosan-arginine was then washed off, the biofilm

scraped into phosphate buffered saline (PBS) and resident bacteria re-suspended using

Dounce homogenizer. The suspended bacteria were serially diluted in PBS and plated onto

LB-agar plates which then were incubated at 37°C for 48 hours. The resulting colonies were

counted and compared to the control with no exposure to chitosan-arginine. The number of

viable bacteria in the treated biofilm were back-calculated and are shown in figure 5. As

shown in figure 5(a), chitosan-arginine significantly killed over 80% in the resident

preformed natural water-based bacterial film. Figure 5(b) demonstrates that chitosan

arginine also kills the resident bacteria ofS epidermidis. The dose response of preformed

biofilms of S. aureus is shown in figure 5(c). At doses as low at100ppm for 1 hr, over 60%

of the bacteria in the biofilm are killed with increasing activity to >95% at 500 ppm. Thus chitosan-arginine exposure to preformed biofilms for periods as short as 1 hour had bacteriocidal activity with increasing activity at increasing doses nearing 100% at 500ppm for

S. aureus.

[0148] Example 6-Bacteriocide of a broad spectrum of bacteria

The bacteriocidal activity of 7% functionalized and 30% functionalized chitosan-arginine

with a MW distribution between 25k Da and 300kDa was tested for a broad range of gram

positive and gram negative bacteria inclu ding A. baumannii, E. coli, P. fluorescens, S.

pyogenes (Staph A), B. subtilis, S. epidermidis, and S. aureus. The different efficacies of the

chitosan-arginine compounds were demonstrated using a fixed concentration of the 7% and

% functionalized chitosan-arginine for a final concentration of 100 ppm, and all bacteria

were tested with a Nanopore water control. All bacteria, except A. baumannii, were

inoculated into Luria Broth (LB) and grown overnight to saturation. . baumanniwas grown

in Nutrient Broth; the final density is about 109 ( OD 600 « 0.7) cells per ml for all strains

tested. The fresh overnight growth was then diluted 1:50 or 1:1000 into RPMI (without

serum) to the final bacterial concentration of 2x10 7 or 106 , respectively ( OD 6 0 0 « 0.1).

Equal aliquots of the diluted bacteria were placed into wells of a 96 well tissue culture plate

and the functionalized chitosan added (100 ppm). The cultures were then incubated at 37°C

with continuous shaking in the presence of 5% atmospheric CO 2 . Bacterial growth was

monitored by periodic recording of the OD6 0 0 for the subsequent 24 and 48 hours. The results

were plotted in figure 6(a) and 6(b) (1:50, cell density 2x10 7 cells/ml) for 24 and 48 hours

respectively and plotted in figure 6(c) and 6(d) (1:1000, cell density 1x10 6 cells/nil). The data clearly shows that the majority of the gram positive strains were profoundly inhibited in growth over the 24 hour period by the presence of functionalized chitosan-arginine, with the exception of S. aureus. The antibacterial resistant and gram negative A. baumannii was dramatically inhibited by both the 7% and 30% chitosan arginine at both dilutions. It is further clear that inhibition occurred in a concentration-dependent manner, whereby the higher concentrations of chitosan-arginine inhibit bacterial growth more than the lower concentrations in most cases. Finally, it is clear that the 30% chitosan arginine has a greater growth-inhibitory effect on these bacteria than the 7% chitosan arginine, thus illustrating the dependency of the growth inhibition on the charge density on the functionalized chitosan.

[0149] Example 7-Bacteriostasis in Cell Culture Media

Two gram positive and one gram negative bacteria (Staphylococcus epidermidis 35984,

Staphylococcus aureus 29213 and Pseudomonas fluorescens 700830, respectively) were

tested for control of growth in cell culture media, RPMI. Bacteria were inoculated into RPMI

and grown overnight to saturation resulting in a final density of about 109 ( OD 6 0 0 « 0.7)

cells per mL for all strains tested. This fresh overnight growth was then diluted 1:100(102),

1:1000 (10-3), 1:10000 (104), 1:100000 (10-1), 1:1000000 (10-6), into RPMI to the final

bacterial concentration of 107, 106, 10s, 104, or 103 cells/mL, respectively. Equal aliquots of

thus diluted bacteria were placed into wells of a 96 well tissue culture plate and the chitosan

argine 30% functionalized was added to the desired final concentration (0-512 ppm) with

Nanopure water as the 0 dose control. The cultures were then incubated at 37°C with

continuous shaking. Bacterial growth was monitored by either periodic recording the OD60 0

for the subsequent 24 hours. The results shown in figure 7(a) demonstrates that at all but the

highest concentrations of S. epidermidis, chitosan-arginine inhibits all growth and kills the

bacteria relative to the control group. The dose response of S. aureus is demonstrated in figure 7(b) where at all bacterial concentrations except 107 and 106 cells/mL show an increasing effectiveness in bacteriostasis as the does increases from 8 ppm to 256 ppm where all cell growth is inhibited, even for the 106 cells/mL. The effect of chitosan-arginine on P.

fluorescens as demonstrated in figure 7(c) to be not as dramatic for this gram negative

bacteria as for the previous two gram positive bacteria, but the dose response is much more

uniform over all concentrations and shows significant inhibition of growth at all

concentrations.

[0150] Example 8--Cytotoxicity in Cell Culture Media

Chitosan-arginine was used to limit bacterial contamination of mammalian cell

culture. Toxicity tests of chitosan-arginine with both 7% and 30% functionalization

demonstrated that these moieties were non-toxic to human cells in culture. MTT assays,

which are a standard assay to determine cytotoxicity, were used on HeLa cells. HeLa ( human

epithelial cells) cells were plated into 96 well tissue culture plates at a final density of 5x10 4

cells per ml of RPMI with 10% serum and allowed to adhere to the bottom of the culture

dish, overnight. The following day, the media was replaced with media containing the

indicated amounts of chitosan arginine (final concentration 0-500ppm). One micromolar

phenylarsine oxide (PAO) and taxol were used as positive controls for cytotoxicity. Cells

were incubated for 48 hours in 5% CO2 at 37 °C. At the end of the 48 hour period, MTT

reagent was added to a final concentration of 0.5 mM and cells were allowed to incubate for

additional 2 hours. During this period, mitochondria in the healthy cells convert the yellow

MTT into dark blue formazan crystals while the dead cells are unable to do so. Media was

removed and the cells & crystals dissolved with DMSO. The resulting deep blue color was

measured at 490nm using a plate reader. As shown in figure 8, chitosan-arginine exhibited

very low toxic activity across a broad range of concentrations. The efficacy of controlling bacterial growth in cell culture media was then demonstrated using chitosan-arginine at concentrations that were sub-lethal to mammalian cells in culture. This effect is demonstrated in figure 8(a) where for the most potent chitosan-arginine (30%), 90% HeLa viability remains up to 750 ppm, well above all targeted antibacterial concentrations.

Furthermore, the 7% chitosan-arginine exhibited no toxicity at any concentration up to 1500

ppm. In both cases, Imicromolar concentration of both PAO and taxol killed -100% of all

HeLa cells.

[0151] Example 9 -Ability of chitosan-arginine surfaces to bind bacteria (Pseudomonas

fluorescens)

In order to demonstrate the ability of chitosan-arginine treated surfaces to bind bacteria

tightly, chitosan-arginine was chemically cross-linked to glass, a surface that lends itself to

electron microscopy but has minimal bacterial binding activity. Chitosan-arginine was

covalently bonded to glass via the following protocol: glass slides (12mm glass cover slips)

were cleaned in ethanol for 1 hour. The glass was then air dried and flame sterilized. The

glass was then "amonized" by treatment with 1% aminopropyltriethoxysilane (APTES) in

acetone. After 15 minutes the glass was washed with 100% ethanol and air dried. The glass

was then coated with chitosan-arginine in phosphate buffered saline (1mg/ml) in the presence

of 1% glutaraldehyde. After 30 minutes, unbound material was washed away with water. The

chitosan-arginine (7% functionalized) on glass plates were exposed to a solution of 109

Pseudomonas fluorescens, rinsed with water, then treated for and subjected to scanning

electron microscopy. As shown in figure 9(a), the APES modified glass is smooth whereas it

is slightly rougher with rounded aggregates upon covalently bindind chitosan-arginine as

shown in figure 9(b). P. fluorescens attaches only very weakly as shown in figure 9(c).

However, the binding of the rodlike and fairly uniform sized bacteria onto the chitosan arginine modified surface is shown in figure 9(d). The density of bacteria relative to the control demonstrates the efficiency of chitosan-arginine binding of a gram-negative bacteria to a surface.

