US 2017.0056513A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0056513 A1 Hedrick et al. (43) Pub. Date: Mar. 2, 2017

(54) THERAPEUTIC COMPOSITIONS Publication Classification COMPRISING N-ALKYL-HYDROXY POLYMERS (51) Int. Cl. A6II 47/48 (2006.01) (71) Applicant: INTERNATIONAL BUSINESS A6IR 48/00 (2006.01) MACHINES CORPORATION, A63L/73 (2006.01) Armonk, NY (US) (52) U.S. Cl. CPC ...... A61K 47/48192 (2013.01); A61 K3I/713 (72) Inventors: James Hedrick, San Jose, CA (US); (2013.01); A61K 48/00 (2013.01) Dylan BODAY, San Jose, CA (US); Jeannette GARCIA, San Jose, CA (US); Rudy WOJTECKI, San Jose, (57) ABSTRACT CA (US); Yi Yan YANG, The Nanos (SG); Chuan Yang. The Nanos (SG) The disclosure describes methods and therapeutic composi tions comprising polymers modified with N-alkyl-hydroxy (21) Appl. No.: 14/836,614 groups comprising one or more carbon atoms. The compo sitions are useful for gene delivery, and exhibit broad (22) Filed: Aug. 26, 2015 spectrum antiviral activity and low toxicity in vitro. Patent Application Publication Mar. 2, 2017 Sheet 1 of 2 US 2017/0056513 A1

10. 0.15 eq. Paraformaldehyde 2 9. 8

s 5 4. c 3. 2

s g & S As ss N. Concentration (ng) Figure 1A

1. 0.30 eg. Paraformaidehyde 9 - 8 f S 5 a 30 2 . xx 22---- 2

S. $, $ so tie is is S s & 9 y & Sy sys s & S Concentration (ng) Figure 1B Patent Application Publication Mar. 2, 2017. Sheet 2 of 2 US 2017/0056513 A1

0.75 eg. Paraformaldehyde

3 -

Concentration (ing) Figure 1C

1 O 3. 8

80 5 a - 3. C - is SiA, Concentration (gi. Figure 1D US 2017/0056513 A1 Mar. 2, 2017

