Self-assembling amphiphilic polymers as Anti-covid-19 agents

Abstract

There are provided improved amphiphilic comb polymers, comprising a hydrophilic backbone with regularly-spaced pendant hydrophobic moieties, having well-controlled molecular weights, structures, and end groups. The polymers self-assemble into core-corona nanoparticles in aqueous environments, which are capable of disrupting viral coat proteins, and which are capable of encapsulating antiviral drugs and prodrugs. Regularly-spaced targeting moieties optionally mediate the adherence of the nanoparticles to the viral coat. The compositions of the invention are useful as treatments for viral infection, including infections with SARS-CoV-2.

Description

SELF-ASSEMBLING AMPHIPHILIC POLYMERS AS ANTI-COVID-19 AGENTSFIELD OF THE INVENTION

[0001] The present invention relates to the fields of amphiphilic block copolymers, and more specifically to the use of such copolymers for drug delivery. The invention also relates to the field of targeted antiviral agents.BACKGROUND

[0002] Amphiphilic block copolymers comprising a hydrophobic block and a hydrophilic block have been well studied in recent years, because of their capacity for self-assembly into a variety of nanostructures as the surrounding solvent is varied. See Cameron et al, Can. J. Chem. / Rev. Can. Chim. 77:1311-1326 (1999). In aqueous solutions, the hydrophobic compartment of an amphiphilic polymer has a tendency to self-assemble in order to avoid contact with water and to minimize the free interfacial energy of the system. At the same time, the hydrophilic blocks form a hydrated “corona” in the aqueous environment, and so the aggregates maintain a thermodynamically stable structure. The result is a stable, latex-like colloidal suspension of polymer aggregate particles having hydrophobic cores and hydrophilic coronas.

[0003] Comb-type amphiphilic co-polymers differ from block co-polymers in that the backbone is largely hydrophobic or hydrophilic, with polymer chains of opposite polarity pendant from the backbone rather than incorporated into it. Comb-type copolymers have been prepared with hydrophobic backbones and hydrophilic branches (Mayes et al., US Patent No. 6,399,700), and also with hydrophilic backbones and hydrophobic branches (Watterson et al., U.S. Patent No. 6,521,736). The former were used to provide multivalent presentation of ligands for cell surface receptors, while the latter were used to solubilize drugs and deliver them to cells.

[0004] Amphiphilic polymer aggregates have been studied as carriers for solubilizing insoluble drugs, targeted drug delivery vehicles, and gene delivery systems. They spontaneously self-assemble into a core-corona structure that is more stable than conventional low-molecular- weight micelles, due to chain entanglement and / or the crystallinity of the interior hydrophobic region. The polymeric nature of the vehicle renders the aggregates relatively immune to the disintegration that ordinary liposomes suffer whendiluted below their critical micelle concentration. The absence of a bilayer membrane enables them to more readily fuse with cell membranes and deliver their payload directly to the cell. The amphiphilic nature of the aggregates also confers detergent-like activity, and appropriately targeted aggregates appear to be capable fusing with and disrupting viral coat proteins.

[0005] Due to the excellent biocompatibility poly(ethylene glycol) (PEG), and the apparent ability of PEG-coated “stealth” particles to evade the reticuloendothelial system, micelles, liposomes, and polymers incorporating PEG have been extensively considered as materials for drug delivery systems. There are many reports of the use of poly (ethylene glycol) (PEG) as the hydrophilic component of PEG-lipids (forming liposomes and micelles); see for example Krishnadas et ctl, Pharm. Res. 20:297-302 (2003). Self-assembling amphiphilic block copolymers, which self-assemble into the more robust “polymersomes”, have also been investigated as vehicles for drug solubilization and delivery (Photos et al, J. Controlled Release, 90:323-334 (2003)). See also Gref et al., Int. Symp. Controlled Release Mater. 20:131 (1993); Kwon et al. , Langmuir , 9:945 (1993); Kabanov et al, J. Controlled Release, 22:141 (1992); Allen et al. , J. Controlled Release , 63:275 (2000); Inoue et al, J. Controlled Release, 51:221 (1998); Yu and Eisenberg, Macromolecules, 29:6359 (1996); Discher etal, Science, 284:113 (1999); Kim et al, U.S. Patent No. 6,322,805; Seo etal,U.S. Patent No. 6,616,941 and Seo et al, European Patent No. EP 0583955. Luo et al, in Macromolecules 35:3456 (2002), describe PEG-conjugated polyamidoamine (“PAMAM”) dendrimers suitable for delivery of polynucleotides.

[0006] Comb-type polymers generated by random functionalization or co-polymerization are mixtures of thousands of different species of differing molecular weights and branching patterns. The absence of a single, consistent structure presents problems in characterization and quality control, and can be an obstacle when regulatory approval is sought. Regular, consistently-structured amphiphilic comb polymers have been introduced to overcome this shortcoming (Diwan et al, U.S. Patent No. 8,173,764), but there remains a need for tight control of the molecular weight of such polymers.

[0007] The recent emergence and global spread of the novel SARS-CoV-2 coronavirus, and the resulting disease designated COVID-19, has created an urgent need for effective chemotherapeutic agents. Although some known anti-viral agents have shown some degree of effectiveness at reducing the severity and length of infections, there remains a particularneed for drugs that are effective for the treatment of COVID-19, and for the novel coronavirus strains that are almost certain to evolve in the future.SUMMARY OF THE INVENTION

[0008] One aspect of the invention relates to a method for the treatment of viral diseases comprising the administration of antiviral agents encapsulated in polyethylene glycol based polymeric micelles. The invention also concerns formulations of polyethylene glycol based polymeric micelles for the treatment of viral diseases and more particularly for the treatment of infections caused by viruses like SARS-CoV-2. The formulations are composed of polyethylene glycol-based polymeric micelles and contain an encapsulated drug effective against the viral disease with good in vitro antiviral activity. Encapsulation according to the present invention provides a marked improvement in the pharmacokinetics of the encapsulated drugs, and improves their aqueous solubility.

[0009] The present invention provides improved biocompatible comb-type polymers of structures (4) and (5) below, and methods for producing the improved polymers:

[0010] In the above structures, each instance of X is individually either OH or NHR, with R being a C10-C18, preferably C14-C16, hydrophobic moiety. The proportion of substitutents X that are OH ranges from 10% to 90%, is preferably 20-65%, and more preferably is 25-60%. Each instance of L is individually either OH or a ligand having specific binding affinity for the surface of a virus. The average value of m ranges from 10 to 100 and is preferably between 20 and 50. The value of n ranges from 5 to 25, and the overall molecular weight of the polymer (4) may range from 2,000 to 25,000 daltons, and is preferably between 5,000 and 15,000 daltons.

[0011] The invention provides aqueous suspensions of core-corona nanoparticles, which self-assemble from the polymer (4), and provides methods for solubilizing antiviral drugs by incorporating such drugs (and prodrugs thereof) in the hydrophobic cores of the polymer particles. The invention further provides hydrophobic prodrugs tailored to be soluble in the hydrophobic cores of the nanoparticles. In some embodiments of the invention, the polymer (4) may be modified by covalent attachment of cell-, tissue-, or virus-specific targeting ligands to provide polymer (5). Attachment of ligands to the repeating units of the polymers of the invention affords multivalent display of the ligand on the polymer (5) chains and on the nanoparticles.