[0152] Example 10-Loss of viral infectivity

In order to demonstrate chitosan-arginine's broad antiviral activity, a wide variety of

viruses were tested in-vitro. The following viruses were utilized in the antiviral studies: 1)

encephalomyocarditis virus (EMCV), B strain, a member of the picornavirus family which

have a positive sense RNA genome, lack enveloped virions and replicate in the cytoplasm of

the host. EMCV can infect a wide variety of cell types and is a model other picornaviral

infections such as rhinovirus, hepatitis A, Coxsackie virus and poliovirus. 2) HINIinfluenza

virus a member of the orthomyxovirus family which have a negative RNA genome, have

enveloped virions and replicate in the nucleus and cytoplasm of the host cell. This is a cell

culture adapted strain of HIN that was isolated from a clinical case of human influenza. 3)

Vaccinia virus a member of the pox family, which has a dsDNA genome, enveloped virions

and replicate in the cytoplasm of host cells. Vaccinia virus (VV) infects a wide variety of

cells leading to a systemic infection which can be lethal. VV is the best characterized model

for both small pox and monkey pox infection of humans. 4) The SP7 strain of Herpes

simplex 1 (HSV1) is a member of herpes family; which has double-stranded DNA genomes,

enveloped virions and replicate in both the nucleus and cytoplasm of the host cell. The SP7

strain is a neuron-virulent strain of HSV1 that was isolated from a fatal neonatal infection. In

this study, 104 infectious units (virions) were exposed to 30% functionalized chitosan

arginine for 15 minutes and then assayed for a loss in infectivity via standard plaque assay.

The following viruses were tested: influenza A (HI, NI), vaccinia virus (Copenhagen strain),

herpes simplex 1 (SP7 strain) and encephlomyocardidits virus (B strain). As shown in figure

, a 2-3 fold reduction in virus titer was observed in all four viruses tested. Thus soluble

chitosan-arginine inactivates both enveloped and non-enveloped viruses.

[0153] Example 11-Inhibition of influenza infection of MDCK cells

The ability of chitosan-arginine to inhibit influenza virus and thereby protect mammalian

cells in culture is shown in figure 11. In this experiment, a known amount of influenza

virions (H1, N) was either mock treated with a control or exposed to 7% functionalized

chitosan-arginine for 5 min. prior the exposing MDCK cells to the virus. The inhibitory

affect of functionalized chitosan on influenza was visualized by light microscopy. When this

stain of influenza virus infects MDCK cells, a characteristic cytopathic effect that includes a

rounding of the infected cells, which eventually separate from and then float off the growth

substrate. As shown in figure 11(a), cells that were pretreated with underivatized ultrapure

chitosan had rounded indicating infection. Figure 11(b) shows that MDCK cells pretreated

for 5 minutes with chitosan-arginine had significantly less rounded cells and the attached

monolayer, demonstrating considerably less early signs of rounding. Un-pretreated MDCK

cells are infected and shown in Figure 11(c) as a control for comparison.

[0154] Example 12-Binding of influenza

In order to demonstrate virus binding to chitosan-arginine treated surfaces, chitosan

arginine was chemically cross-linked to glass, a surface that lends itself to electron

microscopy but has no appreciable viral binding activity. Chitosan-arginine was covalently

bonded to glass via the following protocol: glass slides (12mm glass cover slips) were

cleaned in ethanol for 1 hour. The glass was then air dried and flame sterilized. The glass

was then agonizedd" by treatment with 1% aminopropyltriethoxysilane (APTES) in acetone.

After 15 minutes the glass was washed with 100% ethanol and air dried. The glass was then coated with chitosan-arginine in phosphate buffered saline (Img/ml) in the presence of 1% glutaraldehyde. After 30 minutes, unbound material was washed away with water. The chitosan-arginine (7% functionalized) on glass plates were either exposed to 104 influenza virions or mock treated and then subjected to scanning electron microscopy. The 7% chitosan-arginine had broad molecular weight distribution between 25kDa and 350 kDa. All substrates were exposed to 104 influenza HINI virions for 15 min., washed with isotonic buffer, and then subjected to electron microscopic analysis to detect virion binding. Mock binding followed the same procedure in the absence of virions. As Seen in figure 12(a), the mock modified glass with only APTES did not bind influenza virons. The morphology changes associated with chitosan-arginine binding on the surface were generally minimal with an occasional aggregate as shown in figure 12(b). The binding of the spherical and fairly uniform sized virions onto the chitosan-arginine modified is shown in figure 12(c). The density of virions relative to the controls demonstrates the efficiency of chitosan-arginine binding of viral particles.

[01551 Example 13-Treatment of Infected 3 rd Degree Burns in Mice

Mice were given a 1cm 2 3 d degree dorsal burn that was immediately contaminated with

108 Proteus mirabilis. Mice were treated daily with lOOpL of 1% solution of 7%

functionalized chitosan-arginine dissolved in water, treated with a uniform coating of silver

sulfadiazine cream to coat the surface of the wound or treated with nothing. Survivability is

plotted as a function of hours post burn and infection. Note that all mice without treatment

succumbed and died. With the standard of care treatment of silver sulfadiazine cream, 40% of

the mice survive after 400 hours whereas with the chitosan arginine, 100% improvement is

observed and 80% of the mice survive.

[0156] (B) Chitosan-unnatural amino acid compounds:

Additional control of the selectivity of chitosan-derivative compounds is achieved by

functionalization of the chitosan backbone, (7), below, where X' compounds are unnatural

amino acid shown in structures (8a) through (Sc) and their guanidinylated counterparts as

shown in (9a) through (9c), where r is an integer from 1- 8. The amine in (8a) through (8c) is

protonated as in (2). The guanidine in (9a) through (9c) is protonated as in (3). The position

of X' is independent of distribution along the backbone and m is 0.02 - 0.50, related to the

degree of functionalization of the primary amines. The subscripts p and q represent the

degree of deacetylation such that q is between 0.50 and 0.01. In a preferred embodiment q is

less than 0.20. The sum p + q + m = 1 and s = 1. Note that not all of the monomers on the

chitosan backbone are deacetylated. Given a particular degree of deacetylation the number of

free amines is calculated. Upon reaction with the unnatural amino acid, the degree of

functionalization is determined relative to the number of free amines and is presented as a

percent, m/(1-q) • 100%. The unnatural amino acid structures as taught by the present

invention are distinguished from arginine by having a different carbon chain length. The

variation in r provides control of the location of the charge relative to the chitosan backbone.

The choice of an amine terminal group as shown here in (8a) through (8c) or a guanidine

terminal group as shown here in (9a) through (9c)provides control of charge distribution.

The unnatural amino acid structures are also distinguished from the arginine by having either

a single charge on a amine at the terminal end of an amino acid or as a broad charge

distribution on a resonant guanidinyl group as shown in (2) and (3), above.

OH OH OH

NH2 NH O NH

4 (7)

Where X' is

O , NH2 NH2 (Sa)

NH 2

NH 2 (8b)

NH 2 O) NH 2 (8c), or

NH 2 O) NH HN=( NH 2 (9a)

0 NH2

NH HN=< NH 2 (9b)

NH 2 O NH HN== NH 2 (9c)

[01571 For non-boc-boc-protected unnatural amino acids with terminal amines as shown in

(8a) through (8c), above and non-boc-protected unnatural amino acids with terminal

guanidines as shown in (9a) through (9c), above, polyamino acid functionalization is as

shown in (7) above; where s, a polymerization factor, is between 1 and 10, as described by

comb-like polymers with up to 50% of the total MW fulfilled by X'. One of ordinary skill in

the art will recognize that the functionalized chitosans will have an average degree of

functionalization and polymerization and can polymerize through the alpha amine and/or the

terminal amine. The polymerization facter, s, thus need not be an interger, as s is averaged

over m reactive sites.

[0158] Related chitosan-L/D unnatural amino acid compounds include but are not limited to

2,3-diaminopropionic acid; 2,4-diaminobutyric acid; 2,5-diaminopentanoic acid (ornithine)

and their guanidinylated derivatives.

[0159] (C) Chitosan-acid-amine compounds:

For the purposes of distinguishing the chitosan acid-amine derivative compounds from

the relevant chitosan-amino acid derivative compounds, acid-amine compounds include both

and acid and an amine. In order to functionalize chitosan with an acid-amine group, the a

amine of an amino acid requires protection in the synthesis and subsequent deprotection by

an acid. Higher MW chitosan derivatives are attained by eliminating the deprotection steps.