THERAPEUTIC COMPOSITIONS SUMMARY COMPRISING N-ALKYL-HYDROXY POLYMERS 0005. A therapeutic composition is described having at least one polymer comprising amine end groups, secondary FIELD amine groups, tertiary amine groups, and alkyl-hydroxy groups, wherein at least 5% of the secondary and tertiary 0001. The disclosure relates to methods and therapeutic amine groups comprise N-alkyl-hydroxy groups, and compositions comprising polymers modified with N-alkyl wherein at least 5% of the amine end groups comprise hydroxy groups. The compositions are useful for gene N-alkyl-hydroxy groups. delivery, and exhibit broad-spectrum antiviral activity and 0006. A method for producing a therapeutic composition low toxicity in vitro. is also described that includes forming a reaction mixture comprising a solvent, a polymeric amine and a N-alkyl BACKGROUND hydroxy group precursor, and heating the mixture at a temperature from about 25°C. to about 150° C. to produce 0002 Nucleic acid-based therapies involving gene trans a polymer comprising secondary and tertiary amine groups, ference hold great promise in the treatment of human wherein at least 5% of the secondary and tertiary amine diseases. In principle, faulty and defective genes may be groups are N-alkyl-hydroxy groups. corrected and replaced by functional ones, but redundant gene expression may also be repressed to a normal level by BRIEF DESCRIPTION OF THE DRAWINGS the use of RNA interference. There are two major gene AND FIGURES delivery vectors, viral and non-viral. Viral vectors have Superior transduction capabilities, but the immunogenic and 0007 So that the manner in which the above recited oncogenic potentials of viral vectors have limited their features of the present disclosure can be understood in detail, clinical applications. To solve this problem, non-viral gene a more particular description of the disclosure, briefly sum delivery systems have been developed, and feature number marized above, may be had by reference to embodiments, of advantages including improved biosafety, lower produc some of which are illustrated in the appended drawings and tion costs, ease of transportation and storage, and reproduc figures. It is to be noted, however, that the appended ibility. Despite these advantages, non-viral systems have drawings and figures illustrate only typical embodiments of thus required specific molecular tailoring for specific inter the disclosure and are therefore not to be considered limiting action with certain nucleic acids and cell Surfaces. To of its scope, for the disclosure may admit to other equally improve on the state of the art, there is a need for new effective embodiments. non-viral gene delivery systems that display high therapeutic 0008 FIGS. 1A-1D are charts representing data collected activity, low toxicity and exhibit broad recognition for the according to some embodiments of the disclosure. myriad of nucleic acids and cell Surfaces found in nature. 0003. Many of the desirable characteristics that are DETAILED DESCRIPTION sought after in non-viral gene delivery systems have utility 0009. The present disclosure describes new therapeutic and application in the field of antiviral therapeutics. In recent compositions that are useful for gene delivery, prevention of years, viral infections have emerged as the preeminent viral infection, and are transparent to viral mutation, thus global public health problem because of an increasing mitigating resistance development. The mode of action of human population, aging, global warming, and medical the new therapeutic compositions comprising N-alkyl-hy treatments that Suppress the immune system, including droxy Substituted polymers is related to non-specific Supra irradiation therapy, anti-cancer chemotherapy and organ molecular interactions, such as hydrogen-bonding and elec transplantation. Effective treatment of viral infections is trostatic interactions, between the functional groups of the elusive because of the variance in virus structure (enveloped therapeutic composition polymer(s) and biological com and non-enveloped viruses) together with their ability to pounds such as nucleic acids, genes, proteins, RNA, DNA rapidly mutate and garner resistance. and virus/cell surfaces. The polymers of the therapeutic 0004 To solve the aforementioned problems found with composition may have N-alkyl-hydroxy groups, secondary the current materials and compositions in these fields, new and tertiary amine groups, and charged groups (cationic therapeutic compositions that display efficacy in both gene groups), Such as quaternary amine groups at biological pH. delivery and in antiviral applications have been developed, For the purposes of the disclosure, binding generally refers and are the subject of the present disclosure. The new to chemical interactions including but not restricted to: compositions involve N-alkyl-hydroxy modified polymers, chemical bonding, covalent bonding, hydrogen bonding, which exhibit: a) high gene transfection efficiency in vitro, polar attraction, and dissimilar charge attraction Such as b) display broad-spectrum antiviral activity, c) remain active positive and negative charge attraction. despite viral mutation(s), d) exhibit high virus selectivity, e) 0010. In some embodiments, an N-alkyl-hydroxy substi display low toxicity in vitro; and f), are biodegradable. tuted polymer of the therapeutic composition may form a US 2017/0056513 A1 Mar. 2, 2017 complex with nucleic acids such as therapeutic DNA and/or mers comprising amine groups may have high cationic RNA, and the complex may be further internalized into (positive) charge density at physiological pH, wherein about cells. Upon and after cell uptake, the N-alkyl-hydroxy 50% to about 90% of the amine groups have a positive groups and