[0012] The invention also provides a method for the treatment or prevention of an infection of a human or other animal by a virus, which comprises administering to said animal a suspension of self-assembled nanoparticles which comprise a comb-type polymer having structure (4) or (5). The polymer particles preferably have an antiviral drug or prodrug dissolved or dispersed in the hydrophobic nanoparticle core.

[0013] Even in the absence of a small-molecule antiviral drug or prodrug, the self- assembled nanoparticles have inherent antiviral properties. This antiviral activity is thought to be due to the detergent-like ability of the amphiphilic polymers to disrupt or denature the outer coating of virus particles. This activity is enhanced by the binding affinity of the multiple carboxylate groups and / or ligands L for the surface of the targeted virus.

[0014] The invention further provides methods for the preparation of the polymers, nanoparticles, and drug complexes described herein. The polymers of the invention self- assemble into polymer aggregates that efficiently solubilize, distribute, and deliver drugs in vivo; have inherent antiviral activity; and are non-toxic, biocompatible, and stable.

[0015] The invention also relates to a method for the treatment of viral diseases comprising the administration of antiviral agents encapsulated in the self-assembled nanoparticles of the invention. The invention also concerns pharmaceutical compositions comprising antiviral agents encapsulated in the self-assembled nanoparticles of the invention, and the use of these compositions for the treatment of viral diseases and more particularly for the treatment of infections caused by coronaviruses like SARS-CoV-2. The formulations provide a marked improvement in the pharmacokinetics of the encapsulated drugs and improve their aqueous solubility. The improvements in distribution and solubility enable the administration of a wide variety of prodrugs that would not otherwise be effective.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Fig. 1 is a graph showing the molecular weight of pi-polymer as a function of the ratio of DTT to PEG dimaleate.

[0017] Fig. 2 is a synthetic scheme for preparing the polymers of the invention.

[0018] Fig. 3 is the legend for Fig. 2, and identifies the R groups.

[0019] Fig. 4 is a plot of cell survival as a function of unencapsulated drug or prodrug concentrations.

[0020] Fig. 5 is a plot of cell survival as a function of encapsulated host concentration.

[0021] Fig. 6 is a plot of cell survival as a function of encapsulated guest concentration.DETAILED DESCRIPTION OF THE INVENTION

[0022] Comb polymers having the general structure of the polymers of the present invention have been described in US Patent application serial Nos. 12 / 223,052 and 12 / 518,411 (publication Nos. 2010 / 0260743 and 2010 / 0008938), both of which are incorporated herein by reference in their entireties. It is a feature of these materials, referredto as “pi-polymers”, that the side chains R are neither randomly nor uniformly distributed along the polymer chain, but rather occur in pairs, each pair being spaced more or less regularly along the polymer chain, depending on the degree of monodispersity of the PEG monomer.

[0023] The polymers of the invention have a comb-type architecture, with a backbone formed of alternating branch-point moieties and hydrophilic, water-soluble PEG blocks; and a plurality of hydrophobic side chains R attached to each branch-point moiety, as shown in Formula (4). The hydrophobic side chains R are preferably CIO to Cl 8 alkyl groups, but may incorporate heteroatoms to provide dipole-dipole or hydrogen-bonding interactions with encapsulated drugs or prodrugs. Ether, ester, amide, sulfoxide and sulfonyl groups, for example, can be incorporated into some or all of the groups R.

[0024] The improved polymers of the present invention feature a narrow molecular weight distribution, controlled chain terminal structures, a lowered level of hydrophobic substituents R, and a high density of carboxylate groups, which act as affinity ligands for viral coat proteins. The polymers having lower levels of hydrophobic substitution (e.g., 10%, 20%, 30%, 40% or 50% of R being hydrophobic) have been found to be more water-soluble, and more suitable for injectable formulations.

[0025] Attachment of ligands to the repeating units of the polymers of the invention affords multivalent display of the ligand on the polymer chain and on the nanoparticles surface, which can result in great increases in affinity for the ligands’ target. For example, multivalent antibodies can be far more effective in clearance of their targets than the normal divalent antibodies. Carbohydrate-binding proteins and carbohydrates are known to be multivalent in nature, and ineffective if monovalent. Similarly, multivalent peptide and carbohydrate targeting moieties will be far more effective than the monomer alone. The increase in MW due to attachment to the polymer results in reduced renal clearance rates of peptides and other ligands. In addition, the PEG backbone affords to the peptide benefits similar to those of PEGylation, including evasion of immune surveillance.

[0026] Further, a multivalent targeting moiety will decorate a multivalent target (say, a virus particle) and neutralize it far more effectively than the monomeric targeting moiety.The ability to display multiple (different) peptides in multivalent format will lead to enhanced specificity.

[0027] Broadly, the invention provides a comb polymer having the following structure:where each instance of X is individually either OH or NHR, with R being a C10-C18 hydrophobic moiety, and each instance of L is individually either OH or a ligand having specific binding affinity for the surface of a virus. In structure (5), the average value of m ranges from 10 to 100 and is preferably between 20 and 50, while the value of n ranges from 5 to 25. In view of the methods of synthesis described below, it will be appreciated that structure (5) is an idealized representation, and that any of the depicted carboxy groups, including those at the polymer end caps, may be coupled to the ligand L.

[0028] Each ligand L is may be, for example, one of the following moieties:In the ligands shown, each R1 is individually H or C1-C4 alkyl, and each R2 is individually H, COR1, or CO2RI.

[0029] When dissolved in water or an aqueous medium, the comb polymers described above self-assemble into core-corona type nanoparticles. The invention provides suchnanoparticles having dissolved or dispersed within their hydrophobic cores an antiviral drug or a prodrug thereof.

[0030] The invention provides pharmaceutical compositions, which comprise a pharmaceutically acceptable aqueous carrier and the self-assembled nanoparticles described above, both with and without antiviral drugs, or prodrugs thereof, dissolved therein.

[0031] The invention provides a method of treating or preventing viral infections in humans and other animals, in particular infections caused by coronaviruses, including SARS- CoV-2, by administering effective amounts of the comb polymers and pharmaceutical compositions described above.

[0032] A feature of the present invention is the enhanced combined activity of the polymer and the encapsulated drugs. The polymeric micelle (host) has the property of dismantling the virus by binding to and denaturing its envelope glycoproteins, thus blocking the re-infection part of the virus’s life cycle, i.e., the infection of new cells by newly-released viral particles. The encapsulated drug (guest), meanwhile, is capable of blocking the replication part of the life cycle, in which the virus generates progeny inside cells. With both life cycle processes sufficiently blocked, a synergistic reduction in viral replication and viral load is obtained.

[0033] Another feature of the present invention is the improvement in drug pharmacokinetics and pharmacodynamics provided by the encapsulation of the drug within the polymer nanoparticles. Remdesivir, for example, is known to be more effective when injected as a complex with SBECD The extension of thisconcept to the enhancement of remdesivir' s pharmacokinetics and pharmacodynamics upon encapsulation in polymeric micelles has been discussed in A. Chakraborty, A. Diwan, “Pharmacodynamics of Remdesivir: How to Improve for COVID-19Res. Environ. Sci. (2020) 1(8):431-438A, with in vivo results reported by A. Chakraborty el al. in bioRxiv preprints https: / / doi.org / 10.1101 / 2021.10.22.465399 and https: / / doi.org / 10.1101 / 2021.ll.17.468980.

[0034] The polymers of the invention may be prepared by the process shown in Scheme 1 and in Figure 2.