Furthermore, the a-amine does not contribute significant charge at pH 7 and supports a less reactive carboxyl group. However, the terminal amine is protected by boc before reaction. If the amine is guanidynlation, boc-protection is not necessary. Additional control of the selectivity of chitosan-derivative compounds is achieved by functionalization of the chitosan backbone by X" as shown in (10), below, where X" are acid-amines as shown in structures

(11) or their related guanidinylated counterpart shown in (12), where r represents integers

from 1- 6. The amine in (11) can be protonated as in (2). The guanidine in (12) can be

protonated as in (3). The position of X" is independent of distribution along the backbone

and m is 0.02 - 0.50, related to the degree of functionalization of the primary amines. The

subscripts p and q represent the degree of deacetylation such that q is between 0.50 and 0.01.

In a preferred embodiment q is less than 0.20. The sum p + q + m = 1. Upon reaction with

the acid-amine, the degree of functionalization is determined relative to the number of free

amines and is presented as a percent, m/(1-q) • 100%. The acid-amine structures, as taught

by the present invention, are distinguished from the arginine and natural and unnatural amino

acids by the omission of the a-amine associated with the acid of a biologically relevant

amino acid. This omission also reduces the charge at the most proximal position to the

chitosan backbone. The a-amine has a pKa of -6 so that the a-amine of functionalized

natural and unnatural amino acids are only fractionally charged at physiological pH. The

omission of the a-amine contributes little to the change in positive charge. The acid-amine

structures are also distinguished from the arginine by having a different carbon chain length.

The variation in r provides control of the location of the charge relative to the chitosan

backbone. The choice of an amine terminal group as shown in (11), below, or a guanidine

terminal group as shown in (12), below, provides control of charge distribution.

OH OH OH

H(O O 40 - 4 OHO x44Ho NH 2 NH NH X" O (10) where X" is:

H 2N (11)

O=(

HN )-NH H 2N (12)

Chitosan- acid amine derivative compounds, include but are not limited to, 2-amino-ethanoic

acid, 3-amino-propanoic acid, 4-amino-butanoic acid, 5-amino-pentanoic acid, 6-amino

hexanoic acid and their guanidinylated derivatives.

[01601 (D) Chitosan-natural amino acid compounds:

In accordance with the present invention, additional control of the selectivity of chitosan

derivative compounds is achieved by functionalization of the chitosan backbone by X'" as

shown in (13), below. In a preferred embodiment, X'" is the charged natural amino acid

lysine (pka -10). As shown in (14a), below X"' is L-lysine and as shown in (14b), below

X'" is D-lysine. Additionally X" are also the charged natural amino acids histidine (pka

-6), aspartic acid (asp), and glutamic acid (glu). Here, m is between 0.02 - 0.50 and is

related to the degree of functionalization relative to the number of free amines as m/(1-q)•

100%. The subscripts p and q represent the degree of deacetylation such that q is between

0.50 and 0.01. In a preferred embodiment q is less than 0.20. The sum p + q + m = 1 and s =

1. The upper limit of functionalization is determined by both sterics and electrostatics as

described above. The boc-protected amino acids are similar in size to a glucose monomer,

and have some fairly extensive rotational degrees of freedom. However, the electrostatic

repulsion is difficult to overcome, even in very high salt concentrations. Consequently, an

upper limit of approximately 0.50 fractional functionalization is achieved.

OH OH OH

-0L O 4 0(i&4(OHo O O NH 2 NH NH (13)

Where X"' is

0 NH 2

H 2N (14a)

0 NH 2

H2N

(14b)

[0161] For non-boc-protected amino acids, poly amino acid functionalization is as described

in (13), above, where s, a polymerization factor, is between 1 and 10 as described by comb

like polymers with up to 50% of the total MW fulfilled by X"'. One of ordinary skill in the

art will recognize that the functionalized chitosans-natural amino acid compounds will have

an average degree of functionalization and polymerization. The polymerization factor, s, thus

need not be an integer, as s is averaged over m reactive sites.

[01621 A preferred embodiment of the present invention, shown in (15) below, is the L

stereoisomer of lysine coupled to chitosan. Here, m is between 0.02 - 0.50 and is related to

the degree of functionalization relative to the number of free amines as m/(1-q) • 100%. The

subscripts p and q represent the degree of deacetylation such that q is between 0.50 and 0.01.

In a preferred embodiment q'is less than 0.20. The sum p + q + m = 1 and s = 1. The upper

limit of functionalization is determined by both sterics and electrostatics as described above.

The boc-protected amino acids are similar in size to a glucose monomer, and have some

fairly extensive rotational degrees of freedom. However, the electrostatic repulsion is difficult

to overcome, even in very high salt concentrations. Consequently, an upper limit of

approximately 0.50 fractional functionalization is achieved.

OH OH OH

NH 2 NNH

NH 2

H2 N (15)

[0163] (E) Co-derivatives of chitosan derivative compounds:

In accordance with the present invention, additional wound healing effects, cell

stimulation and molecular tracking are achieved by functionalized chitosan-co-derivative

compounds including, but not limited to, iron chelating molecules and antioxidants, as

shown in (16) through (19), below. In (16), below, X is (Sa), (5b) or (6), above. In (17),

below, X' is (Sa) through (9c), above. In (18), below, X" is (11) or (12), above. In (19),

below, X"' are the natural amino acids: lysine (pka -10), histidine (pka-6), aspartic acid

(asp), and glutamic acid (glu). In (16) through (19), m is related to the degree .of

functionalization of X, X', X", or X"' such that the degree of functionalization is reported

as a percent where m/(1-q) • 100% . In (16) through (19), Y includes, but are not limited to,

desferoxamine (desferrioxamine), alpha hydroxy acids, poly alpha hydroxy acids, retinol,

alpha lipoic acid, flavinoids, coenzmye Q, Q10,, fluorescein, Texas Red, rhodamine, Prodan,

polypeptides and chemical cellular growth factors. V also includes, but are not limited to,

lysine (lys), histidine (his), aspartic acid (asp), glutamic acid (glu), alanine (ala), aspargine

(asn), glutamine (gln), methionine (met), proline (pro), threonine (thr), serine (ser), tyrosine

(tyr), and cysteine (cys). Additionally, Y is a naturally hydrophobic or weakly hydrophobic

amino acids including, but not limited to, tryptophan(trp), phenylalanine (phe), valine (val),

leucine (leu) and isoleucine (ile) where X V Y and X"'# Y. Y is selected such that it is

insoluble until in the lowest physiologically applicable pH. As understood by one of ordinary

skill, Y is not limited to above compounds but includes others in this class of compounds. In

(16) through (19), n is related the degree offunctionalization of Y such that the degree of

functionalization is reported as a percent where n/(1-q) * 100%, where n is 0.001 to .10; p

and q represent the degree of deacetylation such that q is between 0.50 and 0.01. In a

preferred embodiment q is less than 0.20 and p + q + m + a=1.

OH OH OH OH

O O O0 NH 2 NH ,H NH

( (x). YO

or

OH OH OH OH

NH2 (x). Y' O<\ (7

0 or 0N 0~ OH OH OH OH

NH2 NH ,NH NH

Y 0 (18) or

OH OH OH OH

NH 2 NH 1NH NH (9

Co-derivatives of the above defined chitosan-derivative compounds include but are not

limited to desferoxamine (desferrioxamine), alpha hydroxy acids, poly alpha hydroxy acids,

retinol, alpha lipoic acid, flavinoids, coenzmye Q, Q10,fluorescein, Texas Red, rhodamine,

Prodan, cellular growth factors, iron chelating agents, antioxidants, indicating fluorescent

markers, cell growth factors and the natural amino acids listed above;

[01641 (F) Salts of chitosan derivative compounds and of co-derivatives of chitosan

derivative compounds:

In accordance with the present invention, salts of the chitosan-derivative compounds

taught in (A) through (D), above, and salts of the chitsoan-co-derivative as taught in (E),

above, are precipitated with an anionic counterion to form the compounds shown in (20)

through (27), below. Solid chitosan derivative compounds as shown in (4), (7), (10) and (13)

are precipitated with an anionic counterion as shown in (20) through (23), where the

counterion Z includes, but is not limited to chloride, hydroxide, phosphate, carbonate,

acetate, lactate, citrate, cinnamate, oxalate, glutamate in ratios equivalent to or greater than

the positive charge on the chitosan. For chitosan-derivative compounds having a degree of

polymerization, s =1, a=1. For derivatives where s >1, a is a scaling factor that depends on

the pH and the pKa of the functionalizing molecule and is equally balanced by the counterion

charge.

