WO2011057017A1 - Aqueous nanoparticle suspensions for use in drug discovery - Google Patents

Abstract

Methods for evaluating potential active agents are disclosed. A feed stock including a potential active agent and an amphiphilic polymer in a water-miscible solvent is provided, the potential active agent and polymer each being poorly water soluble. The feed stock is combined with an aqueous solution to produce a suspension of nanoparticles that include the potential active agent and the polymer. A liquid-phase assay is performed with an aliquot of the suspension to determine a pre-selected property of the potential active agent. In some embodiments, the polymer is a substituted dextran, such as dextran acetate, dextran propionate, dextran butyrate, dextran isobutyrate, or a mixture thereof. In certain embodiments, the amphiphilic polymer is a functionalized oxidized dextran with at least one oxidized group and an alkyl ester substituent selected from acetate, propionate, butyrate, isobutyrate, and mixtures thereof. In some embodiments, the polymer is a substituted cellulosic.

Description

AQUEOUS NANOPARTICLE SUSPENSIONS FOR USE IN DRUG DISCOVERY

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of United States Provisional Patent Application No. 61/258,690, filed November 6, 2009, and United States Provisional Patent Application No. 61/316,767, filed March 23, 2010, each of which is incorporated herein in its entirety by reference.

FIELD

Embodiments of methods for evaluating potential active agents using an aqueous nanoparticle suspension including an active agent and an amphiphilic polymer are disclosed.

BACKGROUND

Discovery compounds, also referred to herein as potential active agents, are usually stored in dimethylsulfoxide (DMSO) without other additives, and are diluted approximately 1- to 200-fold (to about 50 μg/mL) in an aqueous medium when used in a screening assay (biochemical or cellular) to identify compounds for further study. Poorly water-soluble discovery compounds can precipitate in the now predominantly aqueous media leading to false-negative assay outcomes. Discovery compounds are evaluated for potency in sensitive biochemical assays, so any materials used to improve compound solubility must not impact the assay.

SUMMARY

Embodiments of methods for evaluating potential active agents are disclosed. In one embodiment, a feed stock is provided that includes a potential active agent and an

amphiphilic polymer in a water-miscible solvent, the potential active agent and polymer each being poorly water soluble. As defined herein, a poorly water soluble potential active agent has a solubility of less than 5 mg/mL in water at a temperature of 25°C. As defined herein, a poorly water soluble polymer has a solubility of less than 0.1 mg/mL in water at a temperature of 25°C. The feed stock is combined with an aqueous solution, thereby producing a suspension that includes the potential active agent and the polymer. A liquid- phase assay is then performed with an aliquot of the suspension to determine a pre-selected property of the potential active agent, wherein the pre-selected property depends at least in part upon a dissolved concentration of the potential active agent in the liquid-phase assay. In some embodiments, an aliquot of the suspension provides an increased dissolved

concentration of the potential active agent in the liquid-phase assay compared to an aliquot of an aqueous solution of the potential active agent without the polymer. In other embodiments, the suspension comprises nanoparticles that include the potential active agent and the polymer. In one embodiment, the suspension has a potential active agent to polymer mass ratio of from 1:5 to 1:20.

A feed stock may be formed, for example, by providing a first solution comprising a potential active agent in a first water-miscible solvent, providing a second solution comprising an amphiphilic polymer in a second water-miscible solvent, and combining the first and second solutions to produce the feed stock. Alternatively, the amphiphilic polymer may be added directly to the potential active agent solution (i.e., the first solution).

In some embodiments, the first water-miscible solvent and the second water-miscible solvent are selected independently from dimethylsulfoxide (DMSO), N-methyl pyrrolidinone (NMP), dimethylacetamide (DMAC), dimethylformamide (DMF), acetone, methanol, ethanol, isopropanol, N-propanol, tetrahydrofuran (THF), ethylene glycol, propylene glycol, glycerol, and mixtures thereof. In other embodiments, the first water-miscible solvent and the second water-miscible solvent are selected independently from DMSO, NMP, DMAC, DMF, and mixtures thereof. In other embodiments, at least one of the first water-miscible solvent and the second water-miscible solvent is DMSO.

In some embodiments, the first water-miscible solvent and the second water-miscible solvent are the same. In other embodiments, the first water-miscible solvent and the second water-miscible solvent are different.

In some embodiments, the amphiphilic polymer is a substituted polysaccharide polymer, such as a substituted cellulosic or substituted dextran.

In some embodiments, the amphiphilic polymer is a substituted dextran comprising a substituent selected from acetate, propionate, butyrate, isobutyrate, succinate, and mixtures thereof. In some embodiments, the amphiphilic polymer is selected from dextran acetate, dextran propionate, dextran butyrate, dextran isobutyrate, dextran succinate, dextran acetate propionate, dextran acetate succinate, dextran propionate succinate, dextran acetate propionate succinate, dextran butyrate succinate, dextran isobutyrate succinate, and mixtures thereof. In other embodiments, the amphiphilic polymer is selected from dextran acetate, dextran propionate, dextran acetate propionate, dextran acetate succinate, dextran propionate succinate, dextran acetate propionate succinate, and mixtures thereof. In other embodiments, the amphiphilic polymer may be dextran acetate, dextran propionate, dextran butyrate, dextran isobutyrate, or a mixture thereof. In certain embodiments, the amphiphilic polymer is a functionalized oxidized dextran comprising (a) at least one oxidized group selected from aldehydes, ketones, and mixtures thereof, and (b) an alkyl ester substituent, wherein said alkyl ester substituent is selected from acetate, propionate, butyrate, isobutyrate, and mixtures thereof.

In some embodiments, the amphiphilic polymer is a substituted cellulosic polymer. In certain embodiments, the amphiphilic polymer is selected from methylcellulose,

ethylcellulose, propylcellulose, butylcellulose, cellulose acetate, cellulose propionate, cellulose butyrate, cellulose acetate butyrate, cellulose acetate propionate, methyl cellulose acetate, methyl cellulose propionate, methyl cellulose butyrate, ethyl cellulose acetate, ethyl cellulose propionate, ethyl cellulose butyrate, low-substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate, hydroxypropyl methylcellulose propionate, hydroxypropyl methylcellulose butyrate, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose acetate phthalate, hydroxypropyl methylcellulose acetate trimellitate, ethylcellulose succinate, ethylcellulose phthalate, ethylcellulose trimellitate, carboxymethyl ethylcellulose, cellulose acetate propionate succinate, cellulose acetate succinate, cellulose propionate succinate, cellulose acetate phthalate, carboxymethylcellulose acetate butyrate, and mixtures thereof.

In some embodiments, the amphiphilic polymer is a selected from hydroxypropyl methylcellulose acetate succinate (HPMCAS), hydroxypropyl methylcellulose acetate phthalate (HPMCAP), hydroxypropyl methylcellulose acetate trimellitate (HPMCAT), ethylcellulose succinate (ECS), ethylcellulose phthalate (ECP), ethylcellulose trimellitate (ECT), carboxymethyl ethylcellulose (CMEC), cellulose acetate propionate succinate (CAPrS), cellulose acetate succinate (CAS), cellulose propionate succinate (CPrS), cellulose acetate phthalate (CAP), and carboxymethylcellulose acetate butyrate (CMCAB). In other embodiments, the amphiphilic polymer is selected from HPMCAS, HPMCAP, ECS, ECP, ECT, CAPrS, and CMCAB.

In some embodiments, the amphiphilic polymer is a substituted cellulosic polymer. In certain embodiments, the amphiphilic polymer is selected from methylcellulose,

ethylcellulose, propylcellulose, butylcellulose, cellulose acetate, cellulose propionate, cellulose butyrate, cellulose acetate butyrate, cellulose acetate propionate, methyl cellulose acetate, methyl cellulose propionate, methyl cellulose butyrate, ethyl cellulose acetate, ethyl cellulose propionate, ethyl cellulose butyrate, low-substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate, hydroxypropyl methylcellulose propionate, and hydroxypropyl methylcellulose butyrate. In other embodiments, the amphiphilic polymer is selected from ethyl cellulose, cellulose acetate, cellulose propionate, cellulose butyrate, and cellulose acetate butyrate. In certain embodiments the cellulosic polymer is a non-ionizable, poorly water soluble compound. In certain embodiments, the non-ionizable, poorly water soluble cellulosic polymer is ethyl cellulose.

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating crystallization of active agent in a conventional method of screening discovery compounds.

FIG. 2 is a schematic diagram illustrating formation of a nanoparticle suspension in one embodiment of the disclosed method for screening discovery compounds.

FIG. 3 is a graph of enzyme activity versus compound concentration comparing one embodiment of the disclosed method to a conventional method for screening a discovery compound. FIG. 4 is a graph of product/sum ratio versus log compound concentration, which illustrates that at least one embodiment of the disclosed amphiphilic polymers does not interfere with a biochemical assay.

FIG. 5 is a graph of product/sum ratio versus log compound concentration, which illustrates that at least one embodiment of the disclosed amphiphilic polymers does not interfere with a biochemical assay of staurosporine.

FIGS. 6-8 are transmission electron micrographs of aqueous nanoparticle suspensions prepared by an embodiment of the disclosed method.

FIG. 9 is a graph of activity versus concentration, comparing activity of an active agent to activity of an aqueous nanoparticle suspension of the active agent prepared by an embodiment of the disclosed method.

DETAILED DESCRIPTION

Discovery compounds are compounds that may be potentially useful as active agents. As used herein, by "active agent" is meant a drug, medicament, pharmaceutical, therapeutic agent, nutraceutical, or other compound that may be desired to be administered to a subject. Assays for screening discovery compounds, referred to herein as potential active agents, typically are performed in aqueous media. However, a poorly water-soluble potential active agent may precipitate under the screening assay conditions, producing a false negative result. As defined herein, a poorly water-soluble potential active agent has a solubility of less than 5 mg/mL in water at 25°C. A potential active agent can be evaluated more accurately if it is either soluble or suspended in the aqueous media of the screening assay. Disclosed herein are embodiments of methods for evaluating potential active agents. A potential active agent is combined with an amphiphilic polymer to produce a feedstock. When combined with an aqueous solution, the feedstock produces a suspension including the active agent and the amphiphilic polymer. As used herein, a suspension is a heterogeneous mixture in which small particles (e.g. , with an average diameter of less than 1 μιη) are dispersed substantially uniformly in a liquid. In some embodiments, the suspended particles are nanoparticles having an average diameter of less than or equal to 500 nm. Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term "about." Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that may depend on the desired properties sought and/or limits of detection under standard test conditions/methods. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word "about" is recited. I. Methods for Evaluating Potential Active Agents

Discovery compounds (i.e., potential active agents) often are poorly water soluble (i.e., solubility is less than 5 mg/mL in water at a temperature of 25 °C) and are stored in nonaqueous, water-miscible solutions, such as dimethyl sulfoxide (DMSO). Discovery compounds are screened in various biochemical or cellular assays to determine whether the compounds may be useful as active agents. FIG. 1 illustrates one embodiment of a conventional method for screening potential active agents. A potential active agent is stored as a non-aqueous, water-miscible solution 10. An aqueous assay media is added, producing a supersaturated solution 20 of the active agent. The active agent then crystallizes into a variety of forms and sizes 30. Inset 32 is an optical micrograph of a crystallized discovery compound. Small crystals dissolve more rapidly than large crystals, producing a solution with large crystals 40. The remaining large crystals dissolve very slowly. Hence, the solution typically contains undissolved crystals 50. Because the large crystals slow the dissolution rate of the active agent, it can be difficult to obtain an accurate determination of the concentration of active agent at which 50% of the activity is inhibited (the "IC₅₀") in a bioassay.

Embodiments of the disclosed method produce a suspension of small particles, as illustrated in FIG. 2. A potential active agent and an amphiphilic polymer are dissolved in a non-aqueous, water-miscible solution 60. In one embodiment, addition of an aqueous assay media precipitates nanoparticles 70, forming a nanoparticle suspension 80. Inset 82 is a transmission electron micrograph of precipitated nanoparticles formed when aqueous assay media is added to a non-aqueous solution of a potential active agent and an amphiphilic polymer. A schematic representation of a nanoparticle 90 shows that the nanoparticle has an insoluble, hydrophobic core 92 with tunable surface functionality 94 that allows the hydrophilic and hydrophobic properties of the nanoparticle to be tailored. For example, its properties can be tailored by adjusting the substituents (e.g. , acetate, propionate, butyrate, isobutyrate) of the amphiphilic polymer and/or adjusting the amount and/or ratio of the substituents on the polymer. The nanoparticles include both the potential active agent and the amphiphilic polymer. In contrast to conventional methods, a uniform or substantially uniform aqueous suspension of nanoparticles 100 is formed, which provides a constant, very rapid sourcing of free potential active agent at all dilutions.

When used in a biochemical assay, the nanoparticle suspension improves the dissolved concentration of free potential active agent by rapidly providing potential active agent as it is used in the bioassay. This results in a more meaningful IC₅₀ value, as shown in FIG. 3. Curve A illustrates enzyme activity in the presence of an active agent in a biochemical assay. Curve B illustrates enzyme activity in the presence of the potential active agent, where the potential active agent is combined with an amphiphilic polymer in the form of a nanoparticle suspension. Curve B shows that the active agent is effective at a much lower concentration than would be estimated from an assay in which the potential active agent was evaluated in the absence of the polymer.

Embodiments of the disclosed method have one or more of several advantages over conventional methods for evaluating potential active agents. First, the nanoparticles can source supersaturated concentrations of the potential active agent and rapidly re-source dissolved potential active agent as it is utilized/reacted in the assay. Poorly soluble compounds can be diluted in aqueous solution and assayed without the likelihood of precipitation leading to false-negative results. Second, certain embodiments of the disclosed method expand the accessible chemical space to include low solubility, high log P (P is the partition coefficient, i.e. , the ratio of concentrations of non-ionized solute between two immiscible solvents such as octane and H₂0), and/or high molecular weight compounds and minimize their effects on assay variability. Third, certain embodiments of the disclosed method provide a platform that enables rapid progression of low- solubility compounds into formulations suitable for the clinic or commercial manufacture.

In one embodiment, a method for evaluating a potential active agent comprises (a) providing a feed stock comprising a potential active agent and an amphiphilic polymer in a water-miscible solvent, the potential active agent and polymer each individually being poorly water soluble, (b) combining the feed stock with an aqueous solution, thereby producing a suspension comprising the potential active agent and the polymer, and (c) performing a liquid-phase assay with an aliquot of the suspension to determine a pre-selected property of the potential active agent, wherein the pre-selected property depends at least in part upon a dissolved concentration of the potential active agent in the liquid-phase assay.

In one embodiment, the poorly water soluble potential active agent has a solubility of less than 5 mg/mL in an aqueous solution (over a pH range of 5 to 8 at 25°C) used to perform a liquid-phase assay. As defined herein, a poorly water soluble polymer has a solubility of less than 0.1 mg/mL in water at a temperature of 25 °C. In one embodiment, the poorly water soluble polymer has a solubility of less than 0.1 mg/mL in phosphate-buffered saline at pH 6.5.

In one embodiment, the suspension comprises nanoparticles, the nanoparticles comprising the potential active agent and the polymer.

In one embodiment, the potential active agent and polymer are present in the suspension at a potential active agent:polymer mass ratio of from 9: 1 to 1:999. In another embodiment, the ratio is 1: 1 to 1: 100. In still another embodiment, the ratio is 1:2 to 1:50. In yet another embodiment, the ratio is 1:5 to 1:20. In still another embodiment, the ratio is 1: 10.

In one embodiment, a feed stock comprising a potential active agent and an amphiphilic polymer in a water-miscible solvent is combined with an aqueous solution in a solvent:aqueous solution volume ratio of from 1: 1 to 1: 100. In another embodiment, the ratio is 1: 10 to 1:50. In yet another embodiment, the ratio is 1:20.

In one embodiment, the feed stock is formed by (i) providing a first solution comprising a potential active agent in a first water-miscible solvent, (ii) providing a second solution comprising an amphiphilic polymer in a second water-miscible solvent, the polymer being poorly water soluble, and (iii) combining the first and second solutions to produce a feed stock. In one embodiment, the first water-miscible solvent and the second water- miscible solvent are the same. In another embodiment, the first water-miscible solvent and the second water-miscible solvent are different.

In another embodiment, the feed stock is formed by (i) providing a solution comprising a potential active agent in a first water-miscible solvent, (ii) providing an amphiphilic polymer, the polymer being poorly water soluble, and (iii) combining the solution and the amphiphilic polymer to produce a feed stock.

In another embodiment, the aliquot of the suspension provides an increased dissolved concentration of the potential active agent in the liquid-phase assay compared to an aliquot of an aqueous solution of the potential active agent without the polymer.

In yet another embodiment, the first water-miscible solvent and the second water- miscible solvent are selected independently from dimethyl sulfoxide (DMSO), n-methyl pyrrolidinone (NMP), dimethylacetamide (DMAC), dimethylformamide (DMF), acetone, methanol, ethanol, isopropanol, N-propanol, tetrahydrofuran (THF), ethylene glycol, propylene glycol, glycerol, and mixtures thereof. In another embodiment, the first water- miscible solvent and the second water-miscible solvent are selected independently from DMSO, NMP, DMAC, DMF, and mixtures thereof. In another embodiment, at least one of the first water-miscible solvent and the second water-miscible solvent is DMSO.

In one embodiment, the amphiphilic polymer is a substituted polysaccharide. For example, the amphiphilic polymer may be selected from substituted cellulosics and substituted dextrans. In another embodiment, the amphiphilic polymer is a substituted dextran comprising at least one substituent selected from acetate, propionate, butyrate, isobutyrate, succinate and mixtures thereof. In one embodiment, the amphiphilic polymer is selected from dextran acetate, dextran propionate, dextran butyrate, dextran isobutyrate, dextran succinate, dextran acetate propionate, dextran acetate succinate, dextran propionate succinate, dextran acetate propionate succinate, dextran butyrate succinate, dextran isobutyrate succinate, and mixtures thereof. In another embodiment, the amphiphilic polymer is selected from dextran acetate, dextran propionate, dextran acetate propionate, dextran acetate succinate, dextran propionate succinate, dextran acetate propionate succinate, and mixtures thereof. In another embodiment, the amphiphilic polymer is selected from dextran acetate, dextran propionate, dextran butyrate, dextran isobutyrate, and mixtures thereof. In another embodiment, the amphiphilic polymer is a substituted dextran as described in Section II below and WO 2010/102065 Al, the disclosure of which is incorporated herein by reference.

In still another embodiment, the amphiphilic polymer is a functionalized oxidized dextran comprising (a) at least one oxidized group selected from aldehydes, ketones, and mixtures thereof; and (b) an alkyl ester substituent, wherein said alkyl ester substituent is selected from acetate, propionate, butyrate, isobutyrate, and mixtures thereof. In another embodiment, the amphiphilic polymer is a functionalized oxidized dextran as described in

Section III below and U.S. Provisional Patent Application No. 61/224,358, filed July 9, 2009, the disclosure of which is incorporated herein by reference.

In one embodiment, the amphiphilic polymer is a substituted cellulosic. In another embodiment, the amphiphilic polymer is a substituted cellulosic selected from poorly water soluble versions of methylcellulose, ethylcellulose, propylcellulose, butylcellulose, cellulose acetate, cellulose propionate, cellulose butyrate, cellulose acetate butyrate, cellulose acetate propionate, methyl cellulose acetate, methyl cellulose propionate, methyl cellulose butyrate, ethyl cellulose acetate, ethyl cellulose propionate, ethyl cellulose butyrate, low-substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate, hydroxypropyl

methylcellulose propionate, hydroxypropyl methylcellulose butyrate, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose acetate phthalate, hydroxypropyl methylcellulose acetate trimellitate, ethylcellulose succinate, ethylcellulose phthalate, ethylcellulose trimellitate, carboxymethyl ethylcellulose, cellulose acetate propionate succinate, cellulose acetate succinate, cellulose propionate succinate, cellulose acetate phthalate, carboxymethylcellulose acetate butyrate, and mixtures thereof.

In another embodiment, the amphiphilic polymer is an ionizable, poorly water soluble cellulosic polymer. Exemplary ionizable, poorly water soluble cellulosic polymers include poorly water soluble versions of: hydroxypropyl methylcellulose acetate succinate, hydroxypropyl cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, carboxymethylethyl cellulose, cellulose acetate phthalate, cellulose acetate succinate, methyl cellulose acetate phthalate, hydroxypropyl methylcellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methylcellulose acetate trimellitate, ethyl cellulose succinate, ethyl cellulose phthalate, ethyl cellulose trimellitate, cellulose phthalate succinate, hydroxypropyl methylcellulose phthalate succinate, hydroxypropyl methylcellulose propionate trimellitate, hydroxypropyl methylcellulose propionate phthalate, and

hydroxypropyl methylcellulose propionate succinate. Further details of ionizable, poorly water soluble cellulosic polymers are described in WO 2009/010842 A2, the disclosure of which is incorporated herein by reference.

In still another embodiment, the amphiphilic polymer is an ionizable, poorly water soluble cellulosic polymer selected from poorly water soluble versions of hydroxypropyl methylcellulose acetate succinate (HPMCAS), hydroxypropyl methylcellulose acetate phthalate (HPMCAP), hydroxypropyl methylcellulose acetate trimellitate (HPMCAT), ethylcellulose succinate (ECS), ethylcellulose phthalate (ECP), ethylcellulose trimellitate (ECT), carboxymethyl ethylcellulose (CMEC), cellulose acetate propionate succinate (CAPrS), cellulose acetate succinate (CAS), cellulose propionate succinate (CPrS), cellulose acetate phthalate (CAP), carboxymethylcellulose acetate butyrate (CMCAB), and mixtures thereof. In another embodiment, the ionizable, poorly water soluble cellulosic polymer is selected from poorly water soluble versions of HPMCAS, ECS, ECP, ECT, CAPrS,

CMCAB, and mixtures thereof. In still another embodiment, the ionizable, poorly water soluble cellulosic polymer is HPMCAS. In another embodiment, the ionizable, poorly water soluble cellulosic polymer is ECS. In another embodiment, the ionizable, poorly water soluble cellulosic polymer is ECP. In another embodiment, the ionizable, poorly water soluble cellulosic polymer is ECT. In another embodiment, the ionizable, poorly water soluble cellulosic polymer is CAPrS. In another embodiment, the ionizable, poorly water soluble cellulosic polymer is CMCAB. In yet another embodiment, the ionizable, poorly water soluble cellulosic polymer comprises an ether-linked ethyl substituent, and an ether- or ester-linked ionizable substituent. In still another embodiment, the ionizable, poorly water soluble cellulosic polymer is selected from ethylcellulose succinate, ethylcellulose phthalate, and ethylcellulose trimellitate. In another embodiment, the amphiphilic polymer is a non-ionizable, poorly water soluble cellulosic polymer. Exemplary non-ionizable, poorly water soluble cellulosic polymers include poorly water soluble versions of: methylcellulose, ethylcellulose, propylcellulose, butylcellulose, cellulose acetate, cellulose propionate, cellulose butyrate, cellulose acetate butyrate, cellulose acetate propionate, methyl cellulose acetate, methyl cellulose propionate, methyl cellulose butyrate, ethyl cellulose acetate, ethyl cellulose propionate, ethyl cellulose butyrate, low- substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate, hydroxypropyl methylcellulose propionate, hydroxypropyl methylcellulose butyrate, and mixtures thereof. Further details of non-ionizable, poorly water soluble cellulosic polymers are described in WO 2008/149192 A2, the disclosure of which is incorporated herein by reference.

In another embodiment, the amphiphilic polymer is a non-ionizable, poorly water soluble cellulosic polymer selected from poorly water soluble versions of methylcellulose, ethylcellulose, propylcellulose, butylcellulose, cellulose acetate, cellulose propionate, cellulose butyrate, cellulose acetate butyrate, cellulose acetate propionate, methyl cellulose acetate, methyl cellulose propionate, methyl cellulose butyrate, ethyl cellulose acetate, ethyl cellulose propionate, ethyl cellulose butyrate, low-substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate, hydroxypropyl methylcellulose propionate, hydroxypropyl methylcellulose butyrate, and mixtures thereof. In another embodiment, the amphiphilic polymer is a non-ionizable, poorly water soluble cellulosic polymer selected from poorly water soluble versions of ethyl cellulose, cellulose acetate, cellulose propionate, cellulose butyrate, and cellulose acetate butyrate. In still another embodiment, the non- ionizable, poorly water soluble cellulosic polymer is ethyl cellulose.

In one embodiment, the solution of the potential active agent and the amphiphilic polymer in a water-miscible solvent is converted into an aqueous suspension of nanoparticles with the potential active agent dispersed in the dispersion polymer. A portion of the nanoparticle suspension is then used in a bioassay to measure the activity of the compound.

To provide meaningful results when screening potential active agents, the amphiphilic polymer itself should not interfere with the biochemical assay. For example, when the IC₅₀ concentration is significantly less than the potential active agent's solubility, the same IC₅₀ should be determined in the presence or absence of nanoparticles that include the amphiphilic polymer. In at least some embodiments, the disclosed polymers do not interfere with bioassays. FIG. 4 shows that placebo nanoparticles (i.e., nanoparticles comprised of polymer in the absence of a potential active agent) comprised of dextran propionate produced a substantially constant product/sum ratio over a range of concentrations typically used in drug screening assays. With reference to FIG. 4, the product/sum ratio is the fraction of a substrate that was acted upon by an enzyme. As shown in FIG. 5, staurosporine, a protein kinase inhibitor, demonstrated substantially similar results without polymer (curves labeled A) or formulated into nanoparticles with dextran propionate (curves labeled B).

Nanoparticle suspensions formed by embodiments of the disclosed methods are stable, i.e., the nanoparticles remain intact and discrete over a period of at least several hours. FIGS. 6-8 are transmission electron microscope (TEM) photographs of nanoparticle suspensions prepared by the disclosed method in 4% DMSO:H₂0. The nanoparticles are comprised of 10 wt active agent and 90 wt dextran polymer, relative to the total mass of active agent and polymer, and were photographed after 10 days at room temperature. In FIG. 6, the particle size was 133 + 72 nm initially, and 171 + 92 nm after nine days. In FIG. 7, the particle size was 145 + 80 nm initially, and 189 + 84 nm after nine days. In FIG. 8, the particle size was 166 + 100 nm initially, and 190 + 100 nm after nine days. II. Substituted Dextrans

Substituted dextrans are suitable amphiphilic polymers for use in embodiments of the disclosed method for screening potential active agents. In one embodiment, the dextran is a substituted dextran comprising at least one substituent selected from acetate, propionate, butyrate, isobutyrate, succinate, and mixtures thereof. Suitable substituted dextrans include dextran acetate, dextran propionate, dextran butyrate, dextran isobutyrate, dextran succinate, dextran acetate propionate, dextran acetate succinate, dextran propionate succinate, dextran acetate propionate succinate, dextran butyrate succinate, dextran isobutyrate succinate, and mixtures thereof.

Dextran is an a-D-l,6-glucose-linked glucan. It may have side-chains linked to the backbone of the dextran polymer, with the degree of branching being approximately 5%, and the branches being mostly 1-2 glucose units long. A fragment of the dextran structure is illustrated below.

Substituted dextrans are polymers formed by the derivatization of dextran with ester- linked groups. The groups ester-linked to the dextran may be acetate, propionate, butyrate, isobutyrate, or any combination thereof. A fragment of dextran propionate is illustrated below.

Methods for preparation of ester derivatives of carbohydrates are known. See for example Advances in Polymer Science, 205, Polysaccharides II, Edited by Dieter Klemm (Springer- Verlag, Berlin Heidelberg, 2006). Methods for the preparation of the dextran polymer derivatives of the present invention can be derived from such known methods. Specifically, the dextran polymer derivatives can be prepared as follows. The dextran is first dissolved in a suitable solvent system such as formamide, dimethyl formamide (DMF), or N- methylpyrrolidone (NMP), together with a base, such as pyridine or the sodium salt of the carboxylate corresponding to the alkyl group to be substituted. An anhydride of the alkyl group to be substituted onto the dextran backbone is then added to the mixture. The reaction mixture is then stirred at temperatures ranging from 0 to 100°C for a period of from

30 minutes to 72 hours. When the resulting dextran polymer derivative is poorly aqueous soluble, the reaction can then be quenched by adding water to precipitate the polymer. The resulting precipitate can be collected by filtration. Alternatively, the polymer can be isolated by extraction into a solvent, such as ethyl acetate or methylene chloride, and the extraction solvent removed, for example, by evaporation or spray drying. The polymer can be further rinsed, filtered and dried prior to use.

The degree of substitution of alkyl esters on the dextran polymer may be determined using standard techniques, such as nuclear magnetic resonance (NMR) analysis or high- performance liquid chromatography (HPLC). For example, 13 C NMR analysis may be used to determine the number of alkyl ester groups using the ratio of the peak area of the groups to the peak area of the anomeric carbon in the dextran ring.

III. Functionalized Oxidized Dextrans

Functionalized oxidized dextrans are suitable amphiphilic polymers for use in embodiments of the disclosed methods. For example, the amphiphilic polymer may be a functionalized oxidized dextran comprising (a) at least one oxidized group selected from aldehydes, ketones, and mixtures thereof; and (b) an alkyl ester substituent, wherein said alkyl ester substituent is selected from acetate, propionate, butyrate, isobutyrate, and mixtures thereof.

The dextran is "oxidized", meaning that the backbone of the dextran polymer contains an oxidized substituent. For example, one exemplary oxidized dextran is a polymer wherein one or more of the hydroxyl groups on the dextran backbone have been oxidized to form a carbonyl group such as a ketone, or an aldehyde. Exemplary oxidized structures include the following:

where each R independently is a hydrogen or a C₂ to C₄ alkyl ester group.

The presence of an oxidized group on the dextran may be confirmed by any conventional analytical technique, such as NMR or IR spectroscopy. For example, 13 C NMR analysis may be used to determine the number of oxidized groups using the ratio of the peak area of the oxidized groups (at between 190 and 210 ppm) to the peak area of the anomeric carbon in the dextran ring (at about 90 to 100 ppm). In one embodiment, the degree of substitution of the oxidized form (defined as the average number of oxidized groups per monomer) ranges from 0.01 to 1.0, from 0.05 to 0.8, or even from 0.1 to 0.5.

The oxidized dextran is "functionalized," meaning that the dextran has functional substituents attached to or incorporated into the dextran backbone. As used herein, the term "functional substituents" means a substituent that is attached to the dextran backbone that contains a moiety that affects the physical or chemical properties of the polymer. Exemplary functional substituents include alkyl esters and methylthiomethyl ether substituents.

Exemplary functional groups incorporated into the dextran backbone include alkene groups.

In one embodiment, the functionalized oxidized dextran comprises alkyl ester substituents. By "alkyl ester" substituents is meant that the functionalized oxidized dextran has one or more alkyl group esters linked through one or more hydroxyl groups on the oxidized dextran. In one embodiment, the alkyl ester is selected from acetate, propionate, butyrate, isobutyrate, and mixtures thereof. In another embodiment, the alkyl ester is selected from acetate, propionate, and mixtures thereof. In another embodiment, the alkyl ester consists of a mixture of acetate and propionate.

It should be noted that in the polymer nomenclature used herein, ester- linked substituents are recited after the dextran backbone as the carboxylate. For example, the dextran polymer "dextran propionate" has a propionic acid ester-linked to the dextran backbone. An exemplary structure of an oxidized dextran propionate is as follows:

It should also be noted that a polymer name such as "oxidized dextran propionate" refers to any of the family of oxidized dextrans that have propionate groups attached via ester linkages to a significant fraction of the oxidized dextran' s hydroxyl groups. Generally, the degree of substitution of the alkyl ester can range from 0.01 to 2.95 as long as the other criteria of the polymer are met. "Degree of substitution" or "DS" refers to the average number of functional groups substituted onto each monomer of the dextran polymer. For example, in the oxidized dextran propionate shown above, the average degree of substitution of the oxidized groups is 1.0, the average degree of substitution of the propionate esters is 1.5, and the average degree of substitution of hydroxyl groups is 0.5.

The degree of substitution of the alkyl ester group may range from 0.01 to 2.95. In one embodiment, the degree of substitution of the alkyl ester group is from 1 to 2.95. In another embodiment, the degree of substitution is from 1.5 to 2.8. In still another embodiment, the degree of substitution is from 1.7 to 2.7.

In one embodiment, the alkyl ester group is propionate with a degree of substitution of from 1.5 to 2.8; in another embodiment the degree of substitution is from 1.7 to 2.7. In another embodiment, the alkyl ester group is a mixture of acetate and propionate, with a combined degree of substitution of from 1.5 to 2.8, or even a combined degree of substitution of from 1.7 to 2.7.

In another embodiment, the functionalized oxidized dextran also contains a methylthiomethyl (CH₃-S-CH₂-) substituent that is attached to the dextran backbone through an ether link. The methylthiomethyl substituent may be attached at any one or more of the hydroxyl groups on the dextran backbone. An exemplary structure is as follows:

The degree of substitution of methylthiomethyl-ether (MTME) groups may be determined using 13 C NMR analysis, with the MTME groups showing peaks at about 15 ppm. The degree of substitution of MTME groups may also be determined by elemental analysis. The degree of substitution of MTME groups may range from 0.01 to 1.0, from 0.03 to 0.8, or even from 0.05 to 0.5.

In another embodiment, the functionalized oxidized dextran also contains an alkene substituent. By "alkene substituent" is meant a group that contains a carbon-carbon double bond. The alkene substituent may be present in the dextran repeating unit. An exemplary structure of an oxidized dextran also containing an alkene substituent is as follows:

The degree of substitution of alkene groups may be determined using C NMR analysis, with the alkene groups showing peaks in the 110 to 150 ppm range. The degree of substitution of alkene groups may range from 0.01 to 1.0, from 0.03 to 0.8, or even from 0.05 to 0.5. In one embodiment, the f Formula 1 :

Formula 1

wherein R_1; R₂, and R may be the same or different, and are independently selected from

hydroxyl; carbonyl;

wherein R₄ is a linear or branched d to C₃ alkyl group; and

0-CH₂-S-CH₃.

The dextran used to form the functionalized oxidized dextran has a molecular weight that may range from 1,000 to 1,000,000 daltons. In one embodiment, the dextran used to form the functionalized oxidized dextran has a molecular weight of from 1,000 to 70,000 daltons. In another embodiment, the dextran used to form the functionalized oxidized dextran has a molecular weight of from 1,000 to 20,000 daltons. The resulting dextran alkyl ester may have a molecular weight ranging from 1,400 to 2,000,000 daltons, from 1,400 to 150,000 daltons, or even from 1,400 to 50,000 daltons.

In one embodiment, the functionalized oxidized dextran is poorly water soluble, i.e., it has a solubility of less than 0.1 mg/mL in water at 25°C. In another embodiment, the functionalized oxidized dextran is poorly aqueous soluble. By "poorly aqueous soluble" is meant that when the polymer is added at a solids concentration of 0.2 mg/mL to a phosphate buffered saline solution (PBS) at pH 6.5 at ambient temperature, e.g., a temperature of 25°C, the polymer has a solubility of 0.1 mg/mL or less. By "solids concentration" is meant the amount of solid polymer added to the PBS solution to measure the solubility of the polymer in the PBS solution. An appropriate PBS solution is an aqueous solution comprising 20 mM sodium phosphate (Na₂HP0₄), 47 mM potassium phosphate (KH₂P0₄), 87 mM NaCl, and 0.2 mM KC1, adjusted to pH 6.5 with NaOH. Thus, in one embodiment, the functionalized oxidized dextran has a solubility of 0.1 mg/mL or less in a phosphate buffered saline solution consisting of an aqueous solution of 20 mM sodium phosphate (Na₂HP0₄), 47 mM potassium phosphate (KH₂P0₄), 87 niM NaCl, and 0.2 niM KC1, adjusted to pH 6.5 with NaOH, at ambient temperature.

A test to determine whether a polymer is poorly aqueous soluble may be performed as follows. The polymer is initially present in bulk powder form with average particle sizes of greater than 1 micron. The solid polymer alone is added to a PBS solution to achieve a solids concentration of 0.2 mg/mL polymer in the PBS solution. The PBS solution is stirred for approximately 1 hour at ambient temperature. Next, a nylon 0.45 μιη filter is weighed, and the polymer solution is filtered. The filter is dried overnight (e.g., 16 hours) at 40°C, and weighed the following morning. The amount of polymer dissolved is calculated from the amount of polymer added to the PBS solution minus the amount of polymer remaining on the filter (mg). In one embodiment, when administered at a solids concentration of 0.2 mg/mL to the PBS solution at ambient temperature, the polymer has a solubility of less than

0.09 mg/mL, less than 0.07 mg/mL, less than 0.05 mg/mL, less than 0.03 mg/mL, or even less than 0.01 mg/mL.

The polymer solubility depends, in part, on the ratio of hydrophobic groups (e.g., alkyl ester groups) to hydrophilic groups (e.g., hydroxyl groups) present in the polymer. In order to be poorly water soluble, the functionalized oxidized dextran generally has a sufficient degree of substitution of the alkyl ester groups relative to the hydroxyl groups to limit the water solubility of the polymer. In general, the alkyl ester substituent is present at a degree of substitution of at least 1.0, i.e., on average at least one of the three hydroxyl groups on each monomer of the dextran polymer is replaced with an alkyl ester substituent. For example, the alkyl substituent is present at a degree of substitution of at least 1.5, at least 1.8, 1.0 to 2.95, 1.5 to 2.8, or 1.7 to 2.7.

In one embodiment, a composition comprises functionalized oxidized dextran.

Generally, it is desired that the functionalized oxidized dextran be made and isolated as a relatively pure polymer. In one embodiment, the concentration of impurities in the functionalized oxidized dextran is less than 20 wt%, less than 15 wt%, less than 10 wt%, or less than 5 wt . Some synthetic procedures for making functionalized dextrans may introduce a small amount of oxidized dextran into the product as an impurity, typically through a side reaction. In such cases the properties of the polymer are primarily that of the functionalized dextran, rather than those of the functionalized oxidized dextran impurity. In another embodiment, a composition consists essentially of a functionalized oxidized dextran. In yet another embodiment, a composition consists of a functionalized oxidized dextran.

The dextran may be oxidized using a variety of methods. Exemplary methods include reacting the dextran with oxidizing agents selected from oxygen, hydrogen peroxide, sodium chlorite or bromite, periodic acid and periodates, lead(IV) acetate, nitrogen dioxide, cerium(IV) salts, iron nitrate and permanganate, and dimethylsulfoxide (DMSO).

An alkyl ester may be added to the dextran using procedures known in the art. For example, the dextran may be treated with an alkyl anhydride, an alkyl acid, or an alkyl acid chloride, in the presence of an appropriate catalyst (e.g., a base), resulting in the formation of the alkyl ester substituent.

Functionalized oxidized dextran may be prepared as follows. In one embodiment, the steps of adding the alkyl ester substituent to the dextran backbone and oxidizing the hydroxyl groups occur in a single reaction mixture. The dextran is first dissolved in dimethylsulfoxide (DMSO), together with a base, such as pyridine, tripropyl amine, or sodium propionate. An anhydride of the alkyl group to be substituted onto the dextran backbone is added to the mixture. The reaction mixture is stirred at temperatures ranging from -10 to 100°C for a period of from 5 minutes to 5 days or more. The mixture is then quenched with water. The polymer is isolated and then dissolved in a solvent such as acetone. The dissolved polymer is precipitated in water. Sodium chloride may be added with stirring until the mixture is saturated to salt out the polymer. The resulting precipitate is collected by filtration.

Alternatively, the polymer may be isolated by extraction from brine into a solvent, such as ethyl acetate or methylene chloride, and the extraction solvent removed, for example, by evaporation or spray drying. The polymer may be further rinsed, filtered and dried prior to use.

Thus, in one embodiment, the functionalized oxidized dextran is formed by a process comprising (1) dissolving dextran in an solvent system to form a first mixture, the solvent system comprising dimethylsulfoxide and a base; (2) adding an alkyl anhydride to the first mixture to form a second mixture, resulting in the formation of the oxidized dextran alkyl ester; and (3) recovering the oxidized dextran alkyl ester from the second mixture. Alternatively, the substitution of the alkyl group onto the dextran backbone may occur separately from the oxidation of the polymer. In this embodiment, the dextran is first dissolved in a suitable solvent system, such as formamide, dimethylformamide (DMF), or N- methylpyrrolidinone (NMP), together with a base, such as pyridine, tripropyl amine, or sodium propionate. The solvent system should be substantially free of DMSO, meaning that the solvent system should contain less than 1 wt DMSO. An anhydride of the alkyl group to be substituted onto the dextran backbone is then added to the mixture. The reaction mixture is stirred at temperatures ranging from 0 to 100°C for a period of from 30 minutes to 72 hours. The reaction is quenched by adding water and sodium chloride to precipitate the polymer. The polymer is then dissolved in a mildly oxidizing solvent system, such as DMSO and an alkyl anhydride such as acetic anhydride. The reaction is quenched by addition of water, and the polymer collected by precipitation or by extraction, as previously described.

Thus, in another embodiment, the functionalized oxidized dextran is formed by a process comprising (1) dissolving dextran in an solvent system to form a first mixture, the solvent system comprising a base, and being substantially free of dimethylsulf oxide; (2) adding an alkyl anhydride to the first mixture to form a second mixture, resulting in the formation of the dextran alkyl ester; (3) recovering the dextran alkyl ester from the second mixture; (4) dissolving the dextran alkyl ester in dimethylsulfoxide, together with an alkyl anhydride, to form a third mixture, resulting in the formation of the oxidized dextran alkyl ester; and (5) recovering the oxidized dextran alkyl ester from the third mixture.

IV. Potential Active Agents

Embodiments of disclosed method are used to screen discovery compounds and identify those compounds that may be potentially useful as active agents. The potential active agent may be a "small molecule," generally having a molecular weight of 2000

Daltons or less. The potential active agent may also be a potential "biological active agent." Biological active agents include proteins, antibodies, antibody fragments, peptides, oligonucleotides, vaccines, and various derivatives of such materials. In one embodiment, the potential active agent is a small molecule. In another embodiment, the potential active agent is a potential biological active agent. In still another embodiment, the potential active agent is a mixture of a small molecule and a potential biological active agent.

The potential active agent may be highly water soluble (i.e., greater than 100 mg/mL), sparingly water soluble (i.e., 5-30 mg/mL), or poorly water soluble (i.e., less than 5 mg/mL) at 25 °C. In one embodiment, the potential active agent is "poorly aqueous soluble," and the potential active agent has a solubility of less than 5 mg/mL in an aqueous solution (over the pH range of 5 to 8 at 25°C) used to perform a liquid-phase assay. In another embodiment, the potential active agent has a solubility of less than 5 mg/mL in an aqueous solution (over the pH range of 6.5 to 7.5 at 25°C) used to perform a liquid-phase assay. The potential active agent may have an even lower aqueous solubility, such as less than 1 mg/mL, less than 0.1 mg/mL, and even less than 0.01 mg/mL.

The potential active agent should be understood to include the nonionized form of the potential active agent, pharmaceutically acceptable salts of the potential active agent, or any other pharmaceutically acceptable forms of the potential active agent. By "pharmaceutically acceptable forms" is meant any pharmaceutically acceptable derivative or variation, including stereoisomers, stereoisomer mixtures, enantiomers, solvates, hydrates, isomorphs, polymorphs, pseudomorphs, neutral forms, salt forms and prodrugs.

Examples of classes of active agents include, but are not limited to, compounds for use in the following therapeutic areas: antihypertensives, antianxiety agents, antiarrythmia agents, anticlotting agents, anticonvulsants, blood glucose-lowering agents, decongestants, antihistamines, antitussives, antineoplastics, beta blockers, anti-inflammatories, antipsychotic agents, cognitive enhancers, anti-atherosclerotic agents, cholesterol-reducing agents, triglyceride-reducing agents, antiobesity agents, autoimmune disorder agents, anti-impotence agents, antibacterial and antifungal agents, hypnotic agents, anti-Parkinsonism agents, anti- Alzheimer's disease agents, antibiotics, anti-angiogenesis agents, anti-glaucoma agents, antidepressants, bronchodilators, glucocorticoids, steroids, and antiviral agents.

V. Nanoparticles

Nanoparticles formed by some embodiments of the disclosed methods comprise a potential active agent and an amphiphilic polymer such as a substituted cellulosic polymer, substituted dextran, or a functionalized oxidized dextran. By "nanoparticles" is meant a plurality of small particles in which the average size of the particles is less than or equal to 500 nm. In suspension, by "average size" is meant the effective cumulant diameter as measured by dynamic light scattering (DLS), using for example, Brookhaven Instruments' 90Plus particle sizing instrument (Brookhaven Instruments Corporation, Holtsville, NY). By "size" is meant the diameter if the particles were spherical particles, or the maximum diameter for non-spherical particles. In one embodiment, the average size of the

nanoparticles is less than 400 nm, less 300 nm, less than 200 nm, or even less than 100 nm. In another embodiment, the average size of the nanoparticles is 2 nm to 500 nm, 5 nm to 500 nm, 10 nm to 500 nm, 20 nm to 500 nm, or even 30 nm to 500 nm. In another embodiment, the nanoparticles range in size from 1 nm to 400 nm, from 1 nm to 300 nm, from 1 nm to 200 nm, from 10 nm to 400 nm, or from 30 nm to 400 nm.

The nanoparticles can exist in a number of different configurations. In one embodiment, the nanoparticles comprise a core, the core comprising the potential active agent and the polymer (e.g. , substituted dextran and/or functionalized oxidized dextran). As used herein, the term "core" refers to the interior portion of the nanoparticle. The nanoparticles also have a "surface portion," meaning the outside or exterior portion of the nanoparticle. Thus, the nanoparticles consist of a core (i.e., the interior portion) and a surface portion.

The potential active agent present in the core can exist in pure potential active agent domains (crystalline or non-crystalline), as a thermodynamically stable solid solution of noncrystalline potential active agent distributed throughout the polymer, as a supersaturated solid solution of non-crystalline potential active agent distributed throughout the polymer, or any combination of these states or those states that lie between them. The term "crystalline," as used herein, means a particular solid form of a compound that exhibits long-range order in three dimensions. "Non-crystalline" refers to material that does not have long-range three- dimensional order, and is intended to include not only material that has essentially no order, but also material that has some small degree of order, in which the order is in less than three dimensions and/or is only over short distances. Another term for a non-crystalline form of a material is the "amorphous" form of the material. When the glass-transition temperature (Tg) of the non-crystalline active agent is different from the Tg of the pure polymer by at least 20°C, the core may exhibit a Tg that is different from the Tg of pure non-crystalline active agent or pure polymer.

In one embodiment, less than 20 wt of the potential active agent is present in noncrystalline active agent domains, with the remaining potential active agent distributed throughout the polymer.

In one embodiment, the nanoparticles are homogeneous, meaning that the

composition on the surface of the nanoparticle is essentially the same as in the core of the nanoparticle. In such cases, the nanoparticles may comprise, in one embodiment, a solid amorphous dispersion. In another embodiment, the potential active agent is present as one or more amorphous or crystalline domains throughout each nanoparticle.

In another embodiment, at least 90 wt of the potential active agent in the nanoparticles is non-crystalline. In one embodiment, at least 95 wt of the potential active agent in the nanoparticle is non-crystalline; in other words, the amount of potential active agent in crystalline form does not exceed 5 wt . Amounts of crystalline active agent may be measured by Powder X-Ray Diffraction (PXRD), by Differential Scanning Calorimetry

(DSC), by solid state nuclear magnetic resonance (NMR), or by any other known quantitative measurement.

The non-crystalline potential active agent present in the core can exist in noncrystalline pure potential active agent domains, as a thermodynamically stable solid solution of non-crystalline potential active agent homogeneously distributed throughout the functionalized oxidized dextran, as a supersaturated solid solution of non-crystalline potential active agent homogeneously distributed throughout the functionalized oxidized dextran, or any combination of these states or those states that lie between them. When the glass- transition temperature (T_g) of the non-crystalline active agent is different from the T_g of the pure polymer by at least 20°C, the core may exhibit a T_g that is different from the T_g of pure non-crystalline active agent or pure polymer.

In one embodiment, less than 20 wt of the potential active agent is present in noncrystalline potential active agent domains, with the remaining potential active agent homogeneously distributed throughout the polymer. The potential active agent and polymer are collectively present in the nanoparticle in an amount ranging from 50 wt to 100 wt of the nanoparticle total mass. In one embodiment, the potential active agent and polymer collectively may constitute at least 50 wt%, at least 60 wt%, or even at least 80 wt of the nanoparticle. In one embodiment, the nanoparticles consist essentially of the potential active agent and the functionalized oxidized dextran. By "the nanoparticles consist essentially of is meant that the nanoparticle contains less than 1 wt of any other excipients and that any such excipients have substantially no effect on the performance or properties of the nanoparticle.

The amount of potential active agent in the nanoparticle may range from 0.1 wt to 90 wt . In one embodiment, the amount of potential active agent in the nanoparticle ranges from 1 wt to 80 wt%, from 5 wt to 75 wt%, from 5 wt% to 60 wt%, or even from 5 wt% to 50 wt%.

The amount of polymer may range from 10 wt% to 99.9 wt%. In one embodiment, the amount of polymer in the nanoparticle is at least 15 wt%, at least 20 wt%, or even at least 25 wt%. In another embodiment, the amount of polymer in the nanoparticle is 80% or less.

The mass ratio of potential active agent to polymer in the nanoparticle can range from 1:999 to 9: 1 (that is, from 0.1 wt% active agent to 90 wt% potential active agent relative to the total mass of active agent and polymer in the nanoparticle). In one embodiment, the mass ratio of potential active agent to polymer ranges from 1:99 to 4: 1 (that is, from 1 wt% to 80 wt% potential active agent relative to the total mass of potential active agent and polymer), from 1: 19 to 3: 1 (that is, from 5 wt% to 75 wt%), or even from 1: 10 to 1:5 (that is, from 9 wt% to 60 wt% potential active agent relative to the total mass of potential active agent and polymer in the nanoparticle). In one embodiment, the mass ratio of potential active agent to polymer is less than 9: 1, less than 4: 1, less than 3: 1, or even less than 3:2. In another embodiment, the mass ratio of active agent to polymer is at least 1:999, at least 1:99, or even at least 1: 10.

In one embodiment, the nanoparticles have the following amount of potential active agent and polymer:

5 to 50 wt%, or even 5 to 40 wt% potential active agent; and

40 to 95 wt%, or even 50 to 75 wt% polymer. Without further elaboration, it is believed that one of ordinary skill in the art can, using the foregoing description, utilize the present invention to its fullest extent. Therefore, the following specific embodiments are to be construed as merely illustrative and not restrictive of the scope of the invention. Those of ordinary skill in the art will understand that variations of the conditions and processes of the following examples can be used.

VI. EXAMPLES

Example 1

Nanoparticle Effect on Active Agent Activity

Synthesis of Oxidized Dextran Alkyl Ester Polymer

Nanoparticles were made containing oxidized dextran propionate acetate. To synthesize the oxidized dextran propionate acetate, dextran propionate was first made by dissolving 120.3 g of dextran having a molecular weight of 10,000 daltons (available from Pharmacosmos A/S; Holbaek, Denmark) in 453.6 g formamide in a glass reactor with a Teflon® mixer. The reactor was heated to 50 C, and 42.7 g sodium propionate and 254.0 g propionic anhydride was added to the reaction mixture.

To isolate the product, the reaction mixture was divided into four parts and poured into vessels containing 10 L water to precipitate the polymer. The polymer was recovered by filtering using a Buchner funnel, and washed again with water. The solids were combined and tray dried at 40°C for 14 hours.

Acetate groups were then added to the dextran propionate by dissolving 30.0 g polymer in 105 mL anhydrous DMSO in a round bottom flask, and adding 67.5 mL acetic anhydride. The reaction mixture was stirred at 25 °C for 27 hours. The reaction mixture was quenched by adding 60 mL aliquots to 750 mL of brine solution in a blender, thus

precipitating the polymer. The polymer was recovered and redissolved in 400 mL acetone. The polymer was stirred and precipitated again into brine solution, and rinsed with deionized water. The polymer was redissolved in 250 mL acetone, precipitated into brine, washed with deionized water, filtered and dried.

The dextran propionate acetate above was bleached using the following method. First, 22.62 g was dissolved in 160 mL glacial acetic acid. Next, 23 g sodium hypochlorite solution was added (10 - 13% chlorine; available from Sigma), and the mixture was stirred about 45 minutes. Aliquots were precipitated into 750 mL brine solution and filtered to collect the solids. The solids were added to 750 mL deionized water in a blender, and the precipitate was collected using filtration and dried overnight. The polymer was washed again using deionized water, filtered, and dried for about 3 days under vacuum. The bleaching process was repeated to obtain a white powder.

The degree of substitution (DS) of alkyl esters and oxidized groups on the polymer was determined using 13 C NMR analysis. Samples of the polymer were dissolved in deuterated DMSO at a concentration of 200 mg/mL, and analyzed overnight to obtain spectra with a high signal/noise ratio. NMR peaks were assigned to carbon positions, and integrated to determine peak areas. Alkyl ester or oxidized group concentrations were determined using the ratios of peak areas to the peak area of the anomeric carbon in the dextran ring. The results of this analysis show that the propionate DS was 2.17, acetate DS was 0.14, and the oxidized group DS was 0.37.

Nanoparticle Preparation

Nanoparticles were prepared as follows. First, 0.88 μΐ_^ DMSO was pipetted into the wells of a 96-well plate. Next, 1.62 μL· DMSO solution containing 75 mg/mL of the oxidized dextran propionate acetate described above was added to the wells, followed by 2.50 μΐ_^ DMSO solution containing 3.3 mg/mL kenpaullone (a low-aqueous solubility kinase inhibitor). Nanoparticles were formed by addition of 95 μΐ_^ purified water to each well. Nanoparticles containing kenpaullone were serially diluted in purified water to obtain a range of inhibition concentrations. Enzyme Activity Assay

Phosphopeptide solution, peptide/kinase (cyclin-dependent kinase, CDK2/cyclin A) solution, and ATP (adenosine-5'-triphosphate) were pipetted into the wells to perform a Z'- Lyte Assay (enzyme assay kit available from Invitrogen, Carlsbad, California). This assay is based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. In the primary reaction, the kinase transfers the gamma-phosphate of ATP to a single tyrosine, serine or threonine residue in a synthetic fluorescence resonance energy transfer (FRET) peptide. In the secondary reaction, a site-specific protease (the "Development Reagent") recognizes and cleaves non-phosphorylated FRET-peptides.

Phosphorylation of FRET-peptides suppresses cleavage by the Development Reagent.

Cleavage disrupts fluorescence resonance energy transfer between the donor (i.e., coumarin) and acceptor (i.e., fluorescein) fluorophores on the FRET-peptide, whereas uncleaved, phosphorylated FRET-peptides maintain fluorescence resonance energy transfer. A ratiometric method, which calculates the emission ratio of donor emission to acceptor emission after excitation of the donor fluorophore at 400 nm, is used to quantitate reaction progress.

For this assay, CDK2 concentration was 14 nM, ATP concentration was 25 μΜ, and the highest inhibitor (i.e., kenpaullone) concentration was 2.5 μΜ. The plates were incubated at room temperature for 60 minutes, then development reagent was added and incubation continued at room temperature for an additional 60 minutes. Following incubation, stop reagent was added and fluorescence was measured. Enzyme activity inhibition was calculated and reported as IC₅₀ (log of 50% inhibition concentration, M). Enzyme inhibition of kenpaullone in nanoparticles was compared to enzyme inhibition of kenpaullone alone. The results are shown in FIG 9.

FIG. 9 shows that, at higher inhibitor concentrations, the unformulated inhibitor reaches the solubility limit, resulting in less enzyme inhibition (curve A). In contrast, the nanoparticle inhibitor formulation improves inhibitor solubilization at higher concentrations, and thus allows increased enzyme inhibition (curve B). At the highest inhibitor concentration tested, the CDK2 enzyme activity was about 50% for the unformulated inhibitor, and about 25% with the inhibitor formulated as nanoparticles of the invention. Kenpaullone was calculated to have an IC₅₀ of 2.9 x 10^" M, whereas the nanoparticle formulation (dextran propionate acetate and kenpaullone) had an IC₅₀ of 5.0 x 10^" M.

In one embodiment, a method for evaluating a potential active agent comprises providing a feed stock comprising a potential active agent and an amphiphilic polymer in a water- miscible solvent, the potential active agent having a solubility of less than 5 mg/mL in water at a temperature of 25°C, and the polymer having a solubility of less than 0.1 mg/mL in water at a temperature of 25°C , combining the feed stock with an aqueous solution and producing a suspension comprising the potential active agent and the polymer, and performing a liquid-phase assay with an aliquot of the suspension to determine a pre-selected property of the potential active agent, wherein the pre-selected property depends at least in part upon a dissolved concentration of the potential active agent in the liquid-phase assay. In some embodiments, the feedstock is formed by providing a first solution comprising a potential active agent in a first water-miscible solvent, providing a second solution comprising an amphiphilic polymer in a second water-miscible solvent, and combining the first and second solutions to produce the feed stock.

In either of the above embodiments, the first water-miscible solvent and the second water-miscible solvent may be selected independently from dimethylsulfoxide (DMSO), N- methyl pyrrolidinone (NMP), dimethylacetamide (DMAC), dimethylformamide (DMF), acetone, methanol, ethanol, isopropanol, N-propanol, tetrahydrofuran (THF), ethylene glycol, propylene glycol, glycerol, and mixtures thereof. In some embodiments, the first water- miscible solvent and the second water-miscible solvent may be selected independently from dimethylsulfoxide (DMSO), N-methyl pyrrolidinone (NMP), dimethylacetamide (DMAC), dimethylformamide (DMF), and mixtures thereof. In certain embodiments, at least one of the first water-miscible solvent and the second water-miscible solvent is dimethylsulfoxide (DMSO).

In any or all of the above embodiments, the suspension may have a potential active agent to polymer mass ratio of from 1:5 to 1:20.

In any of all of the above embodiments, the amphiphilic polymer may comprise at least one substituted polysaccharide polymer. In any of all of the above embodiments, the amphiphilic polymer may be selected from substituted cellulosics and substituted dextrans. In any or all of the above embodiments, the suspension may comprise nanoparticles comprising the potential active agent and the polymer.

In any or all of the above embodiments, the amphiphilic polymer may be a substituted dextran comprising at least one substituent selected from acetate, propionate, butyrate, isobutyrate, succinate, and mixtures thereof. In some embodiments, the amphiphilic polymer may be selected from dextran acetate, dextran propionate, dextran butyrate, dextran isobutyrate, dextran succinate, dextran acetate propionate, dextran acetate succinate, dextran propionate succinate, dextran acetate propionate succinate, dextran butyrate succinate, dextran isobutyrate succinate, and mixtures thereof. In certain embodiments, the amphiphilic polymer may be dextran acetate, dextran propionate, dextran butyrate, dextran isobutyrate, or a mixture thereof.

Alternatively, in any or all of the above embodiments, the amphiphilic polymer may be a functionalized oxidized dextran comprising (a) at least one oxidized group selected from aldehydes, ketones, and mixtures thereof; and (b) an alkyl ester substituent, wherein said alkyl ester substituent is selected from acetate, propionate, butyrate, isobutyrate, and mixtures thereof.

Alternatively, in any or all of the above embodiments, the amphiphilic polymer may be a substituted cellulosic polymer. In some embodiments, the amphiphilic polymer may be selected from methylcellulose, ethylcellulose, propylcellulose, butylcellulose, cellulose acetate, cellulose propionate, cellulose butyrate, cellulose acetate butyrate, cellulose acetate propionate, methyl cellulose acetate, methyl cellulose propionate, methyl cellulose butyrate, ethyl cellulose acetate, ethyl cellulose propionate, ethyl cellulose butyrate, low-substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate, hydroxypropyl

methylcellulose propionate, hydroxypropyl methylcellulose butyrate, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose acetate phthalate, hydroxypropyl methylcellulose acetate trimellitate, ethylcellulose succinate, ethylcellulose phthalate, ethylcellulose trimellitate, carboxymethyl ethylcellulose, cellulose acetate propionate succinate, cellulose acetate succinate, cellulose propionate succinate, cellulose acetate phthalate, carboxymethylcellulose acetate butyrate, and mixtures thereof. In some embodiments, the amphiphilic polymer may be a substituted cellulosic polymer selected from hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose acetate phthalate, hydroxypropyl methylcellulose acetate trimellitate, ethylcellulose succinate, ethylcellulose phthalate, ethylcellulose trimellitate, carboxymethyl ethylcellulose, cellulose acetate propionate succinate, cellulose acetate succinate, cellulose propionate succinate, cellulose acetate phthalate, carboxymethylcellulose acetate butyrate, and mixtures thereof. In certain embodiments, the amphiphilic polymer may be selected from methylcellulose, ethylcellulose, cellulose acetate, cellulose propionate, cellulose butyrate, cellulose acetate butyrate, and mixtures thereof.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method for evaluating a potential active agent, comprising:

providing a feed stock comprising a potential active agent and an amphiphilic polymer in a water-miscible solvent, the potential active agent having a solubility of less than 5 mg/mL in water at a temperature of 25°C, and the polymer having a solubility of less than 0.1 mg/mL in water at a temperature of 25°C;

combining the feed stock with an aqueous solution and producing a suspension comprising the potential active agent and the polymer; and

performing a liquid-phase assay with an aliquot of the suspension to determine a preselected property of the potential active agent, wherein the pre-selected property depends at least in part upon a dissolved concentration of the potential active agent in the liquid-phase assay.

2. The method of claim 1 wherein said feed stock is formed by

providing a first solution comprising a potential active agent in a first water-miscible solvent;

providing a second solution comprising an amphiphilic polymer in a second water- miscible solvent; and

combining the first and second solutions to produce the feed stock.

3. The method of claim 1 or claim 2 where said first water-miscible solvent and said second water-miscible solvent are selected independently from dimethylsulfoxide (DMSO), N-methyl pyrrolidinone (NMP), dimethylacetamide (DMAC), dimethylformamide (DMF), acetone, methanol, ethanol, isopropanol, N-propanol, tetrahydrofuran (THF), ethylene glycol, propylene glycol, glycerol, and mixtures thereof.

4. The method of claim 3 wherein said first water-miscible solvent and said second water-miscible solvent are selected independently from dimethylsulfoxide (DMSO), N- methyl pyrrolidinone (NMP), dimethylacetamide (DM AC), dimethylformamide (DMF), and mixtures thereof.

5. The method of claim 3 wherein at least one of said first water- miscible solvent and said second water-miscible solvent is dimethylsulfoxide (DMSO).

6. The method of any one of the previous claims wherein the suspension has a potential active agent to polymer mass ratio of from 1:5 to 1:20.

7. The method of any one of the previous claims wherein said amphiphilic polymer comprises at least one substituted polysaccharide polymer.

8. The method of any one of the previous claims wherein said amphiphilic polymer is selected from substituted cellulosics and substituted dextrans.

9. The method of any one of the previous claims wherein said suspension comprises nanoparticles comprising the potential active agent and the polymer.

10. The method of any one of the previous claims wherein said amphiphilic polymer is a substituted dextran comprising at least one substituent selected from acetate, propionate, butyrate, isobutyrate, succinate, and mixtures thereof.

11. The method of claim 10 wherein said amphiphilic polymer is selected from dextran acetate, dextran propionate, dextran butyrate, dextran isobutyrate, dextran succinate, dextran acetate propionate, dextran acetate succinate, dextran propionate succinate, dextran acetate propionate succinate, dextran butyrate succinate, dextran isobutyrate succinate, and mixtures thereof.

12. The method of claim 10 wherein said amphiphilic polymer is dextran acetate, dextran propionate, dextran butyrate, dextran isobutyrate, or a mixture thereof.

13. The method of any one of claims 1-9 wherein said amphiphilic polymer is a functionalized oxidized dextran comprising (a) at least one oxidized group selected from aldehydes, ketones, and mixtures thereof; and (b) an alkyl ester substituent, wherein said alkyl ester substituent is selected from acetate, propionate, butyrate, isobutyrate, and mixtures thereof.

14. The method of any one of claims 1-9 wherein said amphiphilic polymer is a substituted cellulosic polymer.

15. The method of claim 14 wherein said amphiphilic polymer is selected from

methylcellulose, ethylcellulose, propylcellulose, butylcellulose, cellulose acetate, cellulose propionate, cellulose butyrate, cellulose acetate butyrate, cellulose acetate propionate, methyl cellulose acetate, methyl cellulose propionate, methyl cellulose butyrate, ethyl cellulose acetate, ethyl cellulose propionate, ethyl cellulose butyrate, low-substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate, hydroxypropyl methylcellulose propionate, hydroxypropyl methylcellulose butyrate, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose acetate phthalate, hydroxypropyl methylcellulose acetate trimellitate, ethylcellulose succinate, ethylcellulose phthalate, ethylcellulose trimellitate, carboxymethyl ethylcellulose, cellulose acetate propionate succinate, cellulose acetate succinate, cellulose propionate succinate, cellulose acetate phthalate, carboxymethylcellulose acetate butyrate, and mixtures thereof.

16. The method of claim 14 wherein said amphiphilic polymer is selected from

hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose acetate phthalate, hydroxypropyl methylcellulose acetate trimellitate, ethylcellulose succinate, ethylcellulose phthalate, ethylcellulose trimellitate, carboxymethyl ethylcellulose, cellulose acetate propionate succinate, cellulose acetate succinate, cellulose propionate succinate, cellulose acetate phthalate, carboxymethylcellulose acetate butyrate, and mixtures thereof.

17. The method of claim 14 wherein said amphiphilic polymer is selected from methylcellulose, ethylcellulose, cellulose acetate, cellulose propionate, cellulose butyrate, cellulose acetate butyrate, and mixtures thereof.