[0035] In practice, the chemical reactions illustrated proceed with statistical product distributions and with less-than-perfect efficiency, and it should be understood that the polymer products shown in the schemes and in the claims are ideal representations rather than typical or average structures. For example, in the preparation of (3), the amidation reaction is not quantitative, and in practice it is not desired that it be quantitative. Furthermore, in the treatment of (3) with maleic anhydride (step D in Figure 2), the yield of adduct ranges from 30-50%, which the inventors believe is due to competing Michael addition of side chain hydroxyl groups to form lactone rings. The subsequent reaction with mercaptosuccinic acid (step E in Figure 2) may proceed with yields ranging from 20% to 100%, depending upon the amount of reagent, time, and temperature; these variables can be manipulated to control the density of carboxyl groups on the final product.

[0036] There is a known preference for Michael addition of thiols beta to maleic acid monoesters. Yoon, H.B., et al, “Michael Addition of Thiol Compounds onPoly(ethylene oxide)s: Model Study for the “Site-Specific” Modification of Proteins,” Macromol. Res. 26:194-203 (2018), doi.org / 10.1007 / sl3233-018-6021-4. The regiochemistry is not 100% specific, however, and formulas (2), (3) and (4) should be understood to encompass regioisomers in which any sulfur atom may be attached alpha or beta to any succinyl carboxylate. The invention thus encompasses compositions that are mixtures of regioisomers at the succinate moieties. Due to the asymmetric carbons at the sulfur-bearing carbons, the invention also encompasses polymers which contain mixtures of any or all of the possible regio- and stereo-isomeric possibilities.

[0037] In structures (3) and (4), the hydrophobic moieties R are preferably derived from C8 to C18 aliphatic amines RNH2, and are most conveniently linked to the polymer by amidation of the carboxylic acid groups of polymer (2) as illustrated in Scheme 1. The hydrophobic groups R are preferably C8-C20 hydrocarbon moieties, which may be linear or branched or contain one or more rings. Examples of the group R include but are not limited to n-octyl, 2-ethylhexyl, n-dodecyl, n-hexadecyl, and the like. The solvent power of the hydrophobic core of the self-assembled nanoparticles can be increased by introducing halogen, ether, ester, amide, sulfone, sulfoxide, or nitrile moieties into the hydrophobic group R. As used herein, the term “hydrophobic” when applied to R means that the logP value (octanol-water) of the molecule R-H is greater than 2. In preferred embodiments, the logP of R-H is greater than 2.5.

[0038] In Scheme 1, the end groups of the polymer are controlled by using an excess of DTT, and a capping reaction with maleic acid, in the production of polymer (2). Precise control of the amount of DTT enables production of a targeted molecular weight range, as described below. Amidation to produce polymer (3) can be accomplished with any of a variety of carboxyl activating reagents, which are well known in the field of peptide synthesis. Suitable examples include but are not limited to CDI, DCC, DIC, and EDC. N- hydroxysuccinimide or N-hydroxysulfosuccinimide are preferably used with the carbodiimide reagents. Carbonyl diimidazole (CDI) is a preferred reagent. Esterification with maleic anhydride, followed by addition of mercaptosuccinic acid to the resulting maleoyl groups, is then used to introduce up to six additional carboxyl groups at each branch point moiety, as shown in structure (4).

[0039] In practice, it has been found that quantitative introduction of carboxylate groups is not readily achieved, and the polymer represented by structure (4) may have, on average, anywhere from three to five carboxylic acids per branch-point moiety, depending on the reagents, reactants, and conditions used in the esterification and Michael addition processes. The polymers described herein should therefore be understood to contain some fraction of residual (unreacted) and missing functional groups. The scope of the appended claims, accordingly, extends to polymers having from about 20% to 100% of the depicted level of functionalization, unless specific levels of functionalization are explicitly recited. EXAMPLESGeneral procedures for polymer synthesis

[0040] The invention provides processes for the preparation of the comb polymers of the invention. The key starting material is polyethylene glycol, which is preferably dried before use by stirring under vacuum at an elevated temperature. This may take 8-12 hours, depending on the quality of the PEG. Once dried, the PEG can be stored indefinitely under a dry, inert gas such as nitrogen or argon.

[0041] In order to ensure that the polymers of the invention have reproducible and consistent properties, the PEG is preferably of low dispersity. Most preferable are PEG polymers that are >95% monodisperse, such as are commercially available from Nektar Therapeutics (formerly Shearwater Polymers), Huntsville AL, and Polypure AS, Oslo, Norway. An example of a monodisperse PEG is “PEG-28” from Polypure, which is >95%available from Millipore Sigma, Burlington MA, is also suitable. Polyethylene glycols fromother vendors are expected to be suitable, provided that the certificate of analysis shows a sufficiently narrow molecular weight dispersion.

[0042] Molecular weight control is important because different molecular weights of the polymer (2) will yield different grades and types of the amidated polymer (3), and of any further derived polymers. In the pharmacokinetics of PEG polymers, it is known that smaller polymers are cleared by glomerular filtration via the kidneys, resulting in a lower half life, whereas larger polymers circulate longer and may exhibit fecal excretion, rather than urinary excretion, as the primary elimination pathway.

[0043] In theory, two bifunctional chemicals, when reacted together as in the preparation of polymer (2), can produce extremely high molecular weights if used in equimolar amounts. In practice, the molecular weight is controlled by the Carothers equation, which reflects the fact that an excess of one reactant over the other can be used to control the degree of polymerization. In the present case, because the PEG dimaleate (1) is not purified, it may contain residual maleic acid and / or PEG monomaleate, and the results of polymerization with DTT are not entirely predictable. The present invention, however, provides a method for obtaining a predictable molecular weight.

[0044] Trial reactions on a given batch of PEG dimaleate are first carried out with different DTT molar excess ratios, using the same conditions and reactors of the same geometry as the process equipment. The resulting polymers (2) are purified and their molecular weights are determined by multi-angle light scattering coupled with size exclusion chromatography (SEC-MALS). A plot of the experimental MW vs. the DTT / PEG dimaleate ratio is then prepared, which is characteristic of that particular batch of PEG dimaleate.

[0045] A representative plot is shown in Fig. 1. The DTT / PEG dimaleate ratio required to obtain a desired MW from this particular batch PEG dimaleate can be obtained from this plot, and by using this ratio in the production process, the desired MW is reliably achieved. The plot is considered a specific characteristic associated with the given batch of PEG dimaleate under the specified process conditions. For a different batch of PEG dimaleate, the process is repeated and a new plot generated, so as to provide the operating polymerization characteristic for that batch.