OH OH OH N am

OO -\ O Z: NH 2 NH NH -z-am (X) O - M(20)

or

OH OH OH M am

O O Zam NH 2 NH

- - (21) or

OH OH OH (+)am

O( Ok \ O ~ q[Z -)am NH 2 NH H

-- - (22)

O O Z~a

OH OH OH ()am

OH (X''"), O= NH 2 NH NH

(23)

[01651 Solid chitosan-derivative compounds as shown in (16) through (19), above, are

precipitated with an anionic counterion as shown in (24) through (27) where the counterion Z

includes, but is not limited to, chloride, hydroxide, phosphate, carbonate, acetate, lactate,

citrate, cinnamate, oxalate, glutamate in ratios equivalent to or greater than the positive

charge on the chitosan.

OH OH OH OH ~(am

O O O[Z ]am NH2 INH NH NH (X), Y - Y(24)

or

OH OH OH OH +)am OO OLO am a H2 [NH ( ) O4

(25)

or

OH OH OH OH +)am O-0 OO am am NH2 NH

(26)

or

OH OH OH OH +)am

O O O O [Z~]am NH NH NH NH

- N(27)

[0166] The chitosan-derivative compounds are synthesized with modifications to known

standardized aqueous phase peptide coupling schemes that are, in turn, known variations of

organic based synthetic schemes. As is understood by one of ordinary skill, the selection of

coupling techniques is dictated by the unique solubility properties of chitosan relative to

polypeptides and other acid-amines, and the requirements for biocompatible solvents. In

particular, chitosan is insoluble in organic solvents and most aqueous solutions. It is most soluble in acetic acid, which is incompatible with the amino acid peptide coupling schemes as its.carboxylate group would react. Chitosan is similarly mostly insoluble in a variety of non carboxylate acids, inorganic acids, unless at high acid concentrations. Chitosan is reacted in hydrochloric acid. The preferred protocol involves solubilizing the chitosan in HCI at a pH of

2-3 and adding sodium hydroxide (base) until the pH is 5-7. When the pH is increased

rapidly, the amines on the chitosan are fractionally deprotonated; however, the chitosan

remains solvated for a limited time as the kinetics of precipitation for this highly solvated

polyelectrolyte are very slow. Consequently, the deprotonated amines are available to react

with the electrophilic carboxyl group that has been activated by the coupling agents. The

methodology for forming the chitosan-arginine compounds and the chitosan-derivative

compounds and the reaction pathway for the compounds is discussed below.

[0167] The amino acids in the above-described compounds, natural and unnatural, are either

D or L. The naturally occurring amino acids have L stereochemistry. Naturally occurring

enzymes do not have activity relative to the D stereochemical amino acids. Consequently,

chitosan-derivative compounds where X or X"' is a D-amino acid will not be degraded by

naturally occurring enzymes. The resistance to degradation results in longer activity of the

chitosan-derivative compounds.

[01681 Salts of above defined chitosan derivative compounds include but are not limited to

chloride, hydroxide, phosphate, carbonate, acetate, lactate, citrate, cinnamate, oxalate,

glutamate

[0169] (G) Chitosan-guanidine and related chitosan-guanidine derivatives

The chitosan-derivative compounds of the present invention include chitosan-guanidine

and related chitosan-guanidine derivative compounds. Chitosan-guanidine is shown in (28),

below, where the guanidinyl group can be protonated as shown in (3). The position of each of the m monomers with the guanidine functionalized primary amine is distributed along the chitosan backbone where m is 0.02 - 0.75, related to the degree of functionalization of the primary amines. In (28), p and q represent the degree of deacetylation such that q is between

0.50 and 0.01 and the position of each of the p or q monomers is distributed along the

backbone. In a preferred embodiment q is less than 0.20. The sum p + q + m = 1. Note that

not all of the monomers on the chitosan backbone are deacetylated. Given a particular degree

of deacetylation the number of free amines is calculated. The degree offinctionalization is

determined relative to the number of free amines and is presented as a percent, m/(1-q) •

100%. The upper limit of functionalization is determined by both sterics and electrostatics as

discussed above. The quanidinyl group is relatively small. However, the distributed positive

charge with its extensive resonance over three atoms as shown in (3) provides an electrostatic

repulsion that is difficult to overcome, even in very high salt concentrations. Consequently,

an upper limit of approximately 0.75 fractional functionalization is achieved. One of ordinary

skill in the art will recognize that the functionalized chitosans will have an average degree of

functionalization.

OH OH OH

NH 2 NH NH

NH 2 (28)

[0170] In accordance with the present invention, chitosan is functionalized with a guanidinyl

group because of the stable positive charge residing on the terminal guanidinium moiety on

an arginine molecule. The pKa of this species is similar to that of arginine, but are

distinguished due to the need for significantly fewer chemical processing steps and the molecular weight of the material is better preserved due to the elimination of the high acid deprotection step, as discussed below. For this method of functionalization, the primary amine of chitosan is converted directly into a guanidinium ion as shown in (3). Placing this guanidinyl moiety in such close proximity to the chitosan backbone of the polymer, as shown in (28) above, mimics more closely the properties of chitosan. Particularly, at low pH where the amine is protonated, the charge is permanent and distributed for the guanidinyl moiety, rather than a point charge. The positive charge is also located very close to the chitosan backbone, and has significant antibacterial efficacy, similar to the chitosan-arginine.

[0171] Examples 14 and 15, below demonstrate in-vitro activity of chitosan-guanidine

compounds. These compounds have a preferred MW between 25kDa and 400kDa and have

broad antimicrobial activity against both gram-negative and gram-positive bacteria. Chitosan

guanidine also exhibits higher activity against gram negative bacteria than does the chitosan

arginine. This is likely due to the fact that chitosan-guanidine, as shown in (28), above, has

the positive guanidinium group positioned a shorter distance from the chitosan backbone and

possesses a higher molecular weight than chitosan-arginine. As is understood by one of

ordinary skill, the activity of chitosan-guanidine exhibits different efficacy with different

bacteria.

[0172] ExamDle 14-Bacteriocidal activity of chitosan-guanidine

Both gram positive (S. aureus, S. pyogenes (Strep A), S. epidermidis, B. subtilis ) and

gram negative (A. baumannii, P. fluorescens andE. coli) bacteria were exposed to chitosan

guanidine (<5% functionaized and broad MW distribution having a peak near 370kDa) in

planktonic growth.S. aureus, S. pyogenes (StrepA), B. subtilis, A. baumannii,S. epidermidis,

P. fluorescens and K coli were inoculated into LB (Luria Broth) (except A. baumannii

which were grown in Nutrient Broth) and grown overnight to saturation resulting in a final density of about 10 9 cells per ml ( OD6 0 0 « 0.7). All strains were then diluted 1:50, mixed with or without 150ul of 0.1% chitosan-guanidine to give a final concentration of 50 ppm and incubated at 37°C with continuous shaking for 24 hours. OD measurements at 600 nm were used to represent relative live bacterial concentrations. The two bars in the data indicate the initial OD at 0min that contains both live and dead cells and the OD at 30 minutes after settling which indicates the remaining live bacteria. The results of the bacteriocidal assay demonstrated in figures 18(a) through 18(g) show broad efficacy for chitosan-guanidine.

Chitosan-guanidine is most effective at these doses against Strep A and S. epidermidis as

demonstrated in figures 18(b) and 18(e). Chitosan-guanidine inhibits, but not as dramatically

A. baumanni, P. fluorescens, E coli and B. subtilis as shown in figures 18(d), 18(g), 18(a)

and 18(c). At these conditions, chitosan-guandine has no inhibitory effect on S. aureus.

[01731 Example 15-Antibacterial activity of chitosan-guanidine at short exposure

times

Both gram positive and gram negative bacteria were exposed to chitosan-guanidine (<5%

functionalized and broad MW distribution similar to the starting material with a peak

-370kDA). S. aureus, S. pyogenes (Strep A), A. baumannii, S. epidermidis, P. fluorescens

and E. coli were inoculated into LB (except A. baumannii which were grown in Nutrient

Broth) and grown overnight at 37°C to saturation resulting in a final density of about 109 cells

per mL ( OD 60 0 « 0.7) for all strains tested. Cells were diluted 1:100 and were mixed with

arginine-guanidine to the final concentration of 400ppm ( 0.4mg/ml), incubated at 37°C with

continuous shaking in the presence of chitosan-guanidine for one hour only, washed & serial

dilutions plated onto LB-agar plates. The number of cells surviving the treatment was back

calculated from the colonies that grew on the plates within 24 hours. The percentage of

inhibition was calculated and is shown in figure 19(a). The percentage inhibition was calculated as a percent where Inhibition % ={1 - (CFUwith chitosan guanidine)/(CFUwithoutchiotsan-guandine)]*100%. The highest inhibition rates are demonstrated for E. coli, P. fluorescens and Strep A, those which tended to be inhibited but more mildly by chitosan-arginine. Note that this level of bacteriocide occurred in only one hour treatment. The actual colony counts used to produce the summary chart for S. aureus, S.

pyogenes (StrepA), A. baumannii, S. epidermidis, P. fluorescens and E. coli are demonstrated

in figures 19(b) through 19(g), respectively against their controls without exposure to

chitosan guanidine.