the secondary and tertiary amine groups of the charge due to the binding of a proton (H). Specifically, and nucleic acid/polymer complex may aid, deliver or otherwise in combination with the hydrogen bonding interactions, the enable the release of the nucleic acid from the complex. In positively charged amine groups may engage in electrostatic other embodiments, the therapeutic composition may form interactions with negatively charged nucleic acids, nucleic polymer-virus and polymer-cell assemblies based on the acid residues, and/or proteins over a broad pH range. The aforementioned interactions. The formation of said assem combination of hydrogen bonding and electrostatic interac blies is not affected, negatively impacted, nor dependent on tions may generally result in the strong bonding of thera viral mutation, thus preventing viral resistance to the peutic N-alkyl-hydroxy polymers to nucleic acids and virus/ therapy. For example, the polymer N-alkyl hydroxy groups cell Surfaces. of polymer-virus and polymer-cell assemblies may further 0014. In one embodiment, the therapeutic composition neutralize endosomal pH (absorb or bind H') thus creating an may include more than one N-alkyl-hydroxy substituted inhospitable environment for viral replication and further polymer. For example, the composition may be a mixture of viral infection. N-alkyl-hydroxy modified polymers with differing molar 0011. The aforementioned therapeutic activity may be amounts of N-alkyl-hydroxy substitutions on the polymer realized when at least 5% of the amine groups of the chain. In another embodiment, the therapeutic composition composition are substituted with N-alkyl hydroxy groups, may have at least one N-alkyl-hydroxy substituted polymer, which have at least one carbon atom. The polymers of the which may be linear or branched, or a mixture of N-alkyl therapeutic composition may further include a plurality of hydroxy substituted polymers that may be linear or polymer chain amine end groups (terminal groups) and/or branched. N-alkyl-hydroxy substitution may be found at pendantamine end groups, wherein at least 5% of the groups amine groups integrated in the polymer chain backbone or may be substituted with at least one N-alkyl hydroxy group repeating units, amine groups located at polymer chain ends, comprising at least one carbon atom. The 5% substitution or at sites where the amine groups may be pendant end produces a therapeutic composition that generally comprises groups or are a part of a pendant group or a branch. For the a plurality of secondary and tertiary amine groups with purposes of this disclosure amine groups not integrated in N-alkyl-hydroxy group Substitution, and secondary and ter the polymer backbone may be thought of as amine end tiary amine groups that may be unsubstituted. In some groups. Polymers of the composition may be at least 500 embodiments, all of the amine groups of the polymeric grams/mole in molecular weight (number average) as mea composition may have N-alkyl-hydroxy groups. Sured by techniques such as is gel permeation chromatog 0012. In some embodiments of the disclosure, the raphy (GPC), also known as size exclusion chromatography N-alkyl-hydroxy modification of primary and/or secondary (SEC). Examples of polymers of the therapeutic composi amine groups may decrease or eliminate cell toxicity, and tion amenable to N-alkyl-hydroxy substitution by contact may temper or mediate the buffering capacity of the thera with electrophilic N-alkyl-hydroxy precursors may be peutic composition. For example, the basic nature and selected from the group consisting of polyethylenimine, nucleophilic character of a primary amine may be decreased polyvinylamine, polyallylamine, polyamino acrylates, or modified by the addition of groups such as N-alkyl polyamino methacrylates, copolymers, and salts thereof. hydroxy groups. Significantly, the percent Substitution of Electrophilic N-alkyl-hydroxy precursors suitable for N-alkyl-hydroxy groups, the number of carbon atoms con nucleophilic Substitution upon exposure to the aforemen tained in the N-alkyl-hydroxy groups, and the resultant tioned polymers, and comprising at least one carbon atom, molecular and Supramolecular conformations provide struc may be selected from the group consisting of formaldehyde, aqueous formaldehyde, formalin, 1.3.5-trioxane, metaform tural flexibility and enhanced bonding interactions with aldehyde, paraformaldehyde, glyoxylic acid, MP-glyoxy nucleic acids such as DNA, RNA and/or a virus surface. late, hexamethylentetramine, dimethoxymethane, formyl More specifically, the N-alkyl-hydroxy substituted polymer cation equivalent, and a mixture comprising dimethyl Sul may uniquely bind, bond, adhere, conform, flex, and wrap foxide and a strong acid. The aforementioned group of over, upon, and/or around the unique structural features that compounds may produce a one carbon N-alkyl-hydroxy are found in the myriad of nucleic acids and virus Surfaces, group (carbon attached to a polymer nitrogen atom) that is Such as primary, secondary, tertiary, and quaternary struc referred to as a hemiaminal group. tures, and thus may form a complex with a nucleic acid Such 0015. As illustrated in reaction example 1, an N-alkyl as DNA or RNA, and/or bind to a virus/cell surface and hydroxy/hemiaminal substituted polymer of the therapeutic otherwise render a virus biologically inactive. composition may be produced by the reaction of polyeth 0013 As mentioned prior, the bonding interactions may ylenimine (PEI) and paraformaldehyde ((CHO)) in dim be hydrogen bonding and/or electrostatic interactions ethyl sulfoxide (DMSO) solvent over a time period from involving positive and negative charges. For example, poly about 0.5 hour to about 2 hours: US 2017/0056513 A1 Mar. 2, 2017