[0046] All reactions are carried out under an inert atmosphere such as nitrogen or argon, with magnetic or mechanical stirring.PM2-DTT polymer (2)

[0047] PEG dimaleate (1) (“P10M2”) was prepared from polyethylene glycol 1000 using the method described in US 2010 / 0260743. The polymer was melted under nitrogen at 60- 80°C, water was added to 40-50% w / v final concentration, and the solution was adjusted to pH 6-8.5 by addition of DIPEA. Dithiothreitol (DTT), 1.02 to 1.5 mmol per mmol of maleate double bonds, was added as a solution or as a solid. The molar ratio of DTT to P10M2 was based on the desired MW of the P10M2-DTT polymer (2). The pH of the solution was monitored by a pH probe and viscosity was monitored using in-reactor ultrasonic viscometer probe. The viscosity plateaued in about 15 minutes and remained essentially unchanged for next 30 minutes. Unreacted DTT and terminal sulfhydryl groups were quenched by the addition of maleic acid until the mixture gave a negative test with Ellman’s reagent. The reaction mixture was acidified to pH 2-4 with 6 N hydrochloric acid. The product P10M2- DTT (2) was purified by extraction into dichloromethane (DCM); the low-molecular-weight impurities formed by reaction of DTT with maleic acid and salts remain in the aqueous layer and are removed. The organic layer was further washed with water, and the dichloromethane was distilled off under vacuum, leaving P10M2-DTT polymer (2) suitable for the next step.In an alternative procedure, heptane (2 to 4 times the volume of dichloromethane) was added to precipitate the P10M2-DTT polymer as alow-melting waxy solid. Isolated yield: 70-90% of theory.

[0048] The DP (degree of polymerization) of the resulting polymer ranged from 3 to 14 as desired, based on the amount of DTT employed, and the molecular weight (as determined by SEC-MALS) ranged from 4kDa to 18 kDa.

[0049] The solvent extraction process described above has been found to be superior to the tangential flow membrane filtration used in the prior art, which provided low yields and very dilute solutions of polymer (2), and gave poor separation from contaminants.Amidated PM2-DTT polymer (3)

[0050] Dry polymer (2) (P10M2-DTT) is dissolved in a solvent such as dichloromethane and the carboxyl groups are activated by reaction with activation agents such as diisopropylcarbodiimide (DIC), N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimideN-hydroxysulfosuccinimide are used, particularly with carbodiimide reagents, to minimize side reactions, such as the conversion of reactive O-acyl ureas to unreactive N-acyl ureas.The molar equivalents of activating agent used depends on the alkyl amine substitutiondesired, and maximum amine substitution is not more than the equivalents of activating agent used. The activation time can be between 15 minutes and 2 hrs. For alkyl amines, such as hexadecylamine (HDA), tetradecylamine and other CIO to C18 alkyl amines, 0.1 to 0.2 molar excess of activating agent are used. The amidation reaction is carried out between 20-60°C, preferably between 30-50°C, depending upon the amine. Larger amines were found to react better at higher temperatures, possibly because of reactive group unavailability due to micelle formation in the reaction mixture at lower temperatures. The reaction is quenched with water or acidified water to decompose residual activated carboxy groups, and the amidated polymer (3) is extracted into dichloromethane or a suitable water immiscible solvent, and washed with water and dilute acid to remove water-soluble and basic impurities. The amidated polymer is further freed from residual alkyl amine by treating with a strong cation exchange resin, and the solvent is removed by distillation in vacuo.

[0051] Apparent molecular weights are determined by SEC-MALS. The alkyl amine content is determined by acid hydrolysis of the polymer, followed by estimation of alkyl amine by reaction with a suitable amine reactive reagent such as fluorescamine. Unreacted carboxylic groups are estimated by determination of the polymer’s acid value.P10M2-DTT-C16

[0052] P10M2-DTT (1 mmol carboxy groups) was dissolved in dichloromethane in a reactor set up with a stirrer, a condenser and a thermometer. The solution pH was adjusted to 2-4 with a tertiary base such as triethylamine or diisopropylethylamine. Carbonyldiimidazole (CDI, 0.5 mmol) was added with stirring at a temperature of 10-30°C, controlling the evolution of carbon dioxide generated. The reaction mixture was stirred at ambient temperature for 15-60 minutes to activate the carboxy groups on the polymer. To the activated polymer was then added n-hexadecylamine (HDA, 0.55-0.65 mmol). The reaction was stirred at 20-45°C for 2-24 hours, preferably 18-24 hours, or until TLC or mass spectroscopy of the reaction mixture indicated the desired extent of the reaction. The reaction was terminated by careful addition of aqueous HC1 to decompose residual activated carboxylates. Additional aqueous HC1 was added, and the aqueous layer (containing imidazole, water-soluble salts, and other water-soluble impurities) was removed. The isolated organic phase was washed again with water. Ethanol was added to a 30-60% final concentration, and the polymer solution was treated (in column or in batch mode) with a strong cation exchange resin (H+ form, 3 to 10 equivalents per equivalent of hexadecylamine) to remove the unreacted amine. The efficiency of removal of amine wasfollowed by TLC and mass spectral analysis. The product was isolated by distillation of the dichloromethane-ethanol solvent in vacuo, to give P10M2-DTT-C16 (3) as a waxy solid.

[0053] In an alternative procedure, the reaction mixture as is at the end of the reaction was diluted with dichloromethane to 4-10% w / w of starting polymer content, and the solution was treated (in column or in batch mode) with a strong cation exchange resin (H+ form, 3 to 10 equivalents per equivalent of hexadecylamine) to remove the unreacted amine. The extent of removal of amine was followed by TLC and mass spectral analysis. The product was isolated by distillation of the dichloromethane solvent in vacuo, to give P10M2-DTT-C16 (3) as a waxy solid.Introduction of multidentate carboxylates: P10M2-DTT-C16-(M-MSA); P10M2-DT- (HDA)X(M-MSA)2

[0054] P10M2-DTT-C16 (3) is heated to 90-120°C under a nitrogen atmosphere, to form a stirrable melt, and a solution of excess maleic anhydride in methyl isobutyl ketone is added. The amount of maleic anhydride addded is in at least a 20% stoichiometric excess relative to the calculated quantity of hydroxyl groups present, preferably from 80-140% in excess, and may be as much as 300% or more in excess. Care must be taken to avoid sublimation of maleic anhydride when used without a cosolvent such as MEK, MIBK etc. Therefore, maleic anhydride is added when the polymer temperature reaches approximately 50-80°C, preferably about 60-70°C. As the polymer is being heated with stirring, the maleic anhydride pellets are mixed into the polymer melt. The temperature of the reactor is then further increased to the reaction temperature. The reaction mixture is stirred at 70-140°C, preferably 80-100°C, to form the maleate esters of the DTT hydroxyl groups.

[0055] The reaction mixture is then cooled to about 40-70°C, diluted with water, and the pH raised to 8-9 by addition of DIPEA (diisopropylethylamine) or TEA (triethylamine). Excess mercaptosuccinic acid (1-2 equivalents per equivalent of added maleic anhydride, preferably 1.2-1.8 equivalents, more preferably 1.4-1.6 equivalents) is then added, and allowed to react with the maleate double bonds at pH 8-9. The progress of the reaction may be followed by mass spectroscopy. The reaction mixture is then cooled to room temperature and extracted with 1 : 1 dichloromethane-isopropyl acetate to remove low molecular weight organic contaminants. The pH is adjusted to between 2 and 4 with hydrochloric acid, and the polymer is extracted from water into dichloromethane, and precipitated by addition of 1-4 volumes of n-heptane. The solid is dissolved in butanol or isoamyl alcohol, and re precipitated by addition of n-heptane. The solid is then oven-dried under vacuum or undernitrogen to obtain the product, P10M2-DTT-C16-(M-MSA), also referred to below as P 10M2-DT-(HD A)x(M-MS A)2