[0174 In view of examples 14 and 15 above, and in accordance with the present invention,

chitosan-guanidine has a higher molecular weight, lower charge density and charge closer to

the backbone than chitosan-arginine. Its efficacy against gram negative bacteria is mildly

superior to that of chitosan-arginine and provides the complement in activity to the chitosan

arginine derivatives. As is understood by one of ordinary skill, control of microbial

populations in a variety of environments is achieved by selective application of the chitosan

derivative compounds of the present invention.

[0175] Additional wound healing effects, cell stimulation and fluorescent tracking are

achieved by the functionalized guanidine-chitosan compounds shown in (29) below, having a

co-derivative Y that is a iron chelating molecule or an antioxidant. In (29) below, the degree

of guanidine functionalization is reported as a percent where m/(1-q)• 100%.

OH OH OH OH

O&"X. O O4 O NH 2 NH NH NH NH 2 (29)

[0176] In (29), Y includes, but are not limited to, desferoxamine (desferrioxamine), alpha

hydroxy acids, poly alpha hydroxy acids, retinol, alpha lipoic acid, flavinoids, coenzmye Q, QlO,, fluorescein, Texas Red, rhodamine, Prodan, polypeptides and chemical cellular growth

factors. Y also includes, but are not limited to, lysine (lys), histidine (his), aspartic acid

(asp), glutamic acid (glu), alanine (ala), aspargine (asn), glutamine (gln), methionine (met),

proline (pro), threonine (thr), serine (ser), tyrosine (tyr), and cysteine (cys). Additionally, Y is

a naturally hydrophobic or weakly hydrophobic amino acids including, but not limited to,

tryptophan(trp), phenylalanine (phe), valine (val), leucine (leu) and isoleucine (ile). As

understood by one of ordinary skill, Y is not limited to above compounds but includes others

in this class of compounds.

[01771 In (29), n is related the degree of functionalization of Y such that the degree of

functionalization is reported as a percent where n/(1-q) * 100%. The subscript n is 0.001 to

.10. The subscripts p and q represent the degree of deacetylation such that q is between 0.50

and 0.01. In a preferred embodiment q is less than 0.20 and p + q + m + n =1.

[01781 Solid chitosan guanidine compounds and chitosan-guanidine co-derivatives shown in

(28) and (29), above, are precipitated with an anionic counterion as shown in (30) and (31),

below, where the counterion Z includes, but is not limited to, chloride, hydroxide, phosphate,

carbonate, acetate, lactate, citrate, cinnamate, oxalate, glutamate in ratios equivalent to or

greater than the positive charge on the chitosan.

OH OH OH m

NH 2 NH NH

NH 2

(30)

OH OH OH OH (9m

oH- o OO NH2 0 o O-O NH

[Z-1mNHO i NH NHNH2 -N 2 - (31)

[0179] The chitosan-derivative compounds as taught in (A) through (E) above, their

applications and formulations are summarized in Table III, below.

[01801 Table III

Compounds Applications Chitosan-derivative compounds Soluble Formulations Insoluble Formulations where X is:

Arginine Antibacterial (burns treatment) Antibacterial coatings

(including biofilm

prevention) Charged Amino Acids (where amino Antibacterial (wound treatment, Freeze dried sponge as acid is D orL) including Lysine prosthesis induced infections, absorbent for deep diabetic ulcers, bedsores, bacterial infection

lacerations, scrapes

Unnatural amino acids with amine Antibacterial for topical treatment Hydrogels (tissue or guanidine (where amino acid is of antibacterial resistant strains engineering, cell D or L) growth, stem cell

scaffold) Acid-amines (X = COOH -Cr-NH2, Antibacterial in cell culture media Antiviral binding (in particular for antibacterial surface (mask, clothing, COOH-Cr-(CN3H4))) where r resistant bacteria) gloves, textiles, water 6 filter, etc)

Antiviral (viral in water supplies,

Guanidine influenza)

Chitosan-derivative compounds Control of microflora populations

where X is above and Y is: in gut

desferoxamine, alpha hydroxy Antioxidant (wound healing,

acids, poly alpha hydroxy acids, cosmetic)

retinol, alpha lipoic acid, favinoids,

coenzyme Q, Q1O, formulation of

hydroxy acids, fluroescein and

other other antioxidants,

fluorescent markers and the natural

amino acids

Chitosan -derivative Reduction of bacteria associated

compounds where the with peritonitis

counterionsalt Z is:

chloride, hydroxide, phosphate, Control of enterophathic bacteria

carbonate, acetate, lactate, citrate, in the gut

cinnamate, oxalate, glutamate and

other non toxic or antioxidant

counterions.

[0181] A preferred method for synthesizing chitosan-arginine and related chitosan

derivatives, in accordance with the present invention, is shown and described below:

o~== H3,J NHOH 10__________ NH2 OH

NH2 OH

N Nd N

Y OH INC H 0~ 0

H2N NH NH

OH OH OH o OH OH OH

O0O O F3C OH _ O O NH2P NH NH NH N N N

NH -H

HN 0 HN NH HN=< HN=( NH 2 NH2 (32)

[0182] The method as shown in (32), above, utilizes N-(3-Dimethylaninopropyl)-N'

ethylcarbodiimide (EDC) and N-hydroxysuccinimide, (NHS) to activate the carboxylic acid

group of singly N-Boc-protected arginine forming the N-hydroxysuccinimide ester in aqueous

solution. Other protecting group/deprotection schemes include, but are not limited to, 9

fluorenylmethoxycarbonyl (FMOC)and other water soluble or solubilized benzylic esters.

Other activating/coupling schemes include, but are not limited to, carbodiimides other than

EDC, sulfo-NHS, 2-(lH-Benzotriazole-1-yl)-1,1,3,3-tetranethyluronium

hexafluorophosphate (HBTU), 1-Hydroxybenzotriazole (HoBt). A preferred embodiment of

the present invention includes the process for coupling arginine to chitosan including the

steps of dissolving chitosan in aqueous acetic acid solution (HCl, pH 5 5), and bringing the

chitosan solution to pH 5-7 by dropwise addition of base (NaOH, IM). When the pH is

increased rapidly, the amines on the chitosan are fractionally deprotonated; however, the

chitosan remains solvated for a limited time as the kinetics of precipitation for this highly

solvated polyelectrolyte are very slow. Consequently, the deprotonated amines are available

to react with the electrophilic carboxyl groups that have been activated by the coupling

agents.

[0183] In a separate vessel, the boc-arginine is preactivated by reaction with EDC and NHS

in specific ratios to the free amines on the chitosan that will determine the final arginine functionalization percent relative to the free amines on the chitosan. The formation of an amide bond between the free primary amine of chitosan and boc-arginine is dependent on the activation of the free acid, nucleophilic attack of the primary amine and the ability of the activating group to leave subsequent to the attack. Activation of the free acid is required for nucleophilic attack by the primary amine. This activation is accomplished through the use of

N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysuccinimide, forming the

N-hydroxysuccinimide ester in-situ followed by addition to the dissolved chitosan. It is

important to note that reaction efficiency drops without sufficient pre-activation of the acid.

The ratio of this activated product is directly related to functionalization of the chitosan.

Using two equivalents of activated acid provides approximately 18 %functionalization, while

3 equivalents (or more) provides approximately 30% functionalization. The preactivated

boc-arginine is added dropwise to the chitosan solution to assure uniform distribution of the

mixture into the fairly viscous chitosan solution. Note that other protecting

group/deprotection schemes and other coupling/activating agents are within the scope of the

present invention. The final mixture is allowed to react under ambient conditions for up to 20

hours, with vigorous shaking. Note that stirring does not suffice, as the stir bar only interacts

with a small volume of the solution due to the high viscosity.

[01841 The present invention is also directed to an alternative method for making chitosan

arginine and chitosan-derivatives, as shown in (33), below. This method involves a 1-pot

synthesis where higher ratios of reactants are needed to produce similar results. Chitosan

solution is prepared, as indicated above, to provide soluble chitosan. Arginine, NHS and EDC

are added stepwise, in ratios to amine that determine the final arginine functionalization

percent relative to the free amines on the chitosan. Since the desired NHS-ester formation is

the stable and reactive intermediate, arginine and NHS are added to the solution before the

EDC. The EDC preactivates the carboxyl group for faster reaction with the NHS. The EDC activates the carboxyl group, but may also be hydrolyzed by water, returning the reactants to their original form. Addition of the NHS first minimizes this unwanted side reaction.