REACTION EXAMPLE 1. 0016

O

H ls N N 1N-1N pi N1 NoH 1. N-sh- -> H H 1A 1B

O

H l H T> 100° C. H2O (1) -- H2O (g)

------OH 1C OH O

H l H ------Cool to 20° C. H2O (g) -> H2O (1) HO ! N-l

0017 Reaction example 1 illustrates the step-wise for Solvent, may be isolated by precipitation, wherein the poly mation of N-alkyl-hydroxy/hemiaminal substituted poly mer/DMSO solution is slowly added to a well stirred suit mers (1B-1D) based on PEI (1A). In the scheme, 1A is able precipitation solvent, Such as acetone, followed by contacted with formaldehyde, or a suitable precursor, to filtration, washing, and drying. Diffusion-ordered nuclear produce N-alkyl-hydroxy/hemiaminal polymer 1B (with magnetic resonance spectroscopy (DOSY-NMR) and other chain end group mono-Substitution) at a temperature of at 2D NMR techniques may be used to analyze the polymer least 100° C., to drive off water by-product, and thus drive product and identify the peaks corresponding to N-alkyl the equilibrium to the 1B product side of the equation. hydroxy/hemiaminal moieties. In one example, 0.5 equiva Further equivalents of formaldehyde produce 1C, with bis lents of paraformaldehyde generated a new peak which may N-alkyl-hydroxy/bis-hemiaminal end group Substitution. be identified as a bis-hemiaminal moiety. A desired number Finally, 1D represents a fully substituted N-alkyl-hydroxy/ average molecular weight of at least 500 g/mole may be hemiaminal polymer comprising tertiary amine groups iso measured by gel permeation chromatography or similar lated upon cooling. Reaction example 1 does not restrict the techniques. number, combination, or type of N-alkyl-hydroxy substitu 0018. In some embodiments, a small molecule organic tion. For example, the composition may include polymer base. Such as a tertiary amine, for example TEA, is added to chains with completely substituted amine groups, while the reaction mixture, so that the effective concentration is other polymer chains may be partially substituted. It is from about 0.1 mole/L to about 1 mole/L to suppress the further noted that embodiments of the disclosure do not formation of polymer cross-links (due to hexahydrotriazine restrict the use of a mixture, or mixtures comprising amine formation) and thus promote the formation of the bis polymers to produce N-alkyl-hydroxy modified polymers. hemiaminal end groups. Absent Such a cross-link Suppress Additionally, the practitioner may utilize any copolymer ing agent, a cross-linked polymer may not form effective or comprising amine groups, such as polyethylenimine-graft strongly bound complexes with topologically complex poly(ethylene glycol) (PEI-g-PEG) for example. In some nucleic acids or viral/cell Surfaces, because the cross-linked embodiments, linear or branched PEI, or a mixture thereof, polymer may have restricted freedom of movement as a may be contacted with paraformaldehyde to generate the result of the more rigid structure. requisite N-alkyl-hydroxy/hemiaminal polymer at a tem 0019. In brief summary, reaction example 1 is not meant perature of about 110° C., in the presence of triethylamine to limit or restrict the compositions or methods embodied in (TEA), and wherein the concentration of paraformaldehyde this disclosure, but rather only serves to illustrate some may range from about 0.15 to about 1.2 equivalents. The possible N-alkyl-hydroxy therapeutic compositions that may polymer product, as mixed or dissolved in the DMSO be produced from Such a process. Indeed, the degree of US 2017/0056513 A1 Mar. 2, 2017