[0056] In an alternate purification procedure, when TEA is used as the base catalyst for reaction, the reaction mixture is cooled and then titrated with 3-6 N HC1 to pH 2-3, and the resulting free acid form of the polymer is extracted into dichloromethane. The dichloromethane solution is washed with 1 N HC1 and then with water to remove acid- and water- soluble impurities. The dichloromethane is distilled off under reduced pressure and then under vacuum. In another embodiment, the dichloromethane solution is concentrated under reduced pressure, acetone is added, and the mixed solvents distilled off. The process of replacing dichloromethane with acetone is repeated until dichloromethane is undetectable by gas chromatography. The acetone is dried to completion by vacuum distillation in situ or by oven drying, preferably under nitrogen, to obtain the product, P10M2-DTT-C16-(M-MSA). Ligands

[0057] The representative virus-targeting ligands disclosed below have a primary amino group that is used to conjugate the ligand with the polymer carboxylic acids to give the active drug. The methods illustrated are representative, and other means of attachment will be apparent to those of skill in the art, using any of the many linkers and coupling reactions known in the field of small molecule-polymer conjugates. The ligands presented here fall into a few categories:(a) Ligands containing a nicotinic ester moiety,(b) Ligands containing a caffeic acid moiety,(c) Ligands containing both caffeic acid and nicotinic ester, and(d) Any of the above categories, ami dated with 1-cysteic acid.Nicotinyl esters

[0058] Methyl 6-chloronicotinate is dissolved in THF or MEK as solvent. A molar equivalent of Boc-l-cysteine methyl ester is added followed by addition of potassium carbonate or a tertiary organic base such as triethylamine or DIPEA. Water is added to precipitate the product as a solid, which is isolated by filtration. The filter cake is washed

[0061] Caffeic acid (1 mmole) and H-lysine(boc)-methyl ester hydrochloride (lmmole) are mixed in tetrahydrofuran (THF). Triethylamine is added to adjust the pH between 5-8.5, and N,N’-diisopropylcarbodiimide is added, and the reaction mixture is stirred between 30- 60°C until amidation is complete. The crude caffeoyl-l-lysine(Boc)-methyl ester is de- esterified by treating with 6 N NaOH between 5-40°C. The de-esterified material is extracted into alkali, and acidified to pH 3-5 to precipitate the acid as a solid, which is isolated by

[0066] The above ligands are illustrated as their methyl esters, but ethyl, n-propyl, and butyl esters are contemplated as well. One or both catechol OH groups of caffeic acid may esterified, as carboxymethyl, carboxyethyl), acetate, propionate, and the like.Ligand coupling to polymers

[0067] Standard peptide coupling techniques are used to activate carboxy groups of polymers (4) with carbonyldiimidazole, N,N-diisopropylcarbodiimide, or the like, followed by addition of the desired ligand. Using this approach, the various virus-specific ligands described above can be conjugated to the polymer through amide linkages. The amounts of ligand can be varied as desired. A representative structure (5) is illustrated below, where L represents a ligand coupled via amidation of the polymer carboxyl groups, and X represents a mixture of OH and NHR groups as described above. Coupling to the least sterically hindered carboxyl groups is illustrated, but it will be appreciated that any of the available carboxy groups, including those at the polymer end caps, may be ami dated.Ligand attachment to polymer

[0068] Dry polymer P10M2-DT-(HDA)X(M-MSA)2(4) is dissolved in a suitable inert solvent such as DMF under an atmosphere of nitrogen, at between 15-45°C, and the polymer carboxylic acid groups are activated by addition of CDI (5-30% excess over the number of carboxylic acid groups intended to be amidated.) The activation is carried for 30-60 minutes, and to the activated polymer is added a solution of the ligand L60(OMe)2 in DMF. The pH is maintained at 7.5-9 with TEA or DIPEA. The coupling reaction is continued for 2-20 hrs. After the reaction is complete, the pH is adjusted to 3.5-4.5 with hydrochloric acid, followed by excess water to precipitate the polymer-ligand conjugate. The polymer-ligand conjugate is then purified by either solvent-water extractions or by dialysis or tangential flow filtration with an appropriate cut-off membrane.Drug encapsulation:

[0069] The host polymer and the guest drug are dissolved, in respective proportions from 3: 1 to 40: 1 by mass, preferably from 10: 1 to 20: 1 by mass, in a mutual solvent, such as dimethylsulfoxide (DMSO), ethanol, tetrahydrofuran (THF) or dichloromethane (DCM), and mixed to produce a clear solution. It is known that when combining drugs, their ratios should be close to the ratio of their physiologically effective concentrations that provide a desired extent of effect (e.g. ratios of EC50 (50% effect) or ratios of EC90 (90% effect)), so that their activities are balanced. In general, the physiological EC50 and EC90 cannot be determined accurately, and therefore cell culture-based EC50 and EC90 values are often used. Thus the range of encapsulation ratios for a particular guest and host will be preferably in the range of the ratio of their EC50 values or EC90 values respectively. This solution is then evaporatedin an oven or on a rotary evaporator, or lyophilized, depending upon the solvent used. The dried mixture is then reconstituted in water or suitable buffer to give an emulsion of the guest drug distributed within the self-assembled polymer nanoparticles. The loading ratio of the guest drug is then determined by suitable method such as HPLC or UV -Visible spectroscopy.

[0070] The self-assembled nanoparticles, by virtue of having a hydrophobic core, are capable of dissolving or suspending hydrophobic drugs and pro-drugs that are otherwise not readily formulated into effective pharmaceutical compositions. They enable pro-drugs to be designed for optimal pharmacokinetics, without having to make compromises in the interest of aqueous solubility and / or bioavailability. Many alkyl and alkoxy carbonyl prodrugs are known in the art, and methods for their manufacture are well known and largely routine. Representative examples are provided below, but most known methods can be adapted to a variety of substrates. The use of esters of Cl to C18 aliphatic and aromatic acids, carbonates derived from Cl to Cl 8 aliphatic and aromatic alcohols, and carbamates derived from Cl to C18 aliphatic and aromatic amines, is considered to be within the scope of the invention. The invention makes possible the administration of hydrophobic drugs and prodrugs that might not otherwise be considered as clinical candidates.

[0071] Suitable antiviral drugs, pro-drugs and drug candidates for use in the invention include, but are not limited to, remdesivir, acyclovir, molnupiravir, PF-00835231, ivermectin, colcicine, mebendazole, CDI-45205, and GC-376, and various prodrug esters, amides and carbamates thereof. The new and known drug derivatives (i.e. pro-drugs) of the invention, which are preferably lower alkyl esters or lower alkoxy carbonyl esters (i.e. carbonates) of antiviral drugs known in the art, are prepared by known methods of acylation, or modifications thereof. For example, as is well-known in the art, acid anhydrides, acid chlorides, and activated carboxylic acids may be employed for esterification. Alkoxy carbonyl chlorides may be employed to prepare alkoxy carbonyl esters. Preferred solvents for these reactions are dipolar aprotic solvents such as DMSO, DMF and NMP.

[0072] Representative combinations of drugs and drug derivatives and polymer (4) or (5) are presented in the Tables and 2 below, respectively, along with calculated logD values for the guest species. The calculated parameter logD takes into account the overall partition of both ionized and non-ionized forms of the compound, whereas calculations of logP take into account only the partition of non-ionized (neutral) compounds. H. Kubinyi, “Lipophilicity and drug activity” Prog. Drug Res. (1979) 23:97-198; doi: 10.1007 / 978-3- 0348-7105-l_5.TABLE 1Compounds hosted within polymer (4), P10M2-DT-(HDA)x(M-MSA)2* Log D values determined with MarvinSketch™ software, versions 5.4.0.1 or 6.1.2 (ChemAxon, Budapest, Hungary)TABLE 2Compounds hosted within polymer (5), P10M2-DT-(HDA)x(M-MSA)2 L-60(OMe)2 conjugate* Log D values determined with MarvinSketch™ software, versions 5.4.0.1 or 6.1.2 (ChemAxon, Budapest, Hungary)General procedure for encapsulation.