/-CH 3 N=C==N

H OH OH OH Hac N OH + 0 O 0 Hac OY NH2 NH2 NHq

NH H 2N NH N-OH 0

OH OH OH OH OH OH

O O O Fac OHOOO NH2 NH NH NH2 NH NH

NH 2 -- NH HN- O HN- NH HN=< HN=< NH 2 NH 2

(33)

[0185] It is important to note that the chitosan-arginine coupling is optimized by the ex situ

activation of the boc-arginine. To minimize organic contaminants, this reaction is performed

in aqueous solution. However, this preactivation may also be conducted in organic solvents,

precipitated, cleaned and the activated boc-arginine added as a solid NHS-ester. Other

coupling agents include, but are not limited to, sulfo-NHS, carbodiimides and HoBt. The

organic solvent include, but are not limited to, methanol, dimethylsulfoxide or

dimethylformamide. The activated boc-arginine mixture is then added to a chitosan solution

to initiate the coupling reaction.

[0186] In all preparations, the boc-protected chitosan conjugate is dialyzed in standard

cellulose dialysis membranes with a 5000 molecular weight cut-off. The cut-off value can be

varied, depending on the chitosan molecular weight desired. For these embodiments, the MW

cut off is greater than 5000 Da. The use of counterions in dialysis are significant, particularly

in the final dialysis step described below. In order to prevent precipitation and to assure removal of undesirable by-products such as urea and NHS that result from the first activation step, the chitosan must remain soluble for all dialysis steps. The pH does not need to be adjusted by acid because the protected boc-arginine-chitosanis soluble at pH 7. For low functionalization (below 10%), the protected boc-arginine-chitosanmay be insoluble at pH 7 due to the presence of the boc group and the dialysis solution is lowered to pH 6 by HCl. The low functionalization is soluble when deprotected. The dialysis solution is changed 4 to 5 times using a volume 10-20x that of the reaction volume or until the solution ion concentration does not change. Alternatively, continuous flow ultra-filtration dialysis tubing is used. The boc-protected chitosan-arginine product is lyophilized or concentrated in a cross flow filtration system.

[0187] Deprotection of the boc group from the lyophilized or concentrated product is

achieved by addition of strong acid, such as trifluoroacetic acid (TFA). Other strong acids

such as hydrochloric acid, hydrofluoric acid, or sulfuric acids may be used. In a preferred

embodiment, the lyophilized intermediate is deprotected using a minimal volume of 95%

trifluoroacetic acid, such that all of the lyophilized material is completely dissolved. In

another embodiment, the concentrated intermediate is deprotected using IM HCl. The

deprotection is allowed to proceed for a variety of times between 2 and 24 hours depending

on the desired molecular weight distribution of the product. Because strong acids break the

glycosidic bond between monomeric units of the chitosan, the length of time exposure to

such acids determines the average molecular weight and molecular weight distribution. As

shown in figure 14, as determined by HPLC and 8-angle multi-angle light scattering with

index of refraction, the size distribution of the chitosan before reaction is large and peaks at a

molecular weight of 370,000 kDa. Given a 19 hr deprotection, the chitosan-arginine has a

peak MW of 50,200 kDa with a high end tail trailing to the 370,000 of the parent molecule.

In order to dry the chitosan-arginine, the TFA can be evaporated by blowing pure nitrogen or

purified air over the solution until dry. Alternatively, the solution is neutralized by addition

of NaOH until the solution is between a pH of 3 and 5. The dried residue is taken up in a HCl

solution of 3 to 5 pH.

[01881 Either the dried and acidified solution or the partially neutralized solution is dialyzed

using cellulose dialysis tubing as described above for two days against counterions , for

example, for the chloride salt against 0.01 NaCl or HCl to assure exchange of any remaining

trifluoroacetate. As is understood by one of ordinary skill in the art, any biocompatible salt is

acceptable for displacement of the negative counterions, trifluoroacetate. The dialyzed

chitosan-arginine is lyophilized to produce the solid product.

101891 In a preferred embodiment, the chitosan-arginine solid product is lyophilized as the

hydrochloride salt or phosphate buffer. Depending upon the intended use of the chitosan

arginine solid product, a variety of salts may be used. Common buffering salts include, but

are not limited to, phosphate or carbonate. If the intended use is directed to a wound or

environment where free radicals are present, the compounds containing Z antixodant salts as

described above are the preferred embodiment.

101901 For large scale production or mass manufacture of chitosan-arginine and other related

chitosan-derivative compounds requiring deprotection of an amine protecting group by strong

acid, the process is simplified by the use of ultra-filtration, dialysis filtration (UFDF) in-line

systems. The dialysis steps and/or lyophilization steps are replaced by UFDF. In a preferred

embodiment, the protected chitosan arginine or related chitosan derivative is placed in a

continuous flow UFDF against 0.01M NaCl for up to 100x the total volume of the solution.

The product is lypholilized and returned to the UFDF for final filtration, dialysis and

concentration.

[0191] In another preferred embodiment of the present invention, the protected chitosan

arginine or related chitosan derivative is placed in a continuous flow UFDF against 0.01M

NaCl for over 100x the total volume of the solution. Lyophilization is replaced by

concentration in the UFDF system, the solution is changed to 1-5M HC for deprotection. The

deprotected chitosan-derivatives are placed in gradient flow in the UFDF from 0.01M NaC

to water or Z counterions as described above for at least 100x the total volume of the

solution.

[01921 Chitosan is not soluble at physiological pH; however, chitosan-arginine is soluble at

pH 7. Consequently, the final dialysis step is conducted at a pH of 7. However, the pH of the

final solution will not significantly affect the charge on or solubility of the chitosan-arginine

to pH's above 11. A preferred embodiment of this invention is that the chitosan-arginine and

chitosan derivatives have a broad range of solubility and functionality across a wide range of

pH, thereby allowing it to have applications in a variety of environments. For example,

antibacterial activity is preserved across the entire gut and bowel that starts acidic, as in the

stomach, and becomes more basic, as in the duodenum.

[0193] The synthesis of all chitosan-derivatives follows the same methodology and coupling

scheme as described for chitosan-arginine. The general scheme for synthesis of molecules

with an active amine that requires boc protection to control the reaction is shown in (30). The

methodology of (34), below, applies to (5a), (5b), (Sa) through (Sc), (9a) through (9c), and

(11) as described above.

/-CH 3 OH OH OH H3 c N=C=N - - 0O O

BR-OH H' 1NH 2 NH2 NH H 36

O Boc-R N-OH 0

0

OH OH OH OH OH OH

& o' F3C OH O 0 NHz NH NH NH 2 NH NH Boc R (34)

[0194] The general scheme for synthesis of molecules with a no active amine requiring

chemical protection is shown in (35), below. The methodology of (35) applies to (12) as

described above.

N-CH3 OH OH OH N=C=N H 3 C. OHO HO HO O HaC O NH2 NH2 NH R-OH 2____ O___0_

AN-OH [ 0

OH OH OH

O O O NH2 NH' NH

(35)

[0195] Where R is X, X', X", or X"', as defined above. In accordance with the present

invention, selective bacteriocide and/or bacteriostasis is dependent upon molecular weight.

Molecular weight of the chitosan-derivative compounds is selected by choice of deprotection

time and the molecular weight of the starting chitosan material. In a preferred embodiment, 2

hours of deprotection, at room temperature with constant stirring, is sufficient to remove all

of the boc to levels below detectable by NMR. Longer deprotection times, ie exposure to

strong acid, result in the continual breakdown of the polymer to produce a distribution of molecular sizes, radius of hydration in solution, relative to the starting material with the longer time resulting in a broader distribution and a lower average molecular size. Figure 15 shows the shift in radius of hydration shifts from -28nm (chitosan) to-20nm (CPA12, chitosan-arginine 6%) after 8 hours of deprotection and to -10nm (CPA3, chitosan-arginine

6%) after 19 hrs deprotection. This is a preferred embodiment of the present invention where

a variety of molecular weights is desired for broad spectrum antibacterial activity.

[0196] Molecular weight of the chitosan-derivative compounds of the present invention is

selected by pre-fractionation of original chitosan source by dialysis membranes or purchase

of a particular molecular weight. In a preferred embodiment for high MW chitosan

derivatives, subsequent deprotection is minimized to 2 hours to remove all of the TFA, HC

or similar strong

acids. It is important to note that higher molecular weights are maintained at lower

deprotection times. Selected narrower ranges of MW are obtained by gel permeation

chromatography/size exclusion columns after long deprotection times.