N-alkyl-hydroxy/hemiaminal substitution may be controlled polymer composition. Here, an N-ethyl-hydroxy group is and adjusted by the practitioner to produce a general thera formed, wherein the N-ethyl-hydroxy group has further peutic composition with the desired binding configurations substitution in the form of a —CH2(CH2)CH chain and interactions with complex nucleic acids and virus/cell attached to a hydroxyl carbon. Generally, when an epoxide Surfaces. is reacted with an amine polymer of the disclosure, an 0020. The toxicity profile of the therapeutic compositions N-ethyl-hydroxy group is produced, with unrestricted fur of the disclosure may be adjusted by the N-alkyl-hydroxy/ ther substitution possible at the hydroxyl carbon. There is no hemiaminal Substitution. In some embodiments, therapeutic restriction in the type of epoxide used, nor on the number of compositions comprising N-alkyl-hydroxy/hemiaminal carbon atoms or other atoms or functional groups that modified polymers may exhibit significantly improved cell include the epoxide molecule. For example, the epoxide may viability when exposed to N-alkyl-hydroxy/hemiaminal be monomeric, oligomeric, polymeric and/or multifunc modified PEI in contrast to exposure to unmodified PEI. For tional. example, in some experiments, viable human cells exposed 0022. In other embodiments of the disclosure, the com to N-alkyl-hydroxy/hemiaminal modified PEI may display position may include at least one polymer wherein the greatly decreased cell mortality in contrast to an unmodified N-alkyl-hydroxy groups have differing numbers of carbon PEI control. In further experiments, N-alkyl-hydroxy modi atoms. For example, a polymer may be a mixture of N-alkyl fied PEI with increasing molar amounts of N-alkyl-hydroxy/ hydroxy groups and/or Substitutions that are derived from hemiaminal groups in the order: 0.15, 0.30, 0.75, and 1.2 different N-alkyl-hydroxy group precursors, such as para equivalents were exposed to viable human embryonic kid formaldehyde and 2,3-epoxy-1-propanol (glycidol). In one ney (HEK) 293 cells at differing concentrations, and MTT example, a polymer of the therapeutic composition may (tetrazolium dye) assays were performed. As shown in contain 25% by mole hemiaminal groups derived from FIGS. 1A-1D, MTT assays confirm that cell viability/meta paraformaldehyde and 35% by mole of N-alkyl-hydroxy bolic activity increases with increasing N-alkyl-hydroxy/ groups produced from an epoxide Such as glycidol, thereby hemiaminal group Substitution, as the polymer dosing or producing a composition comprising a polymer with both concentration decreases to 1.95 mg/L. In one embodiment, hemiaminal groups (one carbon atom per hemiaminal) and as illustrated by FIG. 1D, the highest level of substitution propane-diol groups (three carbon atoms per propane-diol (1.20 eq. of paraformaldehyde) renders N-alkyl-hydroxy/ group). In general, the number of combinations or different hemiaminal group modified PEI essentially non-toxic, at a types of N-alkyl-hydroxy substitutions are not restricted by concentration of 1.95 mg/L, and cell viability is comparable the disclosure, and therefore the practitioner may choose any to a control with no polymer added to the mixture. order of addition of such reagents to create the desired mixed 0021. In other embodiments of the disclosure, N-alkyl polymer product. For example, the practitioner may choose hydroxy compositions comprising N-alkyl-hydroxy groups to react glycidol with a polymer first, followed by the that have two or more carbon atoms may be produced from Smaller and more reactive hemiaminal precursor, which has epoxide precursors. In one embodiment, an epoxide, when less steric requirements. contacted or reacted with the aforementioned polymers, may 0023. As illustrated in reaction example 2, N-alkyl-hy produce an epoxide adduct, or N-alkyl-hydroxy group, that droxy/propane-diol Substituted polymers may be prepared may be an N-ethyl-hydroxy group. For example, when the by the reaction of polyethylenimine (PEI) and an epoxide epoxide is ethylene oxide or oxirane, an N-ethyl-hydroxy Such as 2,3-epoxy-1-propanol (glycidol) in dimethylsulfox group may be produced, without further alkyl substitution or ide (DMSO) solvent: attached functionality, other than a hydrogen atom. In another example, PEI is contacted or reacted with 1.2- REACTION EXAMPLE 2 epoxydodecane, to introduce non-polar character to the 0024

H 1. n-n pi N OH N N He 1. N-sh- -> OH 2A 2B

H

-N-n-h--H OH

HO OH

HO 2C US 2017/0056513 A1 Mar. 2, 2017

-continued OH

OH OH -N-n-h---n-n-"OH HO H

N~~HO HO 2D

0025 Reaction example 2 illustrates the step-wise for N-alkyl-hydroxy groups containing 6 carbon atoms and 6 mation of N-alkyl-hydroxy/propane-diol substituted poly hydroxy groups, may be prepared by reaction of branched mers (2B-2D) based on PEI (2A). As shown, 2A is contacted PEI and D-(+)-galactose in water solvent: with the epoxide to produce mono-substituted N-alkyl hydroxy/propane-diol polymer 2B (end group mono-substi REACTION EXAMPLE 3 tution). Polymer 2G, featuring bis-N-alkyl-hydroxy/pro 0028