[0073] The host polymer and the guest antiviral drug or derivative are dissolved, in appropriate proportion from 3: 1 to 20: 1, depending upon the host and the guest in a mutual solvent and mixed to obtain a clear solution. Stirring, or passing through a double barrel or single barrel syringe repeatedly, may achieve the proper mixing. Suitable solvents include but are not limited to dimethylsulfoxide (DMSO), ethanol, tetrahydrofuran (THF), dichloromethane (DCM) and acetone. This solution is then evaporated in a vacuum oven, rotary evaporator, or lyophilizer. The dried mixture is then reconstituted in water or suitablebuffer to give a nanoemulsion of the guest species in the polymer. The loading ratio of the guest drug is determined by suitable method such as HPLC or UV -Visible spectroscopy.General procedures for esterification.

[0074] Adenine and uracil 3-hexanoyloxybutoxymethylphosphonates were prepared by reacting the 3-hydroxybutoxymethylphosphonates with hexanoic anhydride in the presence of catalytic quantities of 4-dimethylaminopyridine (DMAP) in dimethyl sulfoxide. Hydroxyl groups in Boc-protected compounds were also esterified with various acid anhydrides in the presence of 4-DMAP, and then the Boc groups were removed, to give esters with varying degrees of hydrophobicity, suitable for encapsulation. When heterocycle nitrogens prone to acylation were present, acid chlorides instead of acid anhydrides, in N,N-dimethylacetamide, were employed without catalyst.[4-(6-amino-9H-purin-9-yl)cyclopent-2-enyl] methanol (Example 18)

[0075] The process described in EP 1660498 B1 was adapted as follows: 5-amino-4,6- dichloropyrimidine (20 mmol) and (lS,4R)-4-amino-2-cyclopentene-l-methanol hydrochloride (21 mmol) were combined in 1 -butanol (25 mL). Anhydrous sodium bicarbonate (50 mmol) was added and the reaction mixture heated to between 80-90°C. The progress of the reaction was followed by mass spectral analysis. After 2-4 hours the reaction mixture was cooled and filtered to remove inorganic salts and bicarbonate. The residue was washed 3x with 5 mL of 1 -butanol. The organic layer was then washed with water and evaporated to give the product {4-[(5-amino-6-chloropyrimidin-4-yl)amino]cyclopent-2-en- l-yl}methanol in about 90% yield (m / z 241).

[0076] The product from the above reaction (10 mmol) was dissolved in 1 -butanol (20 mL). Trimethylorthoformate (11 mmol) was added, followed by concentrated sulfuric acid (0.5 mmol). The reaction mixture was heated at 80-90°C for about 2 hours, until disappearance of starting compound as determined by MS. The reaction mixture was cooled, sodium bicarbonate was added, and the mixture stirred for 30 minutes to neutralize the acid. The mixture was filtered to remove the salts. Evaporation of solvent provided [4-(6-chloro- 9H-purin-9-yl)cyclopent-2-en-lyl]methanol in about 80% yield (m / z 251).

[0077] This material (10.7 mmol) was dissolved in a mixture of isopropanol (15 mL) and concentrated aqueous ammonia (15 mL) and heated to 70°C in a pressure bottle for 24-72 hours. The solution was then evaporated to dryness. The solid residue was triturated withhexane-acetone to give [4-(6-amino-9H-purin-9-yl)cyclopent-2-enyl]methanol (m / z 232) of greater than 90% purity by HPLC.2-(6-amino-9H-purin-9-yl)-4-hydroxybutanamide

[0078] t-Butyl N-[(t-butoxy)carbonyl]-N-(9H-purin-6-yl)carbamate (10 mmol) wasSodium hydride (20 mmol) was added at room temperature. The suspension was then warmed and stirred at 60°C for 5 minutes to form the sodium salt. 2-bromo-4-hydroxybutyric acid g-lactone (20 mmol) was added, and the reaction mixture refluxed for 3 hours, monitoring by mass spectral analysis. The mixture was cooled to room temperature and methanol added to quench. The reaction mixture was filtered to give t-butyl N-[(tert-butoxy)carbonyl]-N-[9-(3-methyl-2-oxooxolan-3-yl)-9H- purin-6-yl] carbamate, m / z 420. The product (1.41 g) was dissolved in tetrahydrofuran and concentrated aqueous ammonia (5 mL). The reaction mixture was stirred at room temperature for about 2.5 hours and then evaporated to dryness to give the product as a yellow, hygroscopic solid. Mass spectral analysis indicated a mixture of mono-Boc (m / z 337) and bis-Boc (m / z 437) derivatives. Column chromatography on silica with ethyl acetate- methanol provided the mono-Boc derivative as the major product. The N6-Boc protecting group was then removed by treatment with HC1 in dioxane, followed by treatment with concentrated aqueous ammonia, to afford the title compound.

[0080] The above procedures are performed with alkyl amines of various chain lengths, in place of ammonia, to provide N-alkyl butanamides with a variety of LogD values.

[0081] The above procedure is carried out with 2-bromo-2-methyl-4-hydroxybutyric acid g-lactone to afford the title compound.

[0082] The compound, [4-(6-amino-9H-purin-9-yl)cyclopent-2-en-lyl] methanol (0.56 mmol) was dissolved in N,N-dimethylacetamide (1 mL). Hexanoyl chloride (7.15 mmol) was added at room temperature. After about 1 hour at room temperature the reaction mixture was quenched with water to hydrolyze unreacted hexanoyl chloride. The reaction mixture is extracted with ethyl acetate and the organic layer is washed with saturated aqueous sodium bicarbonate solution and then twice with water. The mono hexanoate ester, isolated in 55% yield from the ethyl acetate layer after evaporation, was about 95% pure (HPLC and mass spectral analysis).

[0083] By the above procedure, 3 -deoxy adenosine is reacted with valeric acid to produce the title compound.

[0084] In a dry round-bottomed flask, at ice-cold temperature and under nitrogen blanket, tetrahydrofuran (50 mL) and sodium hydride (50 mmol, 60% suspension in mineral oil) were mixed and stirred for 5 minutes. 4-(2-hydroxyethyl)-2, 2-dimethyl- 1,3-dioxolane (40 mmol) was added and the temperature is raised to room temperature. Diethyl p-toluenesulfonyloxy- methylphosphonate (44 mmol) in tetrahydrofuran (20 mL) was added and the reaction mixture is stirred at room temperature for 2 hours, until mass spectral analysis showed completion of reaction. The product, diethyl {[2-(2,2-dimethyl-l,3-dioxolan-4- yl)ethoxy] methyl }phosphonate (m / z 297), was treated with 1.25 M HC1 in ethanol to remove the ketal protection. The reaction mixture was evaporated to dryness to give the vicinal diol, diethyl [(3,4-dihydroxybutyl)methyl]phosphonate (m / z 257).

[0085] Diethyl [(3,4-dihydroxybutyl)methyl]phosphonate (1 mmol) was then tosylated attriethylamine and dibutyltin oxide (0.02 mmol) at room temperature in dichloromethane. The desired diethyl {3-hydroxy -4-[(4-methylbenzenesulfonyl)oxy]butoxy}methanephosphonate (m / z 411) was isolated by silica gel column chromatography.