[01971 Example 16-Synthesis of chitosan-arginine

The following is an example of the synthesis for a ratio of free amine : boc-arginine : NHS

EDC 1: 3 :3 :3. Chitosan (2.5g, 0.0127 moles) is dissolved in 190 mL 0.1 M HCl solution

by agitation for 30 minutes. In a separate vessel, boc-Arginine-OH (12.54 g, 0.038 moles, 3

equivalents) and N-hydroxysuccinimide (4.39 g, 0.038 equivalents) is dissolved in 200 mL

millipore water. After dissolution of arginine and N-hydroxysuccinimide, N-(3

Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC, 6.75 mL, 0.038 equivalents) added and

stirred for one minute. This activated solution is then added to the dissolved chitosan

solution and agitated overnight at room temperature. This solution is dialysed against 4 L each of solution, 5 steps in 2 days, after the functionalization reaction and after the TFA deprotection where the dialysis conditions are for each step 1) 0.01 M NaCl, 2) 0.01 M NaCl,

3) Millipore ultra pure 18.2Mg-cm water, 4) Millipore ultra pure 18.2MQ-cm water, 5)

Millipore ultra pure 18.2Mn-cm water. No pH adjustment is required on the dialyses.

[0198] In accordance with the present invention, the lyophilized product is broadly

characterized using a variety of techniques. It is important to determine any residual

contaminants, to verify molecular weight and to quantify degree of functionalization. FT-IR

spectroscopy is used to verify the absence of residual TFA. While not quantitative as to the

degree of functionalization, the IR data is helpful in identifying the presence of the shifted

amide bond to easily identify TFA contaminant. As shown in figure 16, an NMR spectrum

with water suppression at 70°C is used to assess the degree of deacetylation and any

remaining contaminant of the starting material chitosan. For quantification of arginine

functionalization, these high temperatures for NMR analysis are not necessary because the

chitosan-arginine methylene peaks are located at ~1.8 ppm and 1.3 ppm, regions on the

chitosan NMR spectrum demonstrated in figure 16 where there is very little proton structure.

The water peak is significantly downfield and not obstructive. An NMR spectrum of

chitosan-arginine taken at 70°C with water suppression is shown in figure 17(a). Note the

additional peaks at -2.21 and 1.7 ppm in addition to a number of downfield peaks. These

peaks represent the protons on the arginine side chain and move up and downfield depending

on the neighboring groups and temperature. Figure 17(b) shows the limited range of proton

peaks between 2.2 and 1.3ppm. Degree of functionalization is determined using the acetate

hydrogens between -2.0 and 1.9 as the comparison unit. The degree of acetylation is known,

and thus the total number of acetates is known. The calculation is conducted by integrating

the acetate hydrogens at 1.95 ppm to a normalized value of 1, and comparing each of the integrated arginine peaks (which are nearly identical) at a ratio of 2/3 to give the degree of functionalization relative to the degree of acetylation. For example 16, with 82% deacetylation, the degree of functionalization is 30+5%. The degree of functionalization relative to free amines is reported. A preferred method of arginine functionalization is by combustion analysis for CHN in which the arginine functionalization is directly determined relative to the starting chitosan to better than 1%. Dynamic light scattering is used to determine the size distribution of chitosan-arginine compounds. Multi-angle light scattering with a gradient gel HPLC column (Shodex) or standard column (TSK) is used to determine average molecular weight and molecular weight distributions. Limulus ameobate lysate assay (kinetic) is used to determine residual endotoxin levels.

[01991 The optimization of the synthesis, according to the present invention, allows for

increased control of the synthetic pathway, control of the degree of functionalization, control

of the molecular weight and provide the basis for addition of any other carbonic acids as is

understood by one of ordinary skill in the art.

102001 The following is a preferred method for synthesizing chitosan-guanidine, in

accordance with the present invention:

The chemical coupling of the chitosan amines to a guanidinyl group involves 1H

pyrazole-l-carboxamidine (HPC) and N,N-Diisopropylethylamine (DJEA). HPC and DIEA

are used to couple guanidine with the amine on the chitosan and to provide a basic

environment for more effective coupling, respectively. The pyrazole is a good, stable leaving

group after the nucleophilic amine reacts with the electrophilic carbon of the carboxamidine.

N. OH OH OH N OH OH OH 40 o 0o HN NH2 4

NH2 NH2 NH NH 2 NH NH NH 2

(36)

[0201] A preferred embodiment of the present invention includes a process for coupling a

guanidinyl group to chitosan, as shown in (36), above. The coupling process includes

dissolving chitosan in water for equal particulate dispersion and dilution in aqueous acidic

acid solution (pH : 5) to produce a soluble starting material. In a separate vessel, the HPC

and DIEA are vigorously mixed in water (by shaking). A two-phase solution occurs as the

DIEA is insoluble in water. The highly emulsified solution is added to the chitosan solution

which results in rapid preciptiation of the chitosan. The pH is rapidly brought down to a pH

of -5, or just to the point where the chitosan is again soluble. The pH is maintained at an

elevated level with concurrent solubility of the chitosan to assure sufficient availability of

active, de-protonated amines for reaction. The formation of a guanidinyl nitrogen requires a

nucleophilic amine. It is important to note that reaction efficiency is enhanced by addition of

simply equimolar DIEA to the chitosan amines. The final mixture is allowed to react under

ambient conditions for up to 20 hours, with vigorous shaking. Note that stirring does not

suffice, as the stir bar only interacts with a small volume of the solution due to the high

viscosity.

[0202] In accordance with the present invention, chitosan-guanidine can be produced by an

alternate process. Here, chitosan guanidine is prepared by reacting formamidine sulfonic acid

as shown in (38), below, that is prepared in one step from commercial materials, as shown in

(37). The conditions necessary for chitosan functionalization are somewhat different than the conditions under which the reagent was originally intended to operate (absolute MeOH, in this case).

0. NH O-OH NH H 2N SO 2 H 0 H2N SO 3 H OH (37)

[0203] Guanidinylation reactions are performed as in (38), below with excess sulfonic acid

with the unreacted reagent being removed via dialysis. The solution is dialysed against

0.01M NaCl 2x and then against ultrapure Millipore water adjusted to pH 7 3x with a

cellulose membrane with a 5kDa cutoff. The chitosan-guanidine product is then lyophilized.

OH OH OH NH OH OH OH

0 0 0 00H 2N 'SO 2 H 0 OO NH 2 NH7 NH NH 2 N' H HN=(N = NH 2 (38)

[0204] The methodology shown in (36) is the preferred embodiement for the process of

guanidinylation of amines of chitosan, of the unnatural amino acids and of the acid-amines

described above.

[02051 Example 17-Synthesis of chitosan-guanidine

Synthesis of 6% functionalized chitosan quanidine while preserving the initial average

molecular weight of chitosan.

Chitosan (1.0 g, 0.005 moles) is added to 15 mL of millipore water. To this solution, 1%

acetic acid is added until the chitosan is completely solublized (30 mL). In a separate vessel

1H-pyrazole-1-carboxamidine (3.66 g, 0.025 moles, 5 equivalents) and NN

Diisopropylethylamine (4.35 mL, 0.025 moles, 5 equivalents) are mixed in 5 mL millipore water. This biphasic solution is vigorously shaken to provide an emulsion. This emulsified solution is added to the chitosan solution, resulting in chitosan precipitation. The pH of this mixed solution is adjusted with 10% acetic acid until the chitosan dissolves (pH 4-5.5). This solution is shaken overnight at ambient temperature and dialysed as normal. The freeze-dried material is analyzed by combustion analysis for C, N and H content and the degree of functionalization determined to be less than 5%.

[0206] 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.

[0207] Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia.