pane-diol end group substitution and thus tertiary amine end 0029. As illustrated in reaction example 3, the branched groups, is produced by additional equivalents of glycidol. PEI comprising sugar moieties may be produced as follows: Finally, 2D represents a substituted N-alkyl-hydroxy/pro D-(+)-galactose (0.209 g, 1.163 mmol), dissolved in 2 mL of pane-diol polymer comprising tertiary amine groups isolated de-ionized (DI) water, may be added drop-wise to a stirred upon cooling. solution comprising 0.1 g (0.01 mmol) of branched PEI in 1 0026. In one embodiment, a substituted N-alkyl-hydroxy/ mL of DI water. The number average molecular weight propane-diol polymer, such as that illustrated by 2D, may be (M), of such a PEI may be from about 5,000 Daltons (Da) produced by reacting and/or contacting a linear PEI of to about 20,000 Da, such as 10,000 Da. The reaction solution approximately 1000 g/mole with 2,3-epoxy-1-propanol may be further heated to a temperature from about 50° C. to (glycidol) in DMSO solvent to generate the requisite about 150° C., such as 70° C., for a time period from about N-alkyl-hydroxy/propane-diol polymer at a temperature 4 hours to about 24 hours. Upon cooling, the solution may from about 50° C. to about 125° C., and wherein the be freeze-dried, and may yield a dark red sticky solid concentration of glycidol may range from about 0.15 to material. The mole ratio for such a reaction may be 1 PEI about 1.5 equivalents. After isolation by precipitation in repeat unit (-CHCH-NH ) to 0.5 D-(+)-galactose. In acetone or another suitable solvent, followed by washing other embodiments, the monosaccharide, D-(-)-arabinose. and drying, the polymer product may be redissolved in a may be reacted with a linear or branched PEI, or both, using suitable deuterated solvent for DOSY-NMR and/or other 2D the aforementioned method to produce a sugar substituted NMR techniques to analyze the polymer product and iden PEI. In a further embodiment, a sugar substituted linear PEI tify the peaks corresponding to the N-alkyl-hydroxy moi may be produced as follows: a linear PEI (M-10.950 Da) eties. GPC may be used to determine the number average may be dissolved in DI water at a water temperature of about molecular weight of the substituted polymer. 70° C., followed by drop-wise addition of D-(+)-galactose. 0027 N-alkyl-hydroxy substituted polymers may also be The solution may be heated for a time period from about 12 produced from carbohydrates, such as Sugars, such as D-(+)- hours to about 24 hours, to produce an N-alkyl-hydroxy galactose. In one embodiment, as illustrated in reaction substituted polymer. Importantly, because linear PEI and/or example 3, an sugar substituted polymer, comprising other polyamines may be insoluble in water at room tem US 2017/0056513 A1 Mar. 2, 2017 perature (s23° C.), it may be necessary to dissolve a linear ylenimine, polyvinylamine, polyallylamine, polyamino PEI in warm water prior to sugar addition. By the same acrylates, polyamino methacrylates, and copolymers and token, Some Sugars may require dissolution in water or other salts thereof. suitable solvents at elevated temperatures for dissolution. 3. The therapeutic composition of claim 1, wherein at 0030 Generally, a polyamine, such as a branched or least a portion of the N-alkyl-hydroxy groups comprise linear PEI, comprising a number average molecular weight hemiaminal groups. from about 5,000 Da to about 20,000 Da, may be reacted 4. The therapeutic composition of claim 1, wherein at with a sugar to produce N-alkyl-hydroxy substituted poly least a portion of the N-alkyl-hydroxy groups contain two or mers. The mole ratio of the PEI repeat unit to the sugar is not more carbon atoms. restricted, and may range from about 1 PEI to about 0.05 5. The therapeutic composition of claim 4, wherein the Sugar, such as about 1 PEI to about 0.25 Sugar. Sugars portion of N-alkyl-hydroxy groups comprises an epoxide suitable for production of N-alkyl-hydroxy substituted poly adduct. mers include, but are not restricted to: monosaccharides (ex. 6. The therapeutic composition of claim 4, wherein the triose, tetrose, pentose, and hexose), disaccharides, oligosac portion of N-alkyl-hydroxy groups comprises a Sugar charides, and polysaccharides. The Sugars may be cyclic or adduct. linear, and may be structural and optical isomers. In one 7. The therapeutic composition of claim 1, wherein the embodiment, a mixture of Sugars, such as glucose and polymer comprises N-alkyl-hydroxy groups that are nucleic fructose, may be used to produce a polymer Substituted with acid, protein or peptide binding groups. a mixture of different Sugar moieties. Once produced, Sugar 8. The therapeutic composition of claim 1, comprising a substituted PEIs may be isolated and/or purified by any nucleic acid bound to the polymer or a therapeutic agent. number of techniques. Such as precipitation in a Suitable 9. The therapeutic composition of claim 7, wherein the Solvent, followed by washing and drying. The polymer nucleic acid is DNA or RNA. product may be redissolved in a suitable deuterated solvent 10. The therapeutic composition of claim 1, wherein the for nuclear magnetic resonance experiments, such as DOSY polymer comprises N-alkyl-hydroxy groups that are virus NMR and/or other 2D NMR techniques to analyze the binding groups. polymer product and identify the peaks corresponding to the 11. The therapeutic composition of claim 9, wherein the N-alkyl-hydroxy moieties. Gel permeation chromatography virus binding groups are virus inactivating groups. (GPC) may also be used to determine the number average molecular weight of the substituted polymer. 