[0086] The methylphosphonate derivative of adenine was prepared by stirringadenine (1 mmol) and sodium hydride (1.5 mmol, 60% suspension in mineral oil) in tetrahydrofuran (5 mL) for 5 minutes, adding diethyl {3-hydroxy-4-[(4-methylbenzene- sulfonyl)oxy]butoxy}methanephosphonate (10 mmol), and refluxing the mixture overnight to complete the reaction. Water (10 mL) was added, and the reaction mixture extracted with ethyl acetate. The ethyl acetate layer was evaporated to dryness. Chromatographic purification provided protected diethyl {[4-(6-amino-9H-purin-9-yl)-3-hydroxybutoxy]methyl}phosphonate. The Boc protection was removed by treatment of the purified product (100 mg) with 4 N HC1 in dioxane at room temperature for several hours at room temperature. The title product (m / z 374) was obtained as a white solid (28.5 mg).Diethyl {[4-(6-amino-9H-purin-9-yl)-3-hexanoyloxybutoxy]methyl}phosphonate (Example 13)

[0087] By the method used in Example 11,9H-purin-9-yl)-3-hydroxybutoxy]methyl}phosphonate, prepared as above, was reacted with hexanoyl chloride to provide the title compound.

[0088] A suspension of uracil (1 mmol) and sodium hydride (1.5 mmol) in N,N- dimethylformamide (1 mL) is stirred at room temperature for 5 minutes. Diethyl {3- hydroxy-4-[(4-methylbenzenesulfonyl)oxy]butoxy}methanephosphonate (1 mmol) in N,N- dimethylformamide (1 mL) is added and the mixture is stirred at 70-110°C for 48 hours. The reaction was quenched by addition of methanol, the solvent was evaporated, and the residue column chromatographed over silica gel. Elution with 7.5% methanol in ethyl acetate provided the title compound as a white solid (100 mg), m / z 351 (M+l), 373 (M+Na), 349 (M-l).

[0089] By the method used in Example 11, diethyl { [4-(2,4-dioxo- 1,2,3, 4-tetrahydro- pyrimidin-l-yl)-3-hydroxybutoxy]methyl}phosphonate, prepared as above, was reacted with hexanoyl chloride to provide the title compound.

[0090] Compound (1 mmol) was dissolved in N,N-dimethylacetamide (2.5 mL) in a dry round bottomed flask under nitrogen. Sodium / -butoxide (1.2 mmol) was added and the mixture stirred at room temperature for 5 minutes. Diisopropyl bromomethylphosphonate (1.1 mmol) was added and the reaction mixture stirred at 50°C. After 4 hours at 50°C additional sodium / -butoxide (1.2 mmol) was added and the reaction mixture stirred 50°C for 1 hour more. After a total of 5 hours at 50°C, mass spectral analysis indicated completion of the reaction, and the reaction mixture was cooled to room temperature and quenched with water (10 mL). The pH was adjusted to 7-7.5 with 1 N aqueous hydrochloric acid, and the solution extracted with three portions of ethyl acetate. The organic layer was washed with water (lx). The ethyl acetate layer was acidified with 1 N aqueous hydrochloric acid and extracted with water (3x) to extract the product as the HC1 salt. The aqueous phase was brought up to pH 7-7.5 with sodium bicarbonate and extracted with ethyl acetate (3x) followed by dichloromethane (lx). The mixed dichloromethane-ethyl acetate phases were evaporated under reduced pressure to give the title compound (65 mg, m / z 410).Acyclovir Esters

[0091] Various ester derivatives of the primary alcohol of acyclovir, from butanoic to palmitic esters as well as benzoate and trimethoxybenzoate esters, were prepared to investigate the efficiency of encapsulation and their antiviral properties. Acyclovir was dissolved in dry DMSO to give about a 10-20 % solution. To this solution, a 5-10 mole % of 4-dimethylaminopyridine (DMAP) was added followed by 1.1 to 2 molar equivalents of the selected acid anhydride. The solution was stirred until the reaction was complete by mass spectral analysis. Water was added to precipitate the acyclovir ester as a white solid, which was washed with water and acetone. Yields were generally >70%.Acyclovir octanoate (Example 15)

[0092] In a 150 ml dry round bottomed flask, acyclovir (2 g, 8.9 mmol) was dissolved in 30 ml of dry DMSO at 20-30°C. To the solution was added DMAP (0.1 g. 0.9 mmol) followed by octanoic anhydride (3.6 g, 13. 3 mmol). The reaction mixture was stirred at room temperature for 1-2 h until the TLC and mass spectrum showed near absence of acyclovir. Water (300 ml) was slowly added to precipitate the product as a white waxy solid. The product was crystallized from acetone to give the octanoate ester (2.25 g, 72% of theory).Remdesivir diacetate

[0093] In a dry reaction flask with drying tube and stir bar, remdesivir (1.2 g, 2 mmol) was dissolved in DMSO (5 mL) to give a clear solution at room temperature. DMAP (25 mg, -0.2 mmol) was added, and the solution stirred until clear. Acetic anhydride (0.214 g, -0.20 ml, 2.1 mmol) was added, and the reaction followed by TQ-MS and TLC. The reaction was complete in less than 1 hour. The reaction mixture was quenched with water, and the white precipitate washed with water to remove acetic acid.Remdesivir dibutanoate (Example 16)

[0094] In a dry reaction flask with drying tube and stir bar, remdesivir (1.2 g, 2 mmol) was dissolved in DMSO (5 mL) to give a clear solution at room temperature. DMAP (25 mg, -0.2 mmol) was added, and the solution stirred until clear. Butyric anhydride (332 mg, 2.1 mmol) was added, and the reaction followed by TQ-MS and TLC. The reaction was complete in less than 1 hour. The reaction mixture was quenched with water, and the white precipitate washed with water.Encapsulation of remdesivir (Example 17)

[0095] A 10-20 % w / w solution of P10M2-DTT-C16-(M-MSA) polymer (4) was prepared in ethyl alcohol. Solid remdesivir, 5 to 20 % by weight of polymer used, was added to the polymer solution. The mixture was well stirred to dissolve remdesivir and then evaporated to dryness under nitrogen at 35-60°C until constant weight was observed. The dried material was then dissolved in PBS or water at pH 6-7, and filter-sterilized for further use. The concentration of remdesivir was determined by HPLC or UV analysis.Efficacy in cell cultures

[0096] For compounds that were soluble in ethanol, the compound (guest) was dissolved in ethanol. It was mixed with the P10M2DT(HDA)x(M-MSA)y polymer (host) solution in ethanol in a guesthost ratio of 1:20 by weight. The solution was evaporated under nitrogen in an oven at about 50°C. The resulting dry film was then redissolved in PBS containing 5% ethanol, and used for the study. It should be noted that some substances did not dissolve properly in lxPBS, settling out upon refrigeration. Ethanol was added to all samples for purposes of uniformity in the study.

[0097] In normal drug formulations, ethanol can be completely avoided. Also, formulations can be made in osmotically balanced solutions, pH adjusted solutions, as the case may be for the different routes of drug administration. For example, physiologically balanced solutions containing mannitol, sodium chloride, or other osmolality balancing agents can be made for injection, infusion, or inhalation purposes. For oral use, typically, a somewhat acidic taste may be preferred, and sweeteners, taste masking agents, flavoring agents, etc. may be added without disturbing the encapsulation.