[0208] According to a first aspect, the present invention provides A method for controlling microbial activity in an individual, comprising administering to the individual a therapeutically effective amount of a functionalized chitosan-derivative in a therapeutically effective excipient, wherein the functionalized chitosan-derivative is a compound of formula: OH OH OH 0 H0 O O OH NH2 ,NH NH (X), O=

wherein m is 0.02-0.50; q is 0.50-0.01; s is 1; p+q+m= 1; the percentage degree of functionalization is m/(-q) • 100%; and X is selected from the group consisting of:

0 O NH 2 NH 2

NH NH HN=( HN=< NH 2 and NH 2

[0209] According to a second aspect, the present invention provides a method for treating or preventing the growth of microbial populations in an individual comprising administering to the individual a therapeutically effective amount of a functionalized chitosan-derivative in a therapeutically effective excipient, wherein the functionalized chitosan-derivative is a compound of formula: OH OH OH 0 H0 O O O NH2 NH NH

wherein m is 0.02-0.50; q is 0.50-0.01; s is 1; p+q+m= 1; the percentage degree of functionalization is m/(1-q) • 100%; and X is selected from the group consisting of:

0 0 NH 2 NH 2

NH NH HN# HN=( NH 2 and NH 2

[0210] According to a third aspect, the present invention provides a method for preventing or inhibiting biofilm formation in an individual comprising administering to the individual a therapeutically effective amount of a functionalized chitosan-derivative in a therapeutically effective excipient, wherein the functionalized chitosan-derivative is a compound of formula: OH OH OH

NH H NH NH

wherein m is 0.02-0.50; q is 0.50-0.01; s is 1; p+q+m= 1; the percentage degree of functionalization is m/(1-q) • 100%; and X is selected from the group consisting of:

86a

0 0 NH 2 NH 2

NH NH HN=( HN# NH 2 and NH 2

[0211] According to a fourth aspect, the present invention provides the use of a therapeutically effective amount of a functionalized chitosan-derivative in a therapeutically effective excipient for the manufacture of a medicament for controlling microbial activity in an individual, wherein the functionalized chitosan-derivative is a compound of formula: OH OH OH ~- 0 0 0 0 O- O O q NH2 ', NH (),

wherein m is 0.02-0.50; q is 0.50-0.01; s is 1; p+q+m= 1; the percentage degree of functionalization is m/(1-q) • 100%; and X is selected from the group consisting of:

0 0 NH 2 NH 2

NH NH HN=( HN# NH 2 and NH 2

[0212] According to a fifth aspect, the present invention provides the use of a therapeutically effective amount of a functionalized chitosan-derivative in a therapeutically effective excipient for the manufacture of a medicament for treating or preventing the growth of microbial populations in an individual, wherein the functionalized chitosan-derivative is a compound of formula: OH OH OH

0 O H0 O H OH- 0 NH H NH NH

86b wherein m is 0.02-0.50; q is 0.50-0.01; s is 1; p+q+m= 1; the percentage degree of functionalization is m/(-q) • 100%; and X is selected from the group consisting of: o 0 NH 2 NH 2

NH NH HN# HN# NH 2 and NH 2

[0213] According to a sixth aspect, the present invention provides the use of a therapeutically effective amount of a functionalized chitosan-derivative in a therapeutically effective excipient for the manufacture of a medicament for preventing or inhibiting biofilm formation in an individual, wherein the functionalized chitosan-derivative is a compound of formula: OH OH OH

NH ,NH NH

wherein m is 0.02-0.50; q is 0.50-0.01; s is 1; p+q+m= 1; the percentage degree of functionalization is m/(1-q) • 100%; and X is selected from the group consisting of:

0 0 NH 2 NH 2

NH NH HN# HN# NH 2 and NH 2

86c

Claims (22)

1. CLAIMS 1. A method for controlling microbial activity in an individual, comprising administering to the individual a therapeutically effective amount of a functionalized chitosan-derivative in a therapeutically effective excipient, wherein the functionalized chitosan-derivative is a compound of formula: OH OH OH H0 L0 0 HO O HO NH2 NH' NH

   wherein m is 0.02-0.50; q is 0.50-0.01; s is 1; p+q+m= 1; the percentage degree of functionalization is m/(1-q) • 100%; and X is selected from the group consisting of:

   O O NH 2 0 NH 2

   NH NH HN# HN# NH 2 and NH 2
2. 2. The method according to claim 1, wherein the microbial activity is in biofilms.
3. 3. The method according to claim 1 or 2, wherein the compound has an average molecular weight between 25,000 and 350,000 Da.
4. 4. The method according to any one of claims 1-3, wherein the compound is soluble at pH between 6.8 and 7.4.
5. 5. The method according to any one of claims 1-4, wherein the degree of functionalization is between 6% and 30%.
6. 6. The method according to any one of claims 1-5, wherein q is less than 0.20.
7. 7. The method according to any one of claims 1-6, wherein the compound is substantially free of compounds having a molecular weight of less than 5,000 Da, and wherein the compound is substantially free of polyarginine polymers.
8. 8. A method for treating or preventing the growth of microbial populations in an individual comprising administering to the individual a therapeutically effective amount of a functionalized chitosan-derivative in a therapeutically effective excipient, wherein the functionalized chitosan-derivative is a compound of formula: OH OH OH H0 0 0 0 O O _OHq NH2 NH' NH (X) 0= wherein m is 0.02-0.50; q is 0.50-0.01; s is 1; p+q+m= 1; the percentage degree of functionalization is m/(1-q) • 100%; and X is selected from the group consisting of:

   O 0 NH 2 0 . NH 2

   NH NH HN=( HN=( NH 2 and NH 2
9. 9. The method according to claim 8, wherein the compound has an average molecular weight between 25,000 and 350,000 Da.
10. 10. The method according to claim 8 or 9, wherein the compound is soluble at pH between 6.8 and 7.4.
11. 11. The method according to any one of claims 8-10, wherein the degree of functionalization is between 6% and 30%.
12. 12. The method according to any one of claims 8-11, wherein q is less than 0.20.
13. 13. The method according to any one of claims 8-12, wherein the compound is substantially free of compounds having a molecular weight of less than 5,000 Da, and wherein the compound is substantially free of polyarginine polymers.
14. 14. A method for preventing or inhibiting biofilm formation in an individual comprising administering to the individual a therapeutically effective amount of a functionalized chitosan-derivative in a therapeutically effective excipient, wherein the functionalized chitosan-derivative is a compound of formula:

    OH OH OH 0 0 0-L0 0 OLOO NH2 NH NH

    wherein m is 0.02-0.50; q is 0.50-0.01; s is 1; p+q+m= 1; the percentage degree of functionalization is m/(1-q) • 100%; and X is selected from the group consisting of:

    O 0 NH 2 . NH 2

    NH NH HN# HN# NH 2 and NH 2
15. 15. The method according to claim 14, wherein the compound has an average molecular weight between 25,000 and 350,000 Da.
16. 16. The method according to claim 14 or 15, wherein the compound is soluble at pH between 6.8 and 7.4.
17. 17. The method according to any one of claims 14-16, wherein the degree of functionalization is between 6% and 30%.
18. 18. The method according to any one of claims 14-17, wherein q is less than 0.20.
19. 19. The method according to any one of claims 14-18, wherein the compound is substantially free of compounds having a molecular weight of less than 5,000 Da, and wherein the compound is substantially free of polyarginine polymers.
20. 20. The use of a therapeutically effective amount of a functionalized chitosan-derivative in a therapeutically effective excipient for the manufacture of a medicament for controlling microbial activity in an individual, wherein the functionalized chitosan-derivative is a compound of formula: OH OH OH H0 0 0 0 O O _OHq NH2 NH' NH wherein m is 0.02-0.50; q is 0.50-0.01; s is 1; p+q+m= 1; the percentage degree of functionalization is m/(1-q) • 100%; and X is selected from the group consisting of: o 0 NH 2 NH 2

    NH NH HN# HN=( NH 2 and NH 2
21. 21. The use of a therapeutically effective amount of a functionalized chitosan-derivative in a therapeutically effective excipient for the manufacture of a medicament for treating or preventing the growth of microbial populations in an individual, wherein the functionalized chitosan-derivative is a compound of formula: OH OH OH H0 L0 0 HO O HO NH2 NH' NH

    wherein m is 0.02-0.50; q is 0.50-0.01; s is 1; p+q+m= 1; the percentage degree of functionalization is m/(1-q) • 100%; and X is selected from the group consisting of:

    0 0 NH 2 NH 2

    NH NH HN# HN=( NH 2 and NH 2
22. 22. The use of a therapeutically effective amount of a functionalized chitosan-derivative in a therapeutically effective excipient for the manufacture of a medicament for preventing or inhibiting biofilm formation in an individual, wherein the functionalized chitosan derivative is a compound of formula:

    OH OH OH 0 0 0-L0 0 O O _OHq NH2 NH NH

    wherein m is 0.02-0.50; q is 0.50-0.01; s is 1; p+q+m= 1; the percentage degree of functionalization is m/(1-q) • 100%; and X is selected from the group consisting of:

    0 0 NH 2 NH 2

    NH NH HN# HN# NH 2 and NH 2

    Synedgen, Inc. Patent Attorneys for the Applicant/Nominated Person

    SPRUSON & FERGUSON