CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. Appln. No. 62/129, 140, filed 6 March 2015, the entirety of which application is incorporated herein by reference for all purposes.

FIELD OF THE INVENTION

The invention relates to co-drugs containing tocopheryloxyacetic acid esters of anticancer agents and antiproliferative agents.

BACKGROUND OF THE INVENTION

Tocopheryloxyacetic acid (TOA) belongs to a new family of anticancer agents: redox-silent derivatives of tocopherol {Zhao Y et al. Vitamin E analogues as

mitochondria-targeting compounds: from the bench to the bedside? Mol Nutr Food Res 2009; 53(1): 129- 139). It was synthesized and reported as a non-cleavable, hydrolytically stable analogue of tocopherol succinate, the best characterized compound in this series (Lawson KA et al. Novel vitamin E analogue decreases syngeneic mouse mammary tumor burden and reduces lung metastasis. Mol Cancer Ther 2003;2:437-444).

D-a-Tocopheryloxyacetic acid (TOA)

Although tocopheryloxyacetic acid and other tocopherol derivatives show promise as anticancer and antiproliferative agents, enhancing their effects with pharmacologically complementary compounds and enabling their effective delivery in active form remains a challenge and improvements in this respect would be welcome.

SUMMARY OF THE INVENTION

In some embodiments, the invention provides a compound including an ester of tocopheryloxyacetic acid with an anticancer or antirestenotic agent selected from the group consisting of SN38, paclitaxel, camptothecin, 7-ethylcamptothecin, etoposide, fenretinide, and lestaurtinib, wherein if the anticancer or antirestenotic agent includes at least two hydroxyl groups, the compound may optionally further include an ester group formed between a carboxylic acid and the anticancer or antirestenotic agent moiety in the compound.

In some embodiments, the invention provides an oil-in-water nanoemulsion wherein the emulsified oily phase includes an oil having dissolved therein a compound as described above. In some embodiments, the invention provides a nanoparticle including a compound as described above.

In some embodiments, the invention provides a method of treating a diagnosed medical condition in a patient, including administering to the patient one or more dosages of the compound, nanoparticles, dispersion of solid nanoparticles, or oil-in-water nanoemulsions described above, wherein the one or more dosages constitute an amount therapeutically effective to treat the medical condition.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the effect of SN38 co-drug-loaded nanoparticles on IMR-32 neuroblastoma cell growth according to the invention, compared with the effect of free SN38, measured 7 days post-treatment.

FIG. 2 shows growth profiles of two neuroblastoma cell types, IMR-32 and BE(2)- C, treated for 24 hours with SN38 in co-drug-loaded nanoparticles according to the invention, compared with untreated cells and cells treated with free SN38 and with blank nanoparticles as controls.

FIG. 3 shows particle size distributions of small (85 nm) and large ( 160 nm) sized polylactide-based nanoparticles containing a D-a-tocopheryloxyacetic acid conjugate with paclitaxel according to the invention.

FIG. 4 shows the antiproliferative effect of 160 nm nanoparticles loaded with D-a- tocopheryloxyacetic acid conjugate with paclitaxel (PTX), according to the invention.

FIG. 5 shows the antiproliferative effect of 85 nm nanoparticles loaded with D-a- tocopheryloxyacetic acid conjugate with paclitaxel (PTX), according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have found that tocopheryloxyacetic acid is uniquely suited for creating rapidly activatable ester-linked co-drug conjugates with anticancer and/or antirestenotic agents having hydroxyl functions available for esterification, non-limiting examples of which include paclitaxel, camptothecin, 7-ethyl-lO-hydroxycamptothecin (SN38), 7-ethylcamptothecin (SN22), etoposide, fenretinide, and lestaurtinib. The accelerated hydrolytic activation of such conjugates is a special property of the tocopheryloxyacetate esters that does not extend to other redox-silent tocopherol derivatives.

Conjugates of α, β, γ and δ-tocopheryloxyacetic acids (any one or more of these) with any hydroxy-functionalized anticancer or antirestenotic agent can be prepared analogously, for example using the methods disclosed in the Examples, and all of these compounds and their therapeutic uses are contemplated according to the invention.