[0098] For compounds that were not sufficiently soluble in ethanol, the guest compound was dissolved in DMSO and mixed with a P10M2DT(HDA)x(M-MSA)y host polymer solution in DMSO in a ratio of 1 :20. The resulting DMSO solution was lyophilized. The lyophilized powder was then dissolved in PBS containing 5% ethanol as above.

[0099] Host polymer and remdesivir (RDV) served as positive controls; with PBS and DMSO as vehicle controls.The drug and prodrug compounds, in DMSO solution or encapsulated in P10M2DT(HDA)x(M- MSA)y nanoparticles dissolved in PBS buffer, were exposed to cultured MRC5 lung fibroblast cells (ATCC CCL-171) in a cell culture plate virally infected by hCoV-229E at different drug concentrations. The polymeric micelle material P10M2DT(HDA)x(M-MSA)y itself has anti- coronavirus activity of its own, and was also employed in PBS buffer as a positive control. Remdesivir, a well-known approved SARS-CoV-2 therapeutic drug with broad-spectrum anti- coronavirus activity, was also used as positive control. The solution vehicle (PBS) was used as negative control. Remdesivir was dissolved in DMSO due to its poor water solubility, and DMSO was also used as a negative control (results not shown). The improvement in cell survival, which is correlated with the reduction in growth of the virus, was read out using the CellTiter-Glo™ assay (Promega Corp., Madison, WI, USA.)Cytotoxicity in cell cultures

[0100] Encapsulated and non-encapsulated compounds were applied to the cell cultures without viral infection, and cell survival was read out using the same CellTiter-Glo™ assay.Results

[0101] Figure 4 plots cell survival against non-encapsulated compound concentrations, and shows the selected compounds’ efficacy as antivirals. Almost all of the compounds showsubstantial concentration-dependent antiviral activity that merits further study.

[0102] Figure 5 plots cell survival against the concentration of host polymer, and shows the compounds’ efficacy as antivirals when encapsulated into P10M2DT(HDA)x(M-MSA)y polymer (4) nanoparticles at a 1:20 ratio. The efficacy of some of these encapsulated compounds, in terms of maximal survival improvement, exceeds that of remdesivir (Example 17). In particular, Examples 10, 11, 12 and 13 show a substantial increase in effectiveness compared to the host polymer (4) itself, and are found to be comparable to or superior to remdesivir.

[0103] Figure 6 plots cell survival against the concentration of encapsulated guest compounds. The plots indicate improvements in survival with increasing amounts of compounds, in contrast to the toxicity of remdesivir (Ex. 17) at increasing concentrations.

[0104] Figure 7 compares the efficacies of encapsulated and non-encapsulated compounds when plotted as guest compound concentrations. The plots clearly indicate significant improvements in the effective activities of the guest compounds upon encapsulation.

[0105] Figure 8 plots uninfected cell survival against encapsulated compound host concentrations. The plots show the high cytotoxicity of encapsulated remdesivir, and the relative non-toxicity of the drugs and prodrugs tested.

[0106] All of the tested compounds, other than remdesivir, were non-toxic up to 200

[0107] Although remdesivir showed superior maximum viral inhibition and at lower concentrations than the synthesized and encapsulated drugs of this invention, its effectiveness rapidly decreases at slightly higher concentrations due to cytotoxicity. Clinical studies of Remdesivir have shown that its clinical effect was limited yet increasing concentrations is not an option due to its toxicity.

[0108] The examples above are non-limiting, representative examples only. The invention contemplates all combinations of known prodrugs, including but not limited to ester, carbonate, ether and carbamate prodrugs, with any known antiviral drugs that are amenable to such derivatization, and the use of any such prodrugs in combination with any of the comb polymers of the invention.

Claims

CLAIMSWe claim:

1. A method for the treatment or prevention of an infection of an animal by a virus, which comprises administering to said animal a comb polymer having the following structure wherein:each group X is individually OH or NHR, where R is a C10-C18 hydrophobic moiety; between 10% and 90% of groups X are OH; each group L is independently OH or a ligand having specific binding affinity for the surface of said virus; the average value of m ranges from 10 to 100; and the average value of n ranges from 5 to 25.

2. The method of claim 1, wherein said comb polymer is in the form of self-assembled core-corona nanoparticles, the cores of said nanoparticles having within them an antiviral drug or a prodrug thereof.

3. The method of claim 1, wherein each group L is a ligand selected from the group consisting of4. The method of claim 3, wherein said comb polymer is in the form of a self-assembled core-corona nanoparticle, and further comprises an antiviral drug or a prodrug thereof contained within said nanoparticle.

5. The method of any one of claims 1-4, wherein the virus is a coronavirus.

6. The method of any one of claims 1-4, wherein the virus is SARS-CoV-2.p g8. A comb polymer having the following structurewherein: each group X is individually OH or NHR, where R is a C10-C18 hydrophobic moiety; between 10% and 90% of groups X are OH; each group L is independently OH or a ligand having specific binding affinity for the surface of said virus; the average value of m ranges from 10 to 100; and the average value of n ranges from 5 to 25.

9. The comb polymer of claim 8, wherein said comb polymer is in the form of self- assembled core-corona nanoparticles, further comprising an antiviral drug or a prodrug thereof contained within said nanoparticles.

10. The comb polymer of claim 7, wherein each group L is a ligand selected from the group consisting ofwherein each R1 is individually H or C1-C4 alkyl, and each R2 is individually H, COR1, or C02R1.

11. The comb polymer of claim 10, wherein said comb polymer is in the form of self- assembled core-corona nanoparticles, further comprising an antiviral drug or a prodrug thereof contained within said nanoparticles.

12. The comb polymer of claim 8, wherein the antiviral drug or prodrug thereof is selected from the group consisting of remdesivir, GS-441524, 3 ’-deoxy adenosine, acyclovir, molnupiravir, [4-(6-amino-9H-purin-9-yl) cyclopent-2-enyl]methanol, 2-(6-amino-9H-purin- 9-yl)-4-hydroxybutanamide, diethyl {[4-(6-amino-9H-purin-9-yl)-3-hydroxybutoxy]methyl} phosphonate, diethyl {[4-(2,4-dioxo-l,2,3,4-tetrahydropyrimidin-l-yl)-3-hydroxybutoxy] methyl }phosphonate, PF-00835231, nirmatrelvir, ritonavir, ivermectin, colchicine, mebendazole, CDI-45205, and GC-376, and prodrugs thereof.

13. The comb polymer of claim 11, wherein the antiviral drug or prodrug thereof is selected from the group consisting of remdesivir, GS-441524, 3 ’-deoxy adenosine, acyclovir, molnupiravir, [4-(6-amino-9H-purin-9-yl) cyclopent-2-enyl]methanol, 2-(6-amino-9H-purin- 9-yl)-4-hydroxybutanamide, diethyl {[4-(6-amino-9H-purin-9-yl)-3-hydroxybutoxy]methyl} phosphonate, diethyl {[4-(2,4-dioxo-l,2,3,4-tetrahydropyrimidin-l-yl)-3-hydroxybutoxy] methyl} phosphonate, PF-00835231, nirmatrelvir, ritonavir, ivermectin, colchicine, mebendazole, CDI-45205, and GC-376, and prodrugs thereof.

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