12. The therapeutic composition of claim 1, comprising a 0031. The terminology used herein is for the purpose of virus bound to the polymer or a therapeutic agent. describing particular embodiments only and is not intended 13. The therapeutic composition of claim 1, wherein the to be limiting of the invention. As used herein, the singular polymer comprises a linear chain. forms “a”, “an and “the are intended to include the plural 14. The therapeutic composition of claim 1, wherein the forms as well, unless the context clearly indicates otherwise. polymer has a number average molecular weight of at least It will be further understood that the terms “comprises' 500 grams per mole. and/or "comprising,” when used in this specification, specify 15. A method for producing a therapeutic composition, the presence of Stated features, integers, steps, operations, comprising: elements, and/or components, but do not preclude the pres forming a reaction mixture comprising a solvent, a poly ence or addition of one or more other features, integers, meric amine and a N-alkyl-hydroxy group precursor, steps, operations, elements, components, and/or groups and thereof. When a range is used to express a possible value heating the mixture at a temperature from about 25°C. to using two numerical limits X and Y (e.g., a concentration of about 150° C. to produce a polymer comprising sec X ppm to Y ppm), unless otherwise stated the value can be ondary and tertiary amine groups, wherein at least 5% X, Y, or any number between X and Y. of the secondary and tertiary amine groups are N-alkyl 0032. The description of the present invention has been hydroxy groups. presented for purposes of illustration and description, but is 16. The method of claim 15, wherein the polymeric amine not intended to be exhaustive or limited to the invention in is selected from the group consisting of polyethylenimine, the form disclosed. Many modifications and variations will polyvinylamine, polyallylamine, polyamino acrylates, be apparent to those of ordinary skill in the art without polyamino methacrylates, and copolymers and salts thereof. departing from the scope and spirit of the invention. The 17. The method of claim 16, wherein the polymeric amine embodiments were chosen and described in order to best has a number average molecular weight of at least 500 grams explain the principles of the invention and their practical per mole. application, and to enable others of ordinary skill in the art 18. The method of claim 15, wherein the N-alkyl-hydroxy to understand the invention. precursor comprises a material selected from the group What is claimed is: consisting of formaldehyde, aqueous formaldehyde, forma 1. A therapeutic composition, comprising: lin, 1.3.5-trioxane, metaformaldehyde, paraformaldehyde, at least one polymer comprising amine end groups, sec glyoxylic acid, MP-glyoxylate, hexamethylentetramine, ondary amine groups, tertiary amine groups, and alkyl dimethoxymethane, formyl cation equivalent, and a mixture hydroxy groups, wherein at least 5% of the secondary comprising dimethyl Sulfoxide and a strong acid. and tertiary amine groups comprise N-alkyl-hydroxy 19. The method of claim 18, wherein a tertiary amine is groups, and wherein at least 5% of the amine end added to the mixture. groups comprise N-alkyl-hydroxy groups. 20. The method of claim 19, wherein the reaction mixture 2. The therapeutic composition of claim 1, wherein the has a concentration of tertiary amine from about 0.1 mole/L polymer is selected from the group consisting of polyeth to about 1.0 mole/L. US 2017/0056513 A1 Mar. 2, 2017

21. The method of claim 15, wherein at least one polymer has a cross-link density of less than 10%. 22. The method of claim 15, wherein the N-alkyl-hydroxy group precursor comprises an epoxide compound. 23. The method of claim 15, wherein the N-alkyl-hydroxy group precursor comprises a carbohydrate compound. 24. The method of claim 23, wherein the carbohydrate compound is selected from the group consisting of mono saccharides, disaccharides, and polysaccharides. 25. The method of claim 24, wherein the carbohydrate compound may comprise a cyclic or linear structure. k k k k k