In contrast, the cleavage kinetics of the aliphatic and certain aromatic esters formed between tocopherol succinate, oxybutyrate or other reported analogs and chemically labile parent drug molecules can be suboptimal for their practical use if it occurs on a timescale similar to that of pharmacophore degradation and inactivation, resulting in markedly lower recovery yields of pharmacologically active products from these co-drugs. And for co-drugs having two cleavable bonds (e.g., tocopheryl succinate, where the linkage between tocopherol and succinate is also labile), further recovery yield loss can result due to hydrolysis of a sizeable fraction of the co-drug via an alternative pathway not leading to co-drug activation.

In comparison, the hydrolysis of conjugates employing the non-cleavable tocopheryloxyacetic moiety favors recovery of both pharmacophores in their functional, unfragmented form with markedly limited competition from side reactions. Notably, the latter property can be advantageous for both aliphatic and aromatic ester co-drugs. In the series of homologous esters based on progressively longer acidic segments

(tocopheryloxyacetic, -propionic, -butyric, etc.), esters based on tocopheryloxyacetic are the only ones enjoying both accelerated and directed hydrolytic activation, thereby providing a unique advantage when such esters are used in co-drug combinations with alcohols, such as paclitaxel, camptothecin, SN38, 7-ethylcamptothecin, CEP-701, fenretinide etc. This increased susceptibility to hydrolytic cleavage is essential for effectively outcompeting the breakdown of the chemically and metabolically unstable pharmacophores and for achieving quantitative recovery of the parent compounds. Of the broad range of complementary pharmacophores whose formulation and delivery can be improved using a tocopheryl oxyacetate moiety in the co-drug, particularly attractive candidates are those incorporating pharmacologically complementary anticancer or antirestenotic agents. The activity of these agents can potentially be enhanced by the therapeutically relevant effects of tocopheryl oxyacetate regenerated as a by-product of the same ester hydrolysis reaction in a synchronized and synlocalized fashion.,

Furthermore, certain compounds having several hydroxyl groups amenable to derivatization offer the possibility of creating "multiple co-drug" structures with high lipophilicity and additionally strengthened pharmacological activity. Thus, the compound may optionally further comprise an ester group formed between a carboxylic acid and the anticancer or antirestenotic agent moiety in the compound.

One example is PhBu-SN38-TOA, shown in Scheme 2 below, where the conjugate of SN38 with tocopheryloxyacetic acid ("SIM38-TOA") is in turn conjugated via its phenolic hydroxyl on the SN38 moiety with 4-phenylbutyric acid, a potent histone deacetylase inhibitor. Another example is SN38-TOA conjugated to a macromolecule, for example a polycarboxylic acid macromolecule. One example is polyglutamate, as shown in Scheme 4 below. The polycarboxylic acid macromolecule may optionally be further esterified with one or more C1-C18 alcohols, which may be used singly or in any combination. If used, the C1-C18 alcohols will typically be linear primary alcohols.

Examples include methanol, ethanol, n-propanol, and n-butanol. Other examples include oleyl alcohol and stearyl alcohol.

The potential therapeutic utility of tocopheryloxyacetic acid ester co-drugs stems from the increased lipophilicity associated with the tocol moiety (logP _0/w of conjugates several units higher compared to that of parent alcohol compounds), which makes the resultant derivatives particularly well-suited for incorporation into cell membranes or encapsulation and delivery in micro- or nanoparticles (NP), thus offering important advantages for treating cancer, arterial restenosis and other proliferative conditions. As an example, in the context of cancer therapy this hydrophobizing effect may facilitate effective intratumoral accumulation and retention of the conjugate by enabling its stable association with cell membrane lipids or with particulate carriers, thus leading to substantially increased site-specificity of the pharmacological effect mediated by the two co-drug forming active moieties. In addition to the ability to favorably change

biodistribution of the co-drug components, the synchronized and synlocalized

regeneration of the two drug molecules exhibiting complementary antiproliferative activities can provide additive or synergistic effects, which in turn can translate into lower therapeutically effective doses, help reduce systemic exposure, and further minimize toxicity.

In some embodiments, the tocopheryloxyacetic acid ester co-drugs may be present as nanoparticles in the form of a nanosuspension; i.e., a suspension in an aqueous medium where the suspended nanoparticles are colloidally stabilized, for example via an ionic or steric stabilizer (e.g., albumin), but in which the nanoparticles do not include a water-insoluble matrix material in addition to the drug substance.

Alternatively, the nanoparticles may additionally include a water-insoluble matrix material, provided that it is biodegradable or bioeliminable, IMonlimiting examples include aliphatic polyesters (e.g ., polylactides and copolymers thereof) and aliphatic polyanhydrides. Exemplary matrix materials include poly(D,L-lactide), poly(D,L-lactide)- poly(ethylene glycol) block copolymer, poly(L-lactide), poly(L-lactide)-poly(ethylene glycol) block copolymer, poly(epsilon-caprolactone), poly(epsilon-caprolactone)- poly(ethylene glycol) block copolymer, poly(lactide-co-glycolide), and poly(lactide-co- glycolide)-poly(ethylene glycol) block copolymer. As used herein, the term "nanoparticle" means a particle whose largest linear dimension is less than 1000 nm. Typically the nanoparticles are approximately spherical and the largest linear dimension is therefore the diameter. The nanoparticles typically have an average diameter less than 200 nm, or less than 150 nm, or less than 100 nm, or less than 75 nm. Typically, the average diameter is at least 10 nm, or at least 20 nm, or at least 40 nm. In some embodiments, the tocopheryloxyacetic acid ester co-drugs may be present in the oil phase of colloidally stable oil-in-water emulsions, particularly nanoemulsions (i.e., particles less than 1 Mm diameter). The co-drug may be dissolved in an oil, thus forming the dispersed internal oily phase of the emulsion. Any of a number of oils can be suitable for dissolving the co-drug, with non-limiting examples being tocopherols (alpha, beta, gamma, delta), tocopherol acetate (also a liquid, but less susceptible to oxidation than tocopherols, thus offering improved stability and longer shelf-life), triolein (glycerol trioleate), and other biocompatible oils suitable for parenteral administration. Suitable nanoemulsions can be prepared using methods known in the art, for example solvent displacement.

EXAMPLES

D-a-tocopheryloxyacetic acid was prepared from D-a-tocopherol and ethyl bromoacetate by the method of Karla A. Lawson, Kristen Anderson, Maria Menchaca, Jeffrey Atkinson, LuZhe Sun, Vernon Knight, Brian E. Gilbert, Claudio Conti, Bob G. Sanders, and Kimberly Kline. Molecular Cancer Therapeutics Vol. 2, 437-444, May

2003.

Conjugate ( 1) of D-a-tocopheryloxyacetic acid and SN38 via 20-OH

Conjugate (1) of D-a-tocopheryloxyacetic acid with SN38 at the 20-OH position was prepared by direct coupling of 10-Boc-SN38 with D-a-tocopheryloxyacetic acid, as shown

Toe = D-a-tocopheryl

Scheme 1. Synthesis of conjugate (1) : SN38-TOA

Preparation of 10-Boc-SN38 was according to the method of Hong Zhao, Belen

Rubio, Puja Sapra, Dechun Wu, Prasanna Reddy, Prakash Sai, Anthony Martinez, Ying Gao, Yoany Lozanguiez, Clifford Longley, Lee M. Greenberger, and Ivan D. Horak. Bioconjugate Chem., Vol. 19, No. 4, 849-859, 2008. A portion of 10-Boc-SN38 (125 mg, 0.25 mmol), D-a-tocopheryloxyacetic acid (142 mg, 0.29 mmol), 4- dimethylaminopyridine tosylate (DPTS) catalyst (150 mg, 0.51 mmol), and l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC) (77 mg, 0.40 mmol) in CH ₂ CI ₂ (2.9 mL) were stirred under argon protection for 7 h at room temperature. After removal of CH ₂ CI ₂ in vacuo, an aqueous 20% solution of NaH ₂ PCy H ₂ 0 (30 mL, acidified with phosphoric acid to pH = 3) was added, the mixture was extracted with ethyl acetate (25 mL), the organic phase was washed with water (2x 30 mL) and dried. The crude product was purified by flash chromatography (silica-gel, chloroform - ethyl acetate, 100 : 0 to 7 : 1), yielding the intermediate 2 (240 mg) . This intermediate was dissolved in dry CH ₂ CI ₂ (2.3 mL), dimethyl sulfide (0.2 mL, 2.70 mmol) and CF ₃ COOH (1.0 mL, 12.8 mmol) were added, the mixture was reacted under argon protection for 3 h at room temperature. The volatiles were removed in vacuo, a 20% aqueous solution of

NaH ₂ P0 ₄ - H ₂ 0 (50 mL) was added, the product was extracted with CHCI ₃ (25 mL) and purified by flash chromatography (silica-gel, chloroform - ethyl acetate, 100: 0 to 1 : 1). Yield of 1 : 185 mg (86%) . ^X H NMR (400 MHz, CDCI ₃ ) of 1 was in agreement with the structure.

Triple conjugate (3) of D-g-tocopheryloxyacetic acid and 4-phenylbutyric acid with SIM38 Triple conjugate (3) of D-o-tocopheryloxyacetic acid and 4-phenylbutyric acid with SN38 was prepared by acylation of SN38 at the 10-OH position with 4-phenylbutyric acid, followed by similar acylation at the 20-OH position in the intermediate 4 with D-o- tocopheryloxyacetic acid, as shown in Scheme 2.

Toe = D-a-tocopheryl

Scheme 2. Synthesis of conjugate (3) : PhBu-SN38-TOA Under an argon atmosphere, SN38 (60 mg, 0.15 mmol), 4-phenylbutyric acid (29 mg, 0.175 mmol) and DPTS catalyst (27 mg, 0.092 mmol) in a mixture of 1-MP (2.0 mL) and CH ₂ CI ₂ (0.7 mL) were cooled in an ice-water bath, and EDC (33 mg, 0.17 mmol) was added. The mixture was stirred in the bath for 15 min, warmed to room temperature (becoming homogeneous) and further stirred for 2h. Another portion of EDC (51 mg, 0.26 mmol) was added, the stirring was continued for an additional 25 h . Aqueous 20% NaH ₂ P0 ₄ -H ₂ 0 (30 mL, acidified with phosphoric acid to pH = 3) was added, the volatiles were removed in the flow of air, the solid reaction product was filtered off, washed with water (30 mL), and dissolved in CHCI ₃ . The crude product was purified by flash chromatography (silica-gel, chloroform - acetonitrile, 100: 0 to 3 : 1). The intermediate 4 (62 mg, 0.115 mmol 77%) in CH ₂ CI ₂ (3.0 mL) was stirred under argon protection with D-a-tocopheryloxyacetic acid (89 mg, 0.182 mmol), DPTS catalyst ( 150 mg, 0.51 mmol) and EDC (74 mg, 0.38 mmol) for 22 h at room temperature. After drying in vacuo, 20% aqueous NaH ₂ P0 ₄ - H ₂ 0 (25 mL, acidified with phosphoric acid to pH = 3) was added, the mixture was extracted with ethyl acetate (20 mL), the organic phase was washed with water (2x 25 mL) and dried . The crude product was purified by flash chromatography (silica-gel, chloroform - ethyl acetate, 100: 0 to 4: 1) Yield of 3 : 90 mg (78%). *H NMR (400 MHz, CDCI ₃ ) of 3 was in agreement with the structure.

Conjugate (5) of D-g-tocopheryloxyacetic acid with fenretinide

Conjugate (5) of D-a-tocopheryloxyacetic acid with fenretinide was prepared as shown in Sche

Fenretinide 5

Toe = D-a-tocopheryl

Scheme 3. Synthesis of conjugate (5) : Fen-TOA

Under an argon atmosphere and protection from light, D-a-tocopheryloxyacetic acid (165 mg, 0.34 mmol), fenretinide ( 113 mg, 0.29 mmol) and DPTS catalyst (113 mg, 0.38 mmol) in CH ₂ CI ₂ (4.0 mL) were cooled in an ice-water bath, and DCC (88 mg,

0.42 mmol) was added . The mixture was stirred in the bath for 15 min, warmed to room temperature, and further stirred for 2h. The reaction mixture was dried in vacuo, 20% NaH ₂ P0 ₄ ' H ₂ 0 ( i0 mL, acidified with phosphoric acid to pH = 3) and ethyl acetate ( 10 mL) were added, the insoluble material was filtered off and washed with ethyl acetate (5 x 2 mL) and with water (5 x 2 mL) . The ethyl acetate phase was washed with water (2 x 20 mL), and d ried . The crude product was purified by flash chromatography (silica- gel, chloroform - hexane - acetonitrile, 50 : 50 : 0 to 50 : 50 :4) Yield of 5: 238 mg (95%) . ¹ H NMR (400 MHz, CDCI ₃ ) of 5 was in agreement with the structure.

Attachment of conjugate (1) to poly-L-qlutamic acid

Conjugate (1) was attached by its phenolic OH to poly-L-glutamic acid via ester bonds as shown in Scheme 4, and the unreacted carboxylic groups on the polymer were then esterified with ethanol .

Scheme 4. Attachment of conjugate (1) to poly-L-glutamic acid : PolyG(SN38-TOA) The sodium salt of poly-L-glutamic acid (Alamanda Polymers, M _n = 15 kDa) was transformed into the free poly-L-glutamic acid by treatment with Dowex-50 (H-form) in dilute aqueous solution, and the water was replaced with N,N-dimethylacetamide

(DMAc), resulting in a solution containing 0.51 mmol/g of carboxylic groups. A portion of the solution (530 mg, containing 0.27 mmol of COOH) was diluted with DMAc ( 1.8 mL) .

Under argon protection, conjugate (1) ( 169 mg, 0.19 mmol), DPTS catalyst (92 mg,

0.31 mmol), EDC (38 mg, 0.19 mmol) and CH ₂ CI ₂ (0.7 mL) were added . The mixture was stirred at room temperature for 24 h, then anhyd rous ethanol (0.37 mL, 6.33 mmol) and another portion of EDC ( 109 mg, 0.57 mmol) were added . The stirring was continued for an additional 25 h, the reaction solution was dried in vacuo, and the residue was triturated with 5% NaH ₂ P0 ₄ - H ₂ 0 (20 mL) . The solid material was filtered off, washed with water ( 15 mL) and dissolved in CH ₂ CI ₂ (20 mL) . The solution was dried over Na ₂ S0 ₄ , filtered from the desiccant, and the solvent was removed in vacuo. The crude polymer (270 mg) was precipitated from the solution in CH ₂ CI ₂ (3 mL) with 2-propanol

(20 mL), and the suspension was concentrated in vacuo to 11 mL. The polymer was filtered off and then washed sequentially with acetonitrile, 2-propanol, and pentane. The procedure was repeated until no more non-polymeric impurities were detected by TLC (silica-gel, heptane - ethyl acetate, 3 : 7) . Yield of the polymeric conjugate : 156 mg . H NMR (400 MHz, CDCI ₃ ) determined ca . 50% of the poly-L-g lutamic acid links modified with 1 (m = 0.5 n, see Scheme 4) .

Conjugate of D-g-tocopheryloxyacetic acid with paclitaxel

D-g-Tocopheryloxyacetic acid (64 mg, 0.13 mmol), paclitaxel ( 100 mg, 0.12 mmol), DPTS catalyst (80 mg, 0.27 mmol) and EDC (48 mg, 0.25 mmol) were reacted in CH ₂ CI ₂ (2.0 mL) under a n argon atmosphere at room temperature for 5h, and the mixture was treated as described above for conjugate 5, omitting the filtration . The crude product was purified by flash chromatography (silica-gel, hexane - ethyl acetate, 4 : 1 to 1 : 1) . Yield of the product : 133 mg (87%) . *H NMR (400 MHz, CDCI ₃ ) was in agreement with the supposed structure of conjugate, confirming the presence of the D- a-tocopheryloxyacetate ester at the 2' hydroxyl of paclitaxel .

Formulation of sub- 100 nm sized polylactide-polyethylene glycol (PLA-PEG) nanoparticles with SN38-TOA (conjugate 1) and its macromolecu lar ( poly-L-glutamic acid based) derivative, PolyG(SN38-TOA)

Biodegradable PEGylated nanoparticles suitable for intravenous administration of SN38 tocopheryloxyacetate-based co-drugs were prepared using a nanoprecipitation method optimized for producing sub- 100 nm sized particulates. Ten mg of the conjugate, 20 mg of PLURONIC® F-68 surfactant and 200 mg of poly(D,L-lactide)-poly(ethylene g lycol) block copolymer ( 15 kDa : 5 kDa) were dissolved in 12 mL of organic solvent (acetone and tetrahydrofuran for SN38-TOA and PolyG(SN38-TOA), respectively) . An 8 mL portion of ethanol was added to the organic phase after complete dissolution of the components. The organic phase was rapidly added to 50 mL of water with magnetic stirring . The mixture was transferred into an evaporation flask, and the solvents were removed by gradually reducing the pressu re from 130 mba r to 40 mbar at 30°C. The formulation was additionally concentrated, glucose was added to the nanoparticle suspension at 5% w/v to adjust the tonicity, and the volume was broug ht to 5.0 mL. The resulting nanoparticles were sterilized by passing them through a 0.22 μιτι filter unit.

The co-drugs were assayed spectrophotometrically against suitable calibration cu rves after nanoparticle dilution with aqueous sodium chloride (5 N) and two-step extraction in 2-butanol or a 1 : 1 : 1 mixture of n-octanol, sec-butanol and acetonitrile, respectively. The co-drug loadings in the nanoparticle formulations were found to be 1.50 and 1.42 mg/mL, respectively. The particle size determined by dynamic light scattering was 80-90 nm.

Nanoemulsion of SN22-TOA dissolved in D-alpha-tocopherol A tocopherol-based nanoemulsion with a 100-nm droplet size containing 7- ethylcamptothecin tocopheryloxyacetate (SN22-TOA) was prepared using a modification of the solvent displacement method. Ten mg of the conjugate, 40 mg of PLURONIC® F- 68 surfactant and 100 mg of D-alpha-tocopherol were dissolved in a mixture of 8 mL of acetone and 12 mL ethanol (alternatively, 20 mL ethanol without acetone could also be used). The organic phase was rapidly added to 50 mL of water with magnetic stirring. The mixture was transferred into an evaporation flask, and the solvents were removed by gradually reducing the pressure from 120 mbar to 40 mbar at 30°C. The formulation was additionally concentrated, glucose was added to the nanoparticle suspension at 5% w/v to adjust the tonicity, and the volume was brought to 5.0 mL. The resulting nanoemulsion was sterilized by passing it through a 0.22 μιη filter unit.

The co-drug was assayed spectrophotometrically against a suitable calibration curve after dilution with aqueous sodium chloride (5 N) and two-step extraction in 2- butanol. The co-drug loading was found to be 1.8-2.2 mg/mL, respectively. The emulsion droplet size determined by dynamic light scattering was 100-130 nm.

Neuroblastoma cell growth inhibition using co-druq-loaded nanoparticles

A luciferase-based method was applied to longitudinally study the effect of the SN38 co-drug-loaded nanoparticles on neuroblastoma cell growth. Human

neuroblastoma IMR-32 cells (a cell line that has features characteristic of high-risk neuroblastoma including MYCN amplification and chromosome lp deletion) were seeded on day -1 on 96-well plates at 40,000 cells per well. On day 0, the cells were incubated with indicated doses of free SN38 (plate 1), blank NP (plate 2), NP loaded with SN38- TOA or NP loaded with PolyG(SN38-TOA) diluted in culture medium. Blank NP were applied as a control at doses equivalent to those of co-drug-loaded NP. After the indicated exposure time, the medium was replaced with fresh culture medium containing 100 Mg/mL of luciferin. The bioluminescent signal from treated and untreated (control) wells was measured at predetermined time points and converted to cell counts. The results are shown in FIG. 1. Both SN38 co-drug-loaded NP formulations and free SN38 applied to cells over 30 min, 4 hr and 24 hr caused profound IMR-32 growth inhibition in the studied dose range (10 to 25 ng/well) measured 7 days post treatment, while the equivalent doses of blank NP had no antiproliferative effect on IMR-32.

Growth profiles of two neuroblastoma cell types, IMR-32 and BE(2)-C treated for 24 hours with 25 ng/well equivalent dose of SN38 in co-drug-loaded NP or free SN38 compared to untreated cells and blank NP controls are shown in FIG. 2. While the less proliferative cell line BE(2)-C was not responsive to NP loaded with PolyG(SN38-TOA), marked growth inhibition was observed with NP loaded with SN38-TOA for both cell types. Formulation of small (85 nm) and large ( 160 nm) sized polylactide-based nanoparticles containing D-a-tocopheryloxyacetic acid conjugate with paclitaxel

Biodegradable NP for intra-arterial delivery of paclitaxel tocopheryloxyacetate co- drug were prepared using a nanoprecipitation protocol optimized for producing differently sized submicronial particles. Ten mg of the conjugate, 100 of mg poly(D,L- lactide) (Mw 63 kDa) and 40 mg PLURONIC® F-68 surfactant were dissolved in a) 10 mL of acetone followed by 10 mL of ethanol after complete dissolution of the components, or b) 8 mL of acetone for making small (85 nm) and large ( 160 nm) NP, respectively. The organic solutions were quickly added to 50 mL of water with magnetic stirring. The resultant mixture was transferred into an evaporation flask, and the solvents were removed by gradually reducing the pressure from 125 mm to 40 mm Hg, 30°C. The formulation was additionally concentrated, glucose was added to the nanoparticle suspension at 5% w/v to adjust the tonicity, and the volume was brought to 5.0 mL. The resulting nanoparticles were sterilized by passing them through a 0.22 pm filter unit. The particle sizes as determined by dynamic light scattering are depicted in FIG. 3.

The co-drug content was assayed spectrophotometrically against a suitable calibration curve after nanoparticle dilution with aqueous sodium chloride (5 N) and two- step extraction in 2-butanol. The co-drug loadings in the 85- and 160-nm sized nanoparticle formulations were found to be 960 and 860 pg/mL, respectively.

Rat aortic smooth muscle cell growth inhibition using paclitaxel co-drug-loaded nanoparticles

Inhibition of vascular smooth muscle cell proliferation contributing to intimal hyperplasia can potentially prevent injury-triggered arterial renarrowing (restenosis) and significantly improve the outcomes of angioplasty procedures used clinically to reopen obstructed blood vessels. Site-specific delivery taking advantage of enhanced

permeability and retention of NP in the injured arterial wall is a promising approach of achieving sustained therapeutically adequate local drug levels while minimizing systemic drug exposure. To evaluate the effectiveness of arterial smooth muscle cell inhibition with NP loaded with D-o-tocopheryloxyacetic acid conjugate with paclitaxel (PTX-TOA), rat aortic smooth muscle cells (A10) exhibiting the defining characteristics of neointimal smooth muscle cells were seeded at 5% confluence on 96-well plates on day -1. On the next day, the cells were treated with the indicated (FIGS. 4 and 5) paclitaxel equivalent doses of the co-drug-loaded NP diluted in cell culture medium (DMEM supplemented with 2% fetal bovine serum and 40 ng per mL of platelet-derived growth factor BB for 1 hr, 4 hr and 24 hr. Blank NP applied at equivalent doses were used as controls. At the end of the incubation period, NP were removed and the cells were incubated under fresh culture medium. Cell viability was determined using the Alamar Blue assay 8 days after treatment. The results, shown in FIG. 4 (160 nm NP) and FIG. 5 (85 nm NP), indicate exposure time-dependent, extensive A10 cell growth inhibition by paclitaxel tocopheryloxyacetate-loaded NP, with a stronger antiproliferative effect exhibited by smaller sized NP.

Although the invention is illustrated and described herein with reference to specific embodiments, the invention is not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims without departing